Datasets:

ManuelAlv

/

Cancer

like 0

breast cancer patients especially those with triple‘negative breast cancer is still grave More effective therapeutic targets are needed to optimize the clinical management of breast cancer Although collagen type VIII alpha chain COL8A1 has been shown to be downregulated in BRIP1‘knockdown breast cancer cells its clinical role in breast cancer remains unknownMethods Gene microarrays and mRNA sequencing data were downloaded and integrated into larger matrices based on various platforms Therefore this is a multi‘centered study which contains breast cancer patients and controls COL8A1 mRNA expression in breast cancer was compared between molecular subtypes In‘house immunohistochemistry staining was used to evaluate the protein expression of COL8A1 in breast cancer A diagnostic test was performed to assess its clinical value Furthermore based on differentially expressed genes DEGs and co‘expressed genes CEGs positively related to COL8A1 functional enrichment analyses were performed to explore the biological function and potential molecular mechanisms of COL8A1 underlying breast cancerResults COL8A1 expression was higher in breast cancer patients than in control samples standardized mean differ‘ence confidence interval [CI] “ Elevated expression was detected in various molecular subtypes of breast cancer An area under a summary receiver operating characteristic curve of CI “ with sensitivity of CI “ and specificity of CI “ showed moderate capacity of COL8A1 in distinguishing breast cancer patients from control samples Worse overall survival was found in the higher than in the lower COL8A1 expression groups Intersected DEGs and CEGs positively related to COL8A1 were significantly clustered in the proteoglycans in cancer and ECM‘receptor interaction pathwaysConclusions Elevated COL8A1 may promote the migration of breast cancer by mediating the ECM‘receptor interac‘tion and synergistically interplaying with DEGs and its positively related CEGs independently of molecular subtypes Several genes clustered in the proteoglycans in cancer pathway are potential targets for developing effective agents for triple‘negative breast cancerKeywords COL8A1 Breast cancer Immunohistochemistry staining MechanismCorrespondence fengzhenbo_gxmu163com chenganggxmueducn Wei Peng and Jian‘Di Li contributed equally as first authors Department of Pathology The First Affiliated Hospital of Guangxi Medical University NO6 Shuangyong Road Nanning Guangxi People™s Republic of ChinaFull list of author information is available at the end of the BackgroundBreast cancer poses a grave threat to female health According to the latest American cancer statistics breast cancer is estimated to be the most common cancer and the second most common cause of cancerassociated The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cPeng a0et a0al Cancer Cell Int Page of deaths in women [] Owing to higher distal metastatic and recurrent rates triplenegative breast cancer TNBC patients exhibit worse overall and diseasefree survival than any other type of breast cancer [“] Etiology investigations suggest that hereditary factors account for nearly onetenth of breast cancer cases Other risk factors such as early or delayed menstruation nulliparity hormone replacement therapy and alcohol consumption also contribute to the prevalence of breast cancer [] Clinical practice guidelines recommend that females aged “ or “ who are at higher risk screen for breast cancer [] Imaging examinations such as bilateral breast Xray imaging positron emission tomography“computed tomography and ultrasound histological findings and especially molecular pathology are the predominant methods of breast cancer diagnosis and assessment [ ] Based on the tumor burden optimal treatments namely breastconserving surgery radiotherapy chemotherapy and endocrine therapy are individually designed for breast cancer patients [] For TNBC patients anthracyclines and taxanes are preferred in the initial treatment and neoadjuvant therapy has been recognized as a standard strategy [“] Unfortunately neither endocrine therapy nor trastuzumab treatment is effective for TNBC Targeted drugs for example vascular endothelial growth factor [VEGF] antibodies epidermal growth factor receptor [EGFR] inhibitors and mammalian target of rapamycin [mTOR] inhibitors are gradually being employed in TNBC treatment even though their therapeutic effects are unsatisfactory [“] Therefore the situation faced by breast cancer”especially TNBC”patients is still grave More effective therapeutic targets are needed to optimize the clinical management of breast cancerMolecular events occurring in breast cancer help us better understand the onset and progression of breast cancer It is generally agreed that chromosome 1q amplification chromosome 16q deletion and PIK3CA mutations are the most common pathways leading to luminal breast cancer [ ] Moreover breast cancer gene BRCA1 and P53 mutations EGFR upregulation and cytokeratin downregulation have been associated with TNBC [ ] It has also been reported that circSEPT9 promotes tumor formation and TNBC progression [] Recently ˆ†Np63 has been found to participate in breast cancer metastasis and dissemination [] On the other hand several genes protect patients from breast cancer progression For example ZNF750 miR5745p and circKDM4C can inhibit breast cancer progression by mediating the epigenetic regulation of prometastatic genes indirectly suppressing SKILTAZCTGF and miR548pPBLD axis regulation [“] Based on these discovered molecular mechanisms some progress has been made in breast cancer treatment Delivering dual microRNA using CD44targeted mesoporous silica nanops proved to be effective in TNBC treatment [] Although many studies provided in a0vitro and in a0vivo a detailed molecular picture of TNBC cancers [“] the underlying cause of breast cancer has not been fully understood Further research is required to elucidate the breast cancer mechanisms and discover effective therapeutic targets for TNBCCollagen type VIII alpha chain COL8A1 also named C3orf7 is located at chromosome and encodes alpha chain in collagen type VIII which is an essential component of extracellular matrix ECM [] Previous studies mainly addressed the relevance between COL8A1 and agerelated macular degeneration ADM as well as cell proliferation [“] Recently limited studies demonstrate the deregulation of COL8A1 in various cancers Elevated COL8A1 expression was found in gastric cancer patients and higher COL8A1 correlated with advanced tumor stages and worse overall survival condition and COL8A1 was selected as a candidate diagnostic biomarker in gastric cancer [“] Additionally upregulation of COL8A1 was also reported in adamantinomatous craniopharyngioma [] Furthermore COL8A1 proved to participate in the progression of colon adenocarcinoma possibly by mediating focal adhesionrelated pathways [] COL8A1 upregulation induced by TGFβ1 was found in renal cell carcinoma carcinogenesis and also correlated with poor prognosis [] Moreover elevated COL8A1 in hepatocellular carcinoma promoted tumor cells proliferation invasion and in a0vivo tumorigenicity [] Thus far only few studies mentioned COL8A1 in breast cancer COL8A1 was one of the key genes restored by epigallocatechin3gallate in a murine breast cancer model [] COL8A1 proved downregulated in both BRIP1knockdown breast cancer cells and MCF10A a0CDH1 noncancer breast cells [ ] However the role of COL8A1 in breast cancer remains unknownConsidering this knowledge gap our study aimed to explore the role of COL8A1 in breast cancer We were focused on investigating the expression of COL8A1 messenger RNA mRNA in various molecular subtypes of breast cancer by analyzing gene microarray and RNA sequencing data sets The COL8A1 protein expression level was validated by immunohistochemistry staining We also aimed to determine prognostic value of COL8A1 in breast cancer to pave the way for future clinical applications Moreover we explored the molecular mechanisms of COL8A1 underlying breast cancer to improve our knowledge of breast cancer carcinogenesis and progression 0cPeng a0et a0al Cancer Cell Int Page of MethodsExpression of a0COL8A1 mRNA in a0breast cancerWe integrated gene microarrays and mRNA sequencing data downloaded from Gene Expression Omnibus The Cancer Genome Atlas TCGA the GenotypeTissue Expression the Sequence Read Archive ArrayExpress and Oncomine The search formula based on MESH terms was as follows Breast OR mammary AND neoplasm OR cancer OR adenoma OR carcinoma OR tumor OR BRCA OR neoplasia OR malignant OR malignancy Studies were screened according to the following criteria i the studied species should be Homo sapiens ii the studied specimens should be tissue dissected from patients or healthy individuals rather than cell lines In the case of duplicated studies or samples the most recent version was retained The exclusion criteria were as follows i expression profiles not including COL8A1 ii breast cancer patients receiving hormone therapy or chemotherapy iii stromal rather than epithelial tumors and iv metastatic rather than primary tumors The included data sets were carefully checked and a log2 transformation was performed if any matrices had not been normalized Additionally the data sets were integrated into larger matrices according to various platforms and batch effects between studies were removed using the limmavoom package in R v361 Subsequently COL8A1 expression values were extracted and grouped according to specimen types Standardized mean difference SMD were calculated to compare the expression of COL8A1 mRNA between breast cancer patients and control samples using STATA v120 Heterogeneity between the included studies was assessed with the I2 statistic Statistical significance was set to an I2 value greater than with a Pvalue less than A random effects model was used in the case of significant heterogeneity Sensitivity analysis was performed to probe the potential source of heterogeneity and a publication bias test was used to evaluate the stability of the SMD results Subgroup analysis was performed to compare the COL8A1 expression levels between molecular subtypes luminal A luminal B human epidermal growth factor receptor 2positive [HER2 ] and TNBCDiagnostic value of a0COL8A1 in a0breast cancerA diagnostic test was performed to assess the clinical significance of COL8A1 in breast cancer Based on the expression value of COL8A1 a receiver operating characteristic ROC curve was plotted to compute the area under the curve AUC using IBM SPSS Statistics v190 AUC values of less than between and and greater than represented weak moderate and strong discriminatory capability of COL8A1 respectively between breast cancer patients and control samples The true positives false positives true negatives and false negatives rates were calculated and the cutoff values were identified A summary receiver operating characteristic sROC curve was drawn using STATA v120 to assess the general discriminatory capability of COL8A1 between breast cancer patients and control samples The significance of the area under the sROC curve was consistent with that of the ROC curve The diagnostic odds ratio DOR sensitivity specificity positive diagnostic likelihood ratio DLR P and negative diagnostic likelihood ratio DLR N were also calculated to precisely determine the accuracy and validity of COL8A1 in distinguishing breast cancer patients from control samplesPrognostic value of a0COL8A1 in a0breast cancerTo explore the relation between COL8A1 mRNA expression and prognosis of breast cancer patients information on clinicopathological parameters was collected The independent samples ttest or oneway analysis of variance was used to identify statistically significant differences in COL8A1 expression between two or more groups A Pvalue of was considered statistically significant Kaplan“Meier curves were used to compare high and low COL8A1 expression groups in terms of survival The logrank test was used to determine whether the prognostic difference was statistically significantInvestigation of a0COL8A1 protein expression in a0breast cancer by a0immunohistochemistry stainingA total of nonspecific invasive breast carcinoma and normal breast tissue specimens were obtained from the First Affiliated Hospital of Guangxi Medical University PRCHINA All patients had previously signed informed consent forms and our research was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University The breast cancer and normal breast tissue specimens were fixed with formalin The two steps immunohistochemistry method was used to determine the protein expression of COL8A1 The primary antibody was polyclonal Antibody to COL8A1 concentrated dilution purchased from Wuhan Pujian COLTD Supervision TM MouseRabbitHRP Broad Spectrum Detection System Product No D300415 was purchased from Shanghai Long Island The experimental procedure conformed to the manufacturer™s instructions The patients™ clinicopathological information was analyzed to determine the relationship between COL8A1 protein expression and prognosisEvaluation of a0genetic alteration and a0mutation landscapesThe cBioPortal for Cancer Genomics httpcbiop ortal proved to be a powerful tool and facilitated our online search and cancer genomics data analysis Using 0cPeng a0et a0al Cancer Cell Int Page of cBioPortal we gained insight into the genetic alterations of COL8A1 in breast cancer patients and obtained information on the association between COL8A1 alterations and breast cancer patient survival We selected the Breast Invasive Carcinoma TCGA Firehose Legacy cohort which contains patients and used a method of mRNA expression zscores relative to diploid samples RNASeqV2 RSEM We also considered the mutation types of COL8A1 in breast cancer patients using the Catalogue of Somatic Mutations in Cancer COSMIC which has been recognized as the most detailed resource for somatic mutations in cancerIdentification of a0differentially expressed genes and a0COL8A1 co‘expressed genes in a0breast cancerTo gain insight into the role of COL8A1 in breast cancer we identified DEGs and COL8A1 CEGs using the limmavoom package The DEG criteria were as follows ilog2FoldChange and ii adjusted Pvalue The CEG criteria were as follows irelation coefficient and ii Pvalue Upregulated DEGs and CEGs positively related to COL8A1 were intersected Similarly downregulated DEGs and CEGs negatively related to COL8A1 were intersectedMolecular mechanisms of a0COL8A1 underlying breast cancerOverlapping genes were used to perform function enrichment to shed light on the potential mechanisms of COL8A1 underlying breast cancer The R clusterProfiler package was used to conduct Gene Ontology GO Kyoto Encyclopedia of Genes and Genomes KEGG Disease Ontology DO and Reactome pathway analyses Proteintoprotein interaction PPI network was constructed using STRING https strin gdb to investigate protein interactions Hub genes and functional modules were identified using Cytoscape v361 Based on breast cancer patients the mutation landscapes of genes clustered in essential pathways were visualized using the TCGAmutations package in R v361ResultsUpregulation of a0COL8A1 mRNA in a0breast cancerAdditional file a0 Figure S1 shows the flow diagram of the study inclusion process A total of studies were included and integrated into larger platform matrices covering breast cancer patients and controls Table a0 COL8A1 was generally upregulated in breast cancer compared to normal breast tissue Thirteen of the twenty platforms showed much higher COL8A1 expression in breast cancer patients than in control samples Additional file a0 Figure S2 Because of significant heterogeneity I2 P a random effects model was used An SMD value of confidence interval [CI] “ showed that COL8A1 expression was significantly higher in breast cancer than in nonbreast cancer tissue Fig a0 Sensitivity analysis indicated that the included studies could not explain the source of heterogeneity Additional file a0 Figure S3a No publication bias existed Additional file a0 Figure S3b Subsequently we compared COL8A1 expression levels between different subtypes of breast cancer COL8A1 expression was universally higher in luminal A luminal B HER2 and TNBC patients than in control samples Additional file a0 Figure S4 Additional file a0 Figure S5 and Additional file a0 Figure S6a Furthermore three platforms showed significantly higher COL8A1 expression in nonTNBC than TNBC while only one showed lower expression in nonTNBC than TNBC Additional file a0 Figure S6b However an SMD of ˆ’ CI ˆ’“ showed no difference in COL8A1 expression between TNBC and nonTNBC patients Additional file a0 Figure S7 Subgroup analysis of four molecular subtypes of breast cancer showed no significant differences in COL8A1 expression levels between them Fig a0Diagnostic and a0prognostic value of a0COL8A1 mRNA in a0breast cancerThe clinical value of COL8A1 in breast cancer was found to be promising Among the thirteen platforms showing high COL8A1 expression twelve platforms indicated the ability of COL8A1 in differentiating breast cancer patients and control samples where four platforms showed strong discriminatory capability of COL8A1 between breast cancer patients and control samples Additional file a0 Figure S8 An area under the sROC curve of CI “ with sensitivity of CI “ and specificity of CI “ displayed moderate capacity in distinguishing breast cancer patients from control samples Fig a03a A DOR of CI “ also highlighted the discriminatory ability of COL8A1 in breast cancer Fig a03b The DLR P and DLR N were CI “ and CI “ respectively Additional file a0 Figure S9 As shown in Additional file a0 Figure S10 and Additional file a0 Table a0S1 elevated COL8A1 expression correlated with race white black molecular subtypes of breast cancer luminal B luminal A TNBC ER PR and HER2 status Moreover Kaplan“Meier curves indicated worse overall survival in high compared to low COL8A1 expression groups Fig a0Expression levels and a0clinical significance of a0COL8A1 protein in a0breast cancerClinical data of breast cancer samples used to perform in immunohistochemistry was summarized 0cPeng a0et a0al Cancer Cell Int Page of ldnaoPESGi abarA iduaSESG ecnarFESG ASUESG inapSESG ecnarFESG ecnarFESG adanaCESGkramneDESGi eropagnSESGl yatIESG ASUESG ynamreGESG inapSESG ASUESGASUESG cil bupeRhcezCESG ldnaerIESGi eropagnSESGASUESG ailartsuAESG iocxeMESGi abarA iduaSESGASUESG ASULPG‘ESGadanaCESG ASUESG adanaCESGASULPG‘ESGASULPG‘ESGadanaCESGianhCESG anhCi ESG ASUESG ASUESGianhCESGl aisyaaMESGi anhCESGadanaCESGi anhCESGASUESGi anhCESGASUESGi eropagnSESGl aisyaaMESGASUESG ESG ESG ESG ESG ESGASUESG ESGASU ESGASU ESGanhCi ESGynamreG ESGASU ESGˆ’‘ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’LPGLPGLPGLPGLPGLPGLPGˆ’LPGLPGLPGLPGLPGLPGLPGxETG‘AGCTESGESGESGESGESGsrebmun latoTseirtnuoC seireSOEG NTNFPFPTleuav‘PfdtlortnoC ACRBnoisseccADSMNDSMN stesataddell a0 orneeht a0fonoitamrofni cisaB elbaT 0cPeng a0et a0al Cancer Cell Int Page of Fig General expression status of COL8A1 in breast cancer BRCA compared to non‘BRCA tissues A standardized mean difference SMD value and 95CI with no overlap of zero indicated COL8A1 was significantly upregulated in BRCA compared to non‘BRCA tissuesAdditional file a0 Table a0 S2 Protein expression confirmed the upregulation of COL8A1 in breast cancer The breast cancer patients whose specimens were analyzed in this study were aged between and mean and their followup durations ranged from to a0 days Immunohistochemistry staining showed varying coloration intensity of COL8A1 in breast cancer and normal breast tissue COL8A1 was negatively weakly moderately or strongly stained in normal breast epithelium Fig a05a“d and breast cancer tissue Fig a0 5e“h According to the staining intensity and color range percentages of breast cancer tissue specimens exhibited low and exhibited high COL8A1 expression whereas of normal breast tissue specimens exhibited low and exhibited high COL8A1 expression A Chi square test confirmed the significantly higher expression of COL8A1 in breast cancer than normal breast tissue χ2 P Moreover elevated COL8A1 expression correlated with estrogennegative ER breast cancer P Genetic alterations and a0mutation kinds of a0COL8A1 in a0breast cancerAlterations and mutations of COL8A1 in breast cancer were relatively frequent Based on cBioPortal COL8A1 was altered in of breast cancer patients Additional file a0 Figure S11 Amplification and high and low mRNA were the main alterations No statistically significant difference in overall and diseasefree survival was found between high and low COL8A1 expression breast cancer groups P Furthermore according to COSMIC substitution missense mutations were the most frequent types Additional file a0 Table a0S3DEGs and a0COL8A1 CEGs in a0breast cancerA total of platform matrices were collected to determine the DEGs The approach has been aforementioned Initially upregulated DEGs downregulated DEGs CEGs positively related to COL8A1 and CEGs negatively related to COL8A1 were identified Additional file a0 Figure S12 shows partial DEGs and CEGs After being intersected 0cPeng a0et a0al Cancer Cell Int Page of Fig Subgroup analysis based on the subtypes of breast cancer The result indicated that the elevated COL8A1 expression shared no significant difference among Luminal A Luminal B HER‘ and Three Negative Breast Cancer TNBC subgroupsthe DEGs and CEGs were divided into two gene sets overlapping upregulated DEGs and CEGs positively related to COL8A1 all genes appeared in no fewer than three data sets and overlapping downregulated DEGs and CEGs negatively related to COL8A1Potential mechanisms of a0COL8A1 underlying breast cancerThe GO KEGG DO and Reactome pathway analyses based on the intersected genes are shown in Additional file a0 Table a0S4 Regarding the overlapping upregulated DEGs and CEGs positively related to COL8A1 the following KEGG pathways were significantly aggregated 0cPeng a0et a0al Cancer Cell Int Page of Fig Diagnostic value of COL8A1 in breast cancer BRCA a Summary receiver operating characteristic sROC curve b Forest plot of diagnostic odd ratio DOR An AUC value and a DOR signified COL8A1 possessed moderate capability in distinguishing BRCA from non‘BRCA patients AUC area under the curve 0cPeng a0et a0al Cancer Cell Int Page of Fig The prognostic value of COL8A1 mRNA in breast cancer tissues a GSE25307 b GSE35629‘GPL1390 c TCGA In the GSE25307 cohort high COL8A1 group possessed poor overall survival condition compared to low COL8A1 group in breast cancer patients 0cPeng a0et a0al Cancer Cell Int Page of See figure on next pageFig Protein expression levels of COL8A1 in breast cancer BRCA and normal breast tissues a‘d normal breast tissues e“h BRCA tissues Magnification × COL8A1 was negatively stained in normal breast epithelium a and BRCA e COL8A1 was weakly stained in normal breast epithelium b and BRCA f COL8A1 was moderately stained in normal breast epithelium c and BRCA g COL8A1 was strongly stained in normal breast epithelium d and BRCA h According to staining intensity and percentage of color range BRCA tissues exhibited low COL8A1 expression and BRCA tissues exhibited high COL8A1 expression While normal breast tissues exhibited low COL8A1 expression and normal breast tissues exhibited high COL8A1 expressionFig a06a proteoglycans in cancer Additional file a0 Figure S13 ECMreceptor interaction Additional file a0 Figure S14 and several cancer pathways such as thyroid cancer colorectal cancer and hepatocellular carcinoma Interestingly genes WNT2 GADD45B FZD2 CDKN1A KRAS LEF1 WNT7B BAK1 BRCA2 CDK4 GRB2 HRAS PIK3R3 and POLK were associated with the breast cancer pathway ID hsa05224 even though is not in the top KEGG pathways Moreover DO analysis indicated that these genes are closely associated with myeloma and bone marrow cancer Fig a06b as well as renal cell carcinoma ovarian cancer renal carcinoma and other types of cancer Furthermore Reactome pathway analysis revealed extracellular matrix anization ECM proteoglycans integrin cell surface interactions and degradation of the extracellular matrix as the top four metabolic pathways Fig a0 6c Regarding GO enrichment extracellular matrix anization extracellular matrix and extracellular matrix structural constituent were the most clustered Biological Process BP Cellular Component CC and Molecular Function MF terms respectively Fig a07a The proteoglycans in cancer and ECMreceptor interaction pathways were selected to construct PPI networks Fig a07b c FN1 and ITGB1 were identified as the hub genes in the two networks respectively The mutation landscapes of the genes in these two important pathways are shown in Fig a0 In particular FN1 was altered in of breast cancer samples where missense mutations accounted for The regulatory networks of COL8A1 and enriched genes in the proteoglycans in cancer and ECMreceptor interaction pathways as well as the top two functional modules are displayed in Additional file a0 Figure S15 On the other hand the enrichment results regarding the overlapping CEGs negatively related to COL8A1 and downregulated DEGs showed no statistical significance these data are therefore not shown Additional file a0 Table a0S5DiscussionThe highlight of this study is that it comprehensively explored the upregulation of COL8A1 mRNA in breast cancer from multiple aspects based on breast cancer patients and controls Our study is multicentered because we collected breast cancer patients from Asia American Europe and Oceania covering different countries This is the first study to investigate the protein expression of COL8A1 in breast cancer using immunohistochemistry staining Moreover this study is the first to assess the clinical prognostic value of COL8A1 in breast cancer Furthermore our study sheds light on the biological function and potential molecular mechanisms of COL8A1 underlying breast cancerThis study demonstrates the upregulation of COL8A1 in breast cancer We found higher COL8A1 expression in breast cancer than normal breast tissue based on large platform matrices integrated from data sets Subgroup analysis based on four molecular subtypes of breast cancer showed that elevated COL8A1 expression is independent of subtypes Further analysis also revealed universally higher COL8A1 expression levels in luminal A luminal B HER2 and TNBC patients than in control samples A comparison of COL8A1 expression between nonTNBC and TNBC patients showed no statistically significant difference Immunohistochemistry staining confirmed the upregulation of COL8A1 protein in breast cancer We thus concluded that COL8A1 expression is higher in breast cancer patients than in control samples and that upregulation is independent of molecular subtypes of breast cancer Though the expression of COL8A1 in tissue cannot reflect the early diagnostic value in breast cancer we assume that if COL8A1 is also differentially expressed in the bodily fluid of patients it will be possible to serve as a potential diagnostic marker for breast cancer Nevertheless no previous studies have demonstrated the expression level of COL8A1 in the bodily fluid of breast cancer patients until nowWe determined the clinical value of COL8A1 in breast cancer for the first time Our diagnostic test showed a moderate discriminatory capability of COL8A1 between breast cancer and normal breast tissue Higher COL8A1 expression levels in breast cancer patients correlated with worse survival outcomes Additionally COL8A1 expression was much higher in patients of the white than of the black race Our TCGA cohort analysis showed lower COL8A1 upregulation levels in TNBC than in the luminal A and B subtypes Furthermore high COL8A1 protein levels were related to ER breast cancer Thus COL8A1 might serve as a prognostic marker for breast cancer 0cPeng a0et a0al Cancer Cell Int Page of 0cPeng a0et a0al Cancer Cell Int Page of Fig Functional enrichment based on overlapping genes of upregulated DEGs and COL8A1 positively related CEGs a Kyoto Encyclopedia of Genes and Genomes b Disease Ontology c Reactcome DEGs differentially expressed genes CEGs co‘expressed genes 0cPeng a0et a0al Cancer Cell Int Page of Fig GO enrichment based on overlapping genes of upregulated DEGs and COL8A1 positively related CEGs a GO analysis b Protein‘to‘protein internet based on KEGG pathway proteoglycans in cancer ID hsa05205 c Protein‘to‘protein internet based on KEGG pathway ECM‘receptor interaction ID hsa04512 GO Gene Ontology DEGs differentially expressed genes CEGs co‘expressed genes KEGG Kyoto Encyclopedia of Genes and GenomesMore importantly our study provides important clues about the role of COL8A1 in breast cancer for the first time The intersected CEGs positively related to COL8A1 and upregulated DEGs were significantly aggregated in several cancer pathways such as thyroid cancer colorectal cancer and hepatocellular carcinoma Interestingly we noticed that genes related to COL8A1 WNT2 GADD45B FZD2 CDKN1A KRAS LEF1 WNT7B 0cPeng a0et a0al Cancer Cell Int Page of Fig Mutation landscapes of COL8A1 and co‘expressed genes clustered in two Kyoto Encyclopedia of Genes and Genomes pathways proteoglycans in cancer and ECM‘receptor interactionBAK1 BRCA2 CDK4 GRB2 HRAS PIK3R3 and POLK were associated with the breast cancer pathway ID hsa05224 even though this pathway is not in

Thyroid_Cancer

In a novel coronavirus SARSCoV2 was found to cause a highly contagious disease characterized by pneumonia The disease COVID19 quickly spread around the globe escalating to a global pandemic In this review we discuss the virological immunological and imaging approaches harnessed for COVID19 diagnosis and research COVID19 shares many clinical characteristics with other respiratory illnessesAccurate and early detection of the infection is pivotal to controlling the outbreak as this enables case identification isolation and contact tracing We summarize the available literature on current laboratory and pointofcare diagnostics highlight their strengths and limitations and describe the emerging diagnostic approaches on the horizonWe also discuss the various research techniques that are being used to evaluate host immunity in laboratoryconfirmed patients Additionally pathological imaging of tissue samples from affected patients has a critical role in guiding investigations on this disease Conventional techniques such as immunohistochemistry and immunofluorescence have been frequently used to characterize the immune microenvironment in COVID19 We also outline the emerging imaging techniques such as the RNAscope which might also aid in our understanding of the significance of COVID19specific biomarkers such as the angiotensinconverting enzyme ACE2 cellular receptorOverall great progress has been made in COVID19 research in a short period Extensive global collation of our current knowledge of SARSCoV2 will provide insights into novel treatment modalities such as monoclonal antibodies and support the development of a SARSCoV2 vaccineKeywordsCOVID19 immunology pathology diagnostics specific T cellsIntroductionIn December a novel respiratory disease named coronavirus disease COVID19 was detected by physicians in Wuhan China The disease was found to be caused by the severe acute respiratory syndrome SARS“CoV2 RNA virus12 Within a matter of weeks COVID19 had spread rapidly and escalated to a global pandemic At the time of writing June million cases had been reported and patients had succumbed to the disease worldwide3 Indeed patients with COVID19 are at high risk of developing a severe and critical disease4 Therefore rapid and accurate diagnostic tests are urgently needed to effectively isolate identify and treat infected individuals and to contain the spread of the virus Failure to do so will inevitably lead to spikes in cases and the resultant overcrowding and collapse of healthcare services5 Moreover research into this novel virus is also critical to understand its pathogenesis and its interaction with the human immune system Insights from such research will guide the design of public health policies and protocols to 1Lee Kong Chian School of Medicine Nanyang Technological University Singapore Singapore2Yong Loo Lin School of Medicine National University of Singapore Singapore Singapore3Institute of Molecular Cell Biology IMCB Agency of Science Technology and Research ASTAR Singapore Singapore4Department of Anatomical Pathology Singapore General Hospital Singapore SingaporeThese authors contributed equallyReceived June and in revised form July Accepted for publication July Corresponding AuthorJoe Poh Sheng Yeong Institute of Molecular Cell Biology IMCB Agency of Science Technology and Research ASTAR College Road Academia Level Diagnostics Tower Singapore Singapore Email yeongpsimcbastaredusg 0c SLAS Technology identify susceptible individuals and diagnostic prognostic and treatment approaches for patientsCurrent diagnostic approaches predominantly involve established virological procedures such as nucleic acid hybridization techniques reversetranscriptase PCR [RTPCR] and recombinase polymerase amplification RPA as well as immunologic approaches like antibody assays Each approach boasts unique strengths and weaknesses For instance while RTPCR demonstrates high sensitivity and specificity its capabilities have been severely limited for practical reasons during this current pandemic due to global shortages of skilled personnel reagents and equipment and a processing time of up to days By contrast immunologic tests such as antibody assays are rapid and require minimal equipment but they have limited utility in the context of acute diagnosis of SARSCoV2 infections This is because it can take several days to weeks following symptom onset for a patient to mount a detectable antibody response6Immunological tools in research include enzymelinked immunosorbent assays ELISAs flow cytometry and mass cytometry CyTOF Imaging techniques for pathological analyses include conventional approaches such as hematoxylin“eosin HE staining immunohistochemical IHC staining or transmission electron microscopy TEM and RNAscope Each of these methods is used to examine the pathophysiology underlying COVID19 from a different perspective each with their own advantages and disadvantages For example it has been established that the entry of SARSCoV2 intro cells depends on the binding of viral proteins with the human receptor angiotensinconverting enzyme ACE2 receptors7 Additionally evidence shows that the type II transmembrane protease TMPRSS2 is also essential for viral entry by priming the viral spike protein for binding to ACE28 Therefore considerable research efforts employing different techniques have been directed at mapping the distribution of ACE2 and TMPRSS2 in tissues and their relationship to the observed manifestations of disease Together the combination of these approaches has advanced our understanding of COVID19In this review we discuss the current approaches in COVID19 diagnosis and research with a focus on findings from virological and pathological imaging methods We also discuss immunological methods which are increasingly recognized as an integral component of the disease processDiagnosticsThe most common symptoms of COVID19 at initial presentation are nonspecific and include a high fever a new and persistent cough and fatigue910 Due to similarities between the clinical characteristics of COVID19 and many other respiratory illnesses the accurate and early detection of infection is pivotal for outbreak control Any delays in diagnosis are increasingly measured in lives lostAccording to the World Health anization WHO the immediate goal for research into COVID19 diagnostics is the development of RNA assays antibody and antigen assays and pointofcare detection11 The intermediateterm priority would be their integration into multiplex diagnostic platforms while the longterm goal would be the investigation of prognostic markersIn this section we summarize the current and emerging diagnostic tools for SARSCoV2 through the lens of immunologyLabBased TestsRTPCR Molecular Testing The detection of viral nucleic acids by RTPCR is the primary method used to confirm a suspected case of COVID19 RTPCR and other nucleic acid hybridization techniques are an integral part of virology and are applied in a broad range of settings including screening diagnosis informing medical and therapeutic decisions and assessing cure rates from therapy12 Chinese officials released the genomic sequence of SARSCoV2 to public databases early in the course of the outbreak13 and the WHO has since published seven protocols for RTPCRbased diagnostics Because of the high sensitivity and specificity of RTPCR it is regarded as the œgold standard for virus detection14 There are two essential steps in the process viral RNA extraction and PCR amplification and probebased detection Multiple largescale highthroughput instruments are available for automating both steps such as the Roche Cobas system which has an advertised throughput of tests per hours15However RTPCRbased testing is costly and timeconsuming requiring up to days using centralized laboratory equipment and skilled personnel furthermore global supply chain challenges have led to significant shortages of essential reagents Lastly falsenegative results due to low sample volumes variable sampling techniques and sampling locations sample degradation during transportation andor improper nucleic acid extraction are a concern16“ In addition the differences in detectable viral material in different sampling locations eg nasopharyngeal vs bronchoalveolar lavage fluid [BALF] vs rectal samples might also explain the falsepositive RTPCR results on repeat testing in œrecovered COVID19 patients Indeed one postmortem case study revealed residual virus in lung tissue despite consecutive negative results on PCR testing from nasopharyngeal swabs19 Separately Winichakoon et al outlined a case of repeatedly negative nasopharyngeal and oropharyngeal swabs in a clinically deteriorating patient where only a BALF PCR test returned positive20Given the high expression of ACE2 on alveolar epithelial cells and negative expression on nasal oral and nasopharynx 0cTan et al cells21 it would be prudent to perform bronchoalveolar lavage on patients to rule out falsenegative results from swabs of upper respiratory tract samples20LabBased Immunological Assays In contrast to RTPCR techniques that detect viral nucleic acids serological and immunological assays aim to detect antibodies against SARSCoV2 or antigenic proteins in infected individuals Neutralization assays are considered the gold standard for assessing neutralizing protective antibodies22 however these assays require specialized biosafety level BSL3 facilities and still take several days to complete Another type of labbased antibody assay is the traditional ELISA which detects all binding antibodies The four principal types of ELISA are direct indirect competitive and sandwich ELISA the indirect ELISA is the most common method for determining antibody concentrations ELISAs have good concordance with neutralization assays for the detection of antibody responses in SARSCoV223 Unfortunately both methods require skilled operators and are limited by low throughput due to the absence of fully automated systemsSerological diagnostics offer several advantages Re quirements for specimen quality are comparatively less stringent than for nucleic acid tests as the antibodies are uniformly distributed in the serum24 Consequently sampling location concerns do not apply here Furthermore good correlation between IgG ELISAs performed on both conventional serum samples and plasma samples have been reported25 of which the latter may be conveniently obtained from residual blood submitted for other routine laboratory testsOne pitfall of antibody assays is their limited utility early in the course of any infection Sparse data are available with regard to the antibody responses produced by patients with COVID19 It seems that SARSCoV2 IgM is detectable at a median of days after symptom onset while IgG is detectable after days26 with the seroconversion rate approaching by day An Italian research group noted that the performance of a commercial VivaDiag COVID19 IgMIgG test was very poor with a sensitivity of only and a negative predictive value of in a cohort of suspected COVID19 patients in the emergency room setting27 As such we believe that for now RTPCR testing is likely more appropriate for diagnosing acute COVID19Notably a longitudinal study examining the IgGIgM profiles of patients found that seroconversion for IgG and IgM occurred in no specific chronological order with a median of days after symptom onset28 all patients achieved seroconversion by day Consequently the detection of both IgG and IgM simultaneously rather than one antibody alone would be idealAnother concern surrounding serologic diagnostics is the production of falsepositive results from crossreactivity due to the high prevalence of the four endemic human coronaviruses in the human population In SARSCoV2 the spike S protein which includes two regions S1 and S2 and the nucleocapsid N protein NP are the major immunogens29 and therefore most diagnostics rely on the detection of antibodies specific for these antigens One work suggests that of the possible targets the S1 subunit antigen is more specific than either the whole S antigen or the N antigen for detecting SARSCoV2 antibodies with no crossreactivity to other coronaviruses except for SARSCoV23 Given that only SARSCoV infections were recorded worldwide30 the risk of false positives from this crossreactivity is miniscule However NP ELISAs are more sensitive than S1 in detecting antibodies in those with a mild infection23 Importantly as in SARSCoV most of the neutralizing antibodies are directed against the S protein31 of which S1 contains a receptorbinding domain RBD responsible for making contact with ACE2 to facilitate viral entry7 Thus theoretically only diagnostics that detect S1specific antibodies are suitable to infer immunity to COVID19 this fact is corroborated by evidence that antiS RBD but not antiNP IgG levels correlated with neutralizing antibody titers in sera from a cohort of recovered patients32 The number of commercial antibody assays is growing detecting either antiNP antibodies antiS1S antibodies or both there is also large variation in their claimed sensitivities and specificities33 Based on the available evidence an ideal serological assay would be a combined test that simultaneously detects both antibodies to NP and S1 antigens assessment of antiNP antibodies has good sensitivity and would be best suited for supporting the diagnosis of infection while the additional antiS1 antibody assay would allow for the determination of immunityRapid TestsPointofCare RTPCR Tests A small number of commercial pointofcare tests utilizing RTPCR have been developed These typically involve the same methodology as conventional RTPCR but implemented with automated and portable benchtopsized instruments that can be operated closer to patient care settings than a centralized laboratory A prominent example is Cepheid™s Xpert Xpress SARSCoV2 run on the Gene Xpert platform This apparatus can provide a result within min Others include the MesaBioTech Accula Test and MicrosensDx RapiPrep COVID19 Despite displaying good sensitivity and specificity these instruments are generally limited by a very low throughput of only one to four tests per run per machine34 and as such are only suited to small laboratories or clinics 0c SLAS Technology Figure Loopmediated isothermal amplification LAMP A LAMP begins when the forward inner primer FIP binds to the A2C region while the forward primer A1 binds to A1C which displaces the FIP complementary strand B The backward inner primer BIP binds B2C while the backward primer B3 binds B3C and displaces the BIP complementary strand C A complementary sequence that initiates loop formation is produced D Loop structures are formed that allow for LAMP with the use of loop primersFigure CRISPR technique Viral RNA is converted to dsDNA using RTRPA recombinase polymerase amplification A The CAS12a nuclease enzyme is activated upon complex binding to the target sequence resulting in cleavage of the target sequence and the fluorescent RNA reporter B T7 transcription converts DNA to complementary RNA Cas13 nuclease enzyme activity is activated upon complex binding to the target sequence resulting in a similar cleavage of the target sequence and the fluorescent RNA reporterImmunological AssaysRapid Antibody Assays Compared with labbased antibody assays rapid assays such as lateral flow immunoassays LFIAs Fig and chemiluminescent immunoassays CLIAs Fig offer the benefits of rapid diagnostic testing at a low cost These assays do not require specialized equipment or expertise35 and are thus excellent candidates for pointofcare testing or deployment on a large scale This an area of intense interest with governments worldwide aiming to order millions of tests to inform policy makers about attack rates in their populations36 LFIAs are predominantly singleuse kits designed for pointofcare use while CLIAs are fully automated analyzers that permit very high testing throughputUnfortunately these tests do not quantify the antibody titers and the performance of LFIAs has been called into question one evaluation of nine commercial LFIAs reported a sensitivity ranging from only to versus RTPCR and to versus ELISA37 Meanwhile the performance of CLIAs is superior with good sensitivity and specificity levels similar to those of ELISA38 Otherwise these tests share the same advantages and drawbacks as the 0cTan et al Table Summary of Diagnostic Approaches for COVID19CategoryType of TestTypical Test Result TimeCharacteristicsExamplesVirologicmolecular RTPCRDaysGold standard high sensitivity WHO RTPCR protocolstests Pointofcare RTPCR“ minLAMP CRISPR hImmunologic testsLFIA for antibodies“ minantigensand specificity high throughput but lab basedRapid good sensitivity and specificity pointofcare testing but low throughputRapid good sensitivity and specificity pointofcare testing but low throughputRapid pointofcare testing but not quantitative poor sensitivityCepheid Xpert Xpress SARSCoV2Sherlock Biosciences SHERLOCKVivaDiag COVID19 IgMIgG rapid testCorisbio COVID19 Ag RespiStripEpitope Diagnostics KT1033 EDI Novel Coronavirus COVID19 ELISA kitRoche Elecsys AntiSARSCoV2 Traditional ELISA“ hGood sensitivity and specificity but lab based not automatedCLIA minRapid good sensitivity and specificity high throughput but lab basedNeutralization assayDaysGold standard high sensitivity Not commercially and specificity able to quantify neutralizing antibodies but requires BSL3 lab facilityavailablelabbased antibody assays discussed above The characteristics and unique advantages and disadvantages of these different methodologies are outlined in Table Antigen Assays An alternative approach to immunological assays is to directly detect SARSCoV2 viral antigens Several commercial pointofcare antigen tests are available but their performance remains to be evaluated These tests may be suitable for making an early diagnosis and are deployable as pointofcare assays However they face the same sampling limitations as RTPCR and are hypothetically hampered by limited sensitivity due to the omission of an amplification process unlike nucleic acid testing For example one multicenter study evaluating the Corisbio COVID19 Ag RespiStrip a lateral flow assay for the SARSCoV2 NP reported a test sensitivity of only Rapid NonPCR Molecular Testing Nucleic acid testing using nonPCR methods is an emerging approach for rapid diagnostics and several assays have received Food and Drug Administration FDA emergency use authorization which facilitates the distribution of unapproved medical products or the offlabel use of approved medical products when certain criteria are met These methods share high sensitivity and specificity on par with RTPCR but with the principal advantages of more rapid testing at a lower cost40“LAMP Fig is one such novel isothermal nucleic acid amplification method that does not require a thermal cycler One example is the ID NOW COVID19 test from Abbott Diagnostics which can deliver results in just min43 and uses a lightweight portable instrument allowing onsite testing of swab samples However it has a limited throughput of only one sample per runThe CRISPR enzymes Cas12 and Cas13 have also been adapted for rapid nucleic acid sensing Fig The DETECTR assay by Mammoth Biosciences45 as well as the SHERLOCK assay by Sherlock Biosciences46 potentially offers sensitivity and specificity comparable to those of RTPCR but can be completed in h However these approaches are still in the early stages of commercialization and current applications are available only as test kits to be run in labs while pointofcare versions exist as proofofconcept demonstrations47 Nonetheless their inherent characteristics hold great potential for diagnosis in the futurePrognostication of DiseaseProfiling of Genetic Susceptibility Work is in progress to ascertain the possible genetic basis for the apparent variations in COVID19 susceptibility and disease severity Cao et al compared expression quantitative trait loci eQTL for ACE2 in different populations finding significantly greater eQTL variants associated with higher ACE2 expression in 0c SLAS Technology Figure Lateral flow immunoassay LFIA A Serum sample deposited on the sample pad B AntiSARSCoV2 antibodies in the sample will bind to the target antigen with a labeled tag C Immobilized antihuman IgM antibodies will capture the SARSCoV2 antibody“antigen complex D Control antibodies are captured by immobilized antibodies in the control lineSerum Prognostic Markers Another application of immunological methods would be to measure markers that enable prognostication in COVID19 Higher titers of antibodies against SARSCoV2 have been associated with more severe disease2350 similar to previous studies in Middle East respiratory syndrome MERS“CoV51 ELISA has been used to provide a quantitative measurement of serum and plasma IgM and IgG antibodies By monitoring the kinetics of IgM and IgG antibodies specific to the N and S proteins on SARSCoV2 it was found that intensive care unit ICU patients had a significantly lower level of SIgG within weeks of symptom onset but a higher level of NIgG antibodies compared with nonICU patients52 This finding highlights the possible utility of SIgG and NIgG as a prognostic tool for COVID19 patientsThe Ddimer level which consists of crosslinked fibrin degradation products that reflect ongoing blood clot formation and breakdown activity in the body is another proposed prognostic marker Modern commercial assays for Ddimers are based on monoclonal antibodies employing either ELISA or microlatex agglutination assays53 Reports have emerged that elevated Ddimer levels suggestive of a hypercoagulable state are associated with drastically worse outcomes A Chinese group reported that Ddimer levels of ‰¥ µgmL on admission were associated with a times increased mortality relative to Ddimer levels of µgmL in a cohort of COVID19 patients54 This finding of Ddimer levels as a negative prognostic marker was also noted in other studies conducted in China455 and the Netherlands56Similarly interleukin IL6 a key component of the cytokine release syndrome is another marker measured by ELISA and has been described to independently predict adverse outcomes in COVID195758 Tumor necrosis factor alpha TNFα another important proinflammatory cytokine has also been found to be strongly correlated with Figure Chemiluminescence enzyme immunoassay CLIA SARSCoV2 antigens will capture IgM and IgG antibodies from the sample serum Secondary antibodies that are conjugated with horseradish peroxidase HRP bind to the captured primary IgM and IgG antibodies and react with a chemiluminescent substrate to generate a strong chemiluminescent signal that is measured in terms of relative light units RLUEast Asian populations but reported no direct evidence supporting the existence of S proteinbindingresistant ACE2 mutants48 out of identified protein altering variants Separately Stawiski et al analyzed ACE2 polymorphisms within a much larger population dataset spanning more than population groups across the world and performed structural predictions to identify variants that might confer protection or rather increase susceptibility to SARSCoV2 S protein binding49 Out of a total of identified proteinaltering ACE2 variants variants were predicted to increase susceptibility while variants were speculated to confer protection however the degree of changes in receptor“virus binding interactions for each structural variant was not quantified These findings represent significant developments in our understanding of population risk profiles for COVID19 and future coronavirus infections 0cTan et al endan damage and mortality even after adjusting for disease severity scores59 Gao et al examined both IL6 and Ddimer levels they proposed a panel comprising tandem testing of these two markers which produced a sensitivity of and specificity of in early prediction of severe COVID1958 Elevated troponin levels a marker of myocardial injury measured with ELISA immunoassays also strongly predict progression to death in patients with severe illness60 These results suggest that multiplex cytokine and serum marker profiling will be a powerful tool in stratifying patients that may guide clinical decisions and resource allocationSummary In sum rapid progress has been made in diagnostics for COVID19 Yet the race against time continues for researchers and biotechnology firms to develop rapid costeffective and reliable test kits that can be deployed on a large scale At the time of writing labbased RTPCR testing has been the dominant diagnostic approach but alternative molecular approaches like isothermal amplification and CRISPR which have clear advantages are on the horizon Immunological tests such as CLIA and LFIA will become increasingly important because of the urgent need for pointofcare diagnostics for mass testing of infected asymptomatic individuals and their close contacts and will be valuable in complementing molecular approaches for confirming infection Furthermore immunological assays will be in great demand by policy makers worldwide for the assessment of immunity to COVID19 However the performance of these serological tests varies significantly particularly their degree of sensitivity and specificity we believe that caution must be taken in the interpretation of these tests Detailed evaluation of the reliability of serological tests will be a key area for future research Lastly given the importance of techniques like ELISA in prognosticating COVID19 immunological methods will undoubtedly occupy a crucial role in achieving all levels of the WHO™s short medium and longterm diagnostic goalsCOVID19 Research ToolsImmunological ApproachesCOVID19 infection has a poor prognosis in individuals with comorbidities and abnormal immune functions Although research surrounding COVID19 is still in its infancy several studies have revealed lymphopenia and the cytokine storm as underlying mechanisms correlating to disease progression Here we discuss the various immunological techniques involved in assessing host immunity in COVID19 patientsELISA As discussed ELISA has also been used to detect the inflammatory cytokines implicated in the cytokine storm seen in patients with severe respiratory failure due to COVID19 One study found that the immune dysregulation in patients with severe respiratory failure was due to a significantly increased production of IL6 and defective lymphoid function because of an IL6mediated decrease in HLADR expression on CD14 monocytes Interestingly interferongamma IFNγ levels were below the detection level in these patients suggesting that T helper Th cells are unlikely to be major players in the overinflammatory response of severe patients61 A similar observation was made in a separate study whereby inflammatory cytokines that mediate major immune responses such as TNFα and IL1β were not significantly elevated in ICU patients62 These findings demonstrate that the immunophenotype of patients with COVID19 can vary depending on presently unclear host immune factors and the severity of their condition This relationship between disease severity and cytokine storm has also been highlighted in other studies that found a significantly elevated plasma concentration of granulocyte colonystimulating factor GCSF IP10 CCL2 and CCL3 in ICU patients compared with nonICU patients63ELISA is also being used as a companion diagnostic tool for therapeutic purposes In a study that explored the use of convalescent plasma therapy from donors as a form of treatment in severe COVID19 ELISA was used to assess the neutralizing activity of the RBDspecific IgM and IgG antibodies found in the donor convalescent plasma64 After the transfusion was complete ELISA was also used to detect IgG IgM and neutralizing antibody titers in the sera of patients to assess the response to treatment65EnzymeLinked Immunosorbent Spot Enzymelinked immunosorbent spot ELISPOT is a sensitive immunoassay that quantitatively measures cytokinesecreting cells at the singlecell level providing insight into immunerelated cellular activities66 Hence it is a promising tool for characterizing specific Tcell immunity in COVID19 patients By IFNγ ELISPOT analysis it was revealed that convalescent COVID19 patients had significantly increased levels of IFNγsecreting T cells when compared with healthy donors A significant correlation between neutralizing antibody titers and NPspecific T cells was identified in these patients suggesting that a combination of humoral and cellular immunity is integral to clearing SARSCoV2 Interestingly it was noted that in convalescent patients weeks IFNγsecreting Tcell numbers had postdischarge decreased suggesting that they may not be maintained for a prolonged period of time even in recovered patients67ELISPOT is also serving a vital role in vaccine development through the detection of potential Tcell epitopes in the S protein RBD of SARSCoV268 One study was able to harness ELISPOT assays to identify three Tcell epitopes that induced a strong adaptive immune response 0c SLAS Technology postimmunization demonstrating the promise of ELISPOT assays in the area of vaccine development32 Recently ELISPOT has also been applied to assess the immunogenicity of newly developed vaccines One such study successfully utilized an IFNγ ELISPOT assay to evaluate Tcell responses to a new SARSCoV2 vaccine in murine splenocytes and rhesus macaque peripheral blood mononuclear cells PBMCs The promising findings from this animal study informed the start of a phase I clinical trial with the same vaccine highlighting the usefulness of ELISPOT in assessing immune responses to new vaccines and promoting vaccine development69Flow Cytometry Unlike ELISA and ELISPOT flow cytometry determines the number of cytokinesecreting cells and has the capacity to immunophenotype based on surface and intracellular markers70 In relation to the current pandemic this technique enables the detection sorting and analysis of multiple subpopulations of immune cells specific to COVID Using flow cytometry researchers detected a cytotoxic immune environment in patient blood samples despite a reduction in the overall lymphocyte population71“ As part of the SARSCoV2 antiviral response peripheral lymphocytes retain the capacity to activate and differentiate into subpopulations such as antibodysecreting cells CD3“CD19CD27hiCD38hi follicular T cells CD4CXCR5ICOSPD1 CD4 Th cells CD38HLADRCD4 cytotoxic T Tc cells CD38HLADRCD8 and regulatory T Treg cells CD3CD4CD25CD127“ These Tc cells harbor large amounts of cytotoxic granules while CD4 Th cells skewed toward a proinflammatory Th1 and Th17 phenotype727375 The overall hyperinflammation and cytotoxic environment supports the notion that a cytokine storm could be liable for the multisystemic insults in patients with severe COVID19Elicitation of antiviral Tcell responses specific to SARSCoV2 is of utmost importance to establishing viral control Many studies have demonstrated robust antiviral responses however there is no known set of markers reported to identify SARSCoV2specific T cells Collectively most groups have characterized SARSCoV2specific T cells based on HLADR CD38 CD69 CD25 CD44 and Ki67 expression T

Thyroid_Cancer

Identification and treatment of obstructive sleep apnea by a primarycare team with a subset focus on chronic painmanagement within the paper and its Supporting InformationfilesFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existBackgroundPatients diagnosed with obstructive sleep apnea OSA who also consume prescription opioids have a greater likelihood of morbidity and mortality This study evaluated whether a primary care team focused on chronic pain care management could use a validatedquestionnaire STOPBang and motivational followup to increase identification and treatment of OSAMethodsThis study was a retrospective dual arm prepost controlled study Participants of thisstudy included the complete chronic pain management sub group treated by this primarycare team Participants were � years old and prescribed daily opioids for treatment ofchronic pain All participants had a multifaceted individualized educational meeting thatincluded completing a STOPBang questionnaire Participants who received a score �three were advised to follow up with their primary care physician Participants were seenquarterly throughout the studyResultsThe primary outcome of this study was that of participants with likely OSA were usingCPAP for a minimum of months range of “ months 18month average postintervention vs CPAPuse in the control group months of observation both groupswere chronic opioid users with OSA This was a relative improvement p Asecondary outcome was that of nonprior CPAP users obtained CPAP post intervention a prepost improvement p x2 with degree of freedom Alsoparticipants who were likely to have OSA STOPBang score � or had a positive polysomnography AHI with comorbidities compared to those unlikely to have OSA STOPPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamBang score or had a negative polysomnography AHI in this study were more likelyto be male have a higher BMI have hypertension have cardiovascular disease andorhave diabetes all typesConclusionTeam based care management for participants taking prescription opioids where STOPBang questionnaires were completed were associated with an increase in the identificationand treatment of OSAIntroductionStudy rationaleThe objective of this study was to evaluate whether a teambased care management intervention would increase identification and treatment of obstructive sleep apnea in participants taking medication for chronic painCurrent knowledgeObstructive sleep apnea OSA is a chronic sleep disorder characterized by episodes of apneasand hypopneas or the complete or partial collapse of the upper airway [] A diagnosis ismade when a patient has or more of these events in an hour or five of these events if otherpredicting symptoms for OSA are present [] Approximately million American adults havethis sleep disorder Patients with moderate to severe OSA are often treated with a continuouspositive airway pressure device or CPAP Treatment can reduce the risks of hypertension coronary artery disease heart failure arrhythmias sudden cardiac death and stroke [] A validated questionnaire STOPBang score can be used as a screening tool to identify patients atrisk for OSA A STOPBang sleep questionnaire is commonly distributed to patients whoshow signs of having potential risk factors for OSA œSTOP questions snoring tirednessobserved apnea and high blood pressure and œBang BMI kgm2 Age years Neckcircumference cm and Gender male [ “] A score of three or higher on STOPBangplaces patients at higher risk of OSA requiring further evaluation which includes an overnightpolysomnogram or sleep study to confirm diagnosis []Prescription opioid use for pain management is still a common practice in primary care settings though the rates of overdose due to these drugs in the United States are at an unprecedented high with deaths in [“] Patients using opiates for chronic painmanagement have also been found to have a greater likelihood of developing central sleepapnea which worsens the severity of OSA and ataxic breathing [] A relationship existsbetween pain and sleep disruption with over of patients who have chronic pain havingreported difficulty sleeping which increases hyperalgesia [ ] An analgesic effect may bepresent where pain creates sleep disorders and a shortage of sleep increases pain [ ]Patients with OSA who are then prescribed opioids have an increased risk of opioidinducedrespiratory depression OIRD [] Despite the potential morbidity of the combination ofOSA central sleep apnea and Opioid use studies have shown that those that have this combination only remain on definitive treatment for their OSA CPAP of the time [] It istherefore of the utmost importance that patients who chronically use opioids are evaluated forOSA and those diagnosed adhere to CPAPPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamIn order to decrease mortality it is necessary to educate patients on the risks and healthconcerns related to taking opioids Brief interventions can influence behavior of patients whoare at high risk for abuse of a substance [] Individualized nurseled management educationis a tool that can be used as a brief intervention within a teambased approach [] These individualized information gathering motivational meetings allow patients to experience empathy as their medical needs are met [ ] Individualized nurseled management educationis a tool that can be used as a brief intervention within a teambased approach [] Instructionand guidance can be coupled with an assessment of a patient™s potential risk factors for comorbidities such as OSA using a STOPBang questionnaireMaterials methodsDesignThis study was a retrospective dual arm prepost controlled study to evaluate whether anintervention STOPBang questionnaire education and motivational followup wouldimprove OSA diagnosis and treatment compliance The first investigation arm compared thecontrol group daily opioid use with OSA to the part of the intervention group with OSA onCPAP or at risk for OSA STOPBang � This arm™s purpose was to determine whether theintervention increased treatment compliance with CPAP The second investigation armcompared the study group to itself prepost intervention This arm evaluated whether applyingthe intervention increased diagnosis and treatment of OSA The single independent variablein this study was a participant™s admission to the chronic pain management subgroup withthe administration of a STOP Bang questionnaire and the dependent variable was CPAP useInclusion criteria consisted of ongoing participation in primary care pain management subgroup daily opioid use and an age � This study occurred between October and January which included monthsfor exposure and then at least months of follow up One of Intermountain Healthcare™s outpatient facilities in Utah served as the site for this study The total number of patients duringthe duration of the study who entered into the pain management sub group � years oldand were taking a daily dose of opioids was Out of the identified participants participants remained in the clinic during the entirety of this study Fig Ethics approvalIRB Number for this study was obtained from the Institutional Review Board ofIntermountain Healthcare Consent was waived for this retrospective studyControlsA Medline and Pubmed basic search from “ was used to identify a historical yet contemporary United States based control group that had Obstructive Sleep Apnea and met theinclusion criteria of being � years old and were taking a daily dose of opioids The controlthat was chosen was comparable to this study™s intervention group with regard to Race AgeGender Morphine Equivalent Daily Dose and Comorbidities Also the control used camefrom a study that lasted a similar duration []SubjectsEach participant received an information gathering and educational session with a registerednurse in the primary care facility at the time of their admittance into the chronic pain management program between “ During this session a full pain history was obtained fromparticipants All participants in the study underwent STOPBang screening All participantswho were at high risk for OSA STOPBang � were referred back to their primary carePLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamFig Participant retention101371journalpone0237359g001physician Participants and physicians made joint decisions during a followup appointmentwhether to pursue polysomnography Reasons why participants did not complete polysomnography or attain treatment for OSA are that the participants refused workup there was ashared decision due to participant frailty had cancer end stage or were noncompliant toCPAP Fig Fig participants at high risk for sleep apnea STOPBang score � who did not undergo diagnostic evaluation orcomplete treatment101371journalpone0237359g002PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamEach participant received initial inperson education with the same registered nurse regarding nonpharmacological treatment of pain selfhelp groups risks and benefits of treatment ofpain management compliance with treatment of pain and its comorbidities and informationabout Naloxone an opioid reversal agent This education was provided in a hour sessionusing the FRAMES Feedback Responsibility Advise Menu for Change Empathy andEnhancing Self Efficacy motivational method [] A chronic pain management agreementwas executed Additional information obtained during this session included a Current OpioidMisuse Measure and a STOPBang questionnaire All participants who received a score greaterthan or equal to three on the STOPBang were told to follow up with their primary care physician All primary care providers were made aware they would receive electronic data onSTOPBang questionnaires and participants with scores � would make a follow up appointment with them Upon completion of this session information was recorded electronically andtransmitted to the primary care physician on record Each participant also had quarterly inperson followup appointments with their primary care physician In addition quarterly inperson or phone visits with the pain management nurse were continued Physician visits varied in length and scope Individualized nursing visits that continued to use the FRAMESmethod and lasted for a duration of minutes were focused on remaining compliant with allongoing therapies The nurse would document the interaction and any concerns about compliance with any therapy in the patient™s EMR Electronic Medical Record at the conclusionof the visits The information was based on the subjective input received from the participantQuarterly followup remained consistent throughout the study Attending all followupappointments both with the primary care physician and the pain management nurse was aprerequisite to remain in the pain management group Remaining in the pain managementwas group was the only avenue to receive controlled pain medication at this facilityData collectionThe baseline biometric parameters of Race Gender BMI Age Tobacco Use Alcohol Use andMorphine Equivalence Daily Dosage MEDD were extracted from each participant™s electronic medical record EMR Collected data also included information on Chronic DiseaseHypertension Cardiovascular Disease Pulmonary Disease Thyroid Disease Diabetes Gastroesophageal Reflux Disease and Chronic Kidney Disease and specific medication typesBenzodiazepines and Pregabalin STOPBang scores were gathered during the initial chronicpain management encounter when participants joined the study Subjective data on CPAPadherence was found in the EMR documentation of the pain management nurse and primarycare physician follow up reports postinclusion in the studyStatistical methodsMeans and standard deviations were calculated for demographic variables The DAgostinoPearson test was used to confirm normality [] Unequal variance ttesting was used in the statistical analysis of both arms of this study to compare Age BMI and Morphine EquivalentDaily Dosing Two proportion Ztesting was used to compare the control group daily opioiduse with OSA to the participants in the intervention group with OSA on CPAP or at highrisk for OSA STOPBang � with regards to CPAP use Race Gender Diabetes Cardiovascular Disease and Hypertension Two proportion Ztesting was also used to compare the participants not likely to have OSA STOPBang or had a negative polysomnography to thosewith OSA on CPAP or at high risk for OSA STOPBang � not completing polysomnography or not compliant with CPAP A x contingency table using McNemar™s test with correction for continuity was used to compare the participants™ CPAP use prepost intervention []PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamIRB approvalIRB Approval was granted from Intermountain Healthcare IRB IRB Number ResultsThe primary outcome of this study was that of participants with likely OSA were usingCPAP for a minimum of months range of “ months 18month average postintervention vs CPAP use in the control group months of observation which were chronic opioid users with OSA This was a relative improvement p A secondary outcomewas that of nonprior CPAP users obtained CPAP post intervention a prepostimprovement p x2 with degree of freedom Fig This resulted in ofintervention participants being treated with CPAP Comparing the average number of intervention participants with OSA on CPAP or at risk for OSA STOPBang � to the control dataset resulted in significant differences for BMI and Male Gender Otherdemographics between these two groups Age p MEDD p Race Caucasianp Hypertension p Cardiovascular Disease p and Diabetes all formsp showed no statistically significant differences between groups Comparing the averagenumber of intervention participants with OSA on CPAP or at risk for OSA STOPBang � to the average number of intervention participants not likely to have OSA STOPBang orhad a negative polysomnography resulted in the following statistically significant differencesBMI p Male Gender p00001 Hypertension p Cardiovascular Diseasep and Diabetes all forms p Other demographics between these two groupsAge p MEDD p Race Caucasian p Benzodiazepine Use p Pregabalin Use p and Active Chronic Disease States Gastroesophageal Reflux p Chronic Kidney Disease Thyroid Disease p and Pulmonary Diseasep showed no statistically significant differences between groups Table DiscussionMain findingsCurrent high death rates compared to healthy subjects of OSA and chronic pain managementwith daily opioid use warrant efforts to improve care [“] This study™s findings can be anFig CPAP use before and after intervention101371journalpone0237359g003PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamTable Comparison of clinical characteristics S1 AppendixDemographicsAverage Number of Participants withOSA on CPAP or at risk for OSASTOPBang � Control DailyOpioid Use withOSAAgeBMI ���Morphine Equivalent mgdat Conclusion of Study � months Pvalue Average Number of Participants Not Likelyto Have OSA STOPBang or NegPolysomnography PrePostPvalueDemographicsAverage Percentage of Participantswith OSA on CPAP or at risk for OSAControl DailyOpioid Use withPvalueAverage Percentage of Participants NotLikely to Have OSA STOPBang orPrePostPValueSTOPBang � OSANeg PolysomnographyRace CaucasianGender Male��Diabetes���Cardiovascular Disease��Hypertension�Gastroesophageal Reflux DiseaseThyroid Disease All FormsPulmonary Disease All Varieties Active CancerChronic Kidney DiseaseConcurrent Benzodiazepine UseConcurrent Pregabalin Use� p value of �� p value of ��� p value of 101371journalpone0237359t001NANANANANANANA NANANANANANANAimportant step towards improving mortality and morbidity when these two diseases are combined The results suggested that using a STOPBang questionnaire accompanied with motivational education initially as well as in followup improved diagnosis and treatment of OSAThese findings suggest that teambased care can positively impact the outcome of patientswith OSA when they are concurrently on chronic daily opioids and CPAP These findings warrant additional research Diagnosed sleep apnea participants who were using CPAP had a significantly higher rate of being male having hypertension having cardiovascular diseasehaving diabetes all types and having a higher BMI The fact that of participants at riskfor sleep apnea STOPBang � did not receive a full evaluation or end up on definitive treatment highlights the need for continued effort in this fieldLimitationsThe data found in this study regarding CPAP adherence was subject to limitations as wellSome participants that joined this teambased care management approach to treat chronicpain had already been prescribed CPAP and their adherence to the treatment was included inthis studies data set possibly overestimating the conclusions Adherence to CPAP in this studywas based on subjective treatment reconciliation at followup appointments with physiciansand nurses and since many participants received their sleep treatment from outside sourcesCPAP adherence data other than confirmation from the patient was not available If a participant indicated that they had not been using their CPAP � of the time between anyPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamfollowup appointments after the initiation of treatment they were considered to be nonadherent This type of data collection is subject to significant recall bias and could potentiallyinflate the percentage of participant adherenceThis preliminary research was done in the format of a prepost dual arm retrospectivestudy This type of study has inherent and significant limitations First due to the nature of theintervention team members nurses and physicians were not blinded and continued to evaluate participants throughout the course of this study and could have recommended evaluationand therapy of sleep apnea for alternative reasons This study could have been affected by aherd mentality since the primary care physicians worked together in one group and theircomposite style could have increased or decreased the identification and treatment of sleepapnea These limitations could be mitigated by performing a large prospective multicenterstudyThis study is also limited due to the nature of using a historical control [] Although the control used mimicked the experimental group in most ways daily opioid use MEDD race age andpercentages of diabetes hypertension and cardiovascular disease it varied in both BMI controlgroup had a higher average BMI and gender control group had a greater number of male participants It has been previously demonstrated that a higher BMI favors adherence to CPAP [] This does not detract from the finding that the intervention group in this study which had alower BMI had a higher adherence to CPAP Gender has also been evaluated in previous CPAPcompliance studies Two studies have shown no difference between genders whereas a differentstudy showed an increase in male noncompliance and another where there was an increase inmale compliance [“] As there is no definitive determination from these studies it is possiblethat the variance between the intervention and control groups were due to the gender differenceIt would seem from the above studies that there was no strong trend resulting from gender making gender unlikely to be the cause of the preponderance of the varianceThere was a loss of followup with primary care physicians after the administration of theSTOPBang questionnaire in this study Fig Patients may have fotten to or chosen notto schedule a followup appointment with their primary care physician after being determinedhigh risk for sleep apnea The participant received education on six other aspects of opioidmanaged pain during the one hour informational meeting Another limitation was that thedata from the intervention session was transmitted enblock to the primary care physicianelectronically These limitations could have been improved by having the nurse set up a specific appointment to consider sleep disorders in the highrisk group and to either verbally orelectronically deliver this information in an isolated approachComparisonsThis study reaffirmed previous studies that showed STOPBang was a tool that could identifyOSA [ “] Other similarities to previous investigations were found In those studies wheredaily opioid dosing for pain management was present “ of patients were found to sufferfrom OSA and of patients were found to be compliant to CPAP [ ] The data rangefor previous studies in regard to OSA incidence is consistent with this study in which ofparticipants were either diagnosed with OSA on CPAP or were at high risk for OSA STOPBang � not completing polysomnography or not compliant with CPAP This study was significantly higher with regards to the percentage of remaining on CPAP suggesting thatdiligence in repetitive diagnosis attempts and continued follow with motivational techniquesmay improve care in this type of patient Also this study like others suggested p thathypertension cardiovascular disease diabetes male gender and higher BMI are more common in patients with OSA []PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamConclusionsLeft untreated sleep apnea has a high rate of morbidity and mortality The use of prescribedopioids as a form of chronic pain management in a primary care setting can increase apatient™s likelihood of having sleep apnea A teambased intervention that included theadministration of a STOPBang questionnaire was associated with an increased diagnosis ofOSA prepost improvement level It was also associated with an increased adherenceto CPAP relative improvement The primary reasons CPAP treatment was not receivedincluded refusal of evaluation nonadherence to CPAP or shared physicianpatient decisionbased on agefrailtySupporting informationS1 Appendix Raw study data setXLSXAuthor ContributionsConceptualization Kathleen Whittington Leigh Simpson Dixie HarrisData curation Kathleen Whittington Michael ClayFormal analysis Kathleen Whittington Michael Clay Dixie HarrisInvestigation Joanna TierneyProject administration Joanna TierneySupervision Dixie HarrisWriting “ original draft Kathleen WhittingtonWriting “ review editing Kathleen Whittington Leigh Simpson Michael Clay JoannaTierney Dixie HarrisReferencesFarney Robert J œThe STOPBang equivalent model and prediction of severity of obstructivesleep apnea relation to polysomnographic measurements of the apneahypopnea index Journal ofclinical sleep medicine JCSM official publication of the American Academy of Sleep Medicine vol “65B 105664JCSM1306 PMID Manne Mahesh B œObstructive Sleep Apnea Who Should Be Tested and How ClevelandClinic Journal of Medicine Aug wwwmdedgecomccjmarticle105363cardiologyobstructivesleepapneawhoshouldbetestedandhow Abrishami A Khajehdehi A Chung F A systematic review of screening questionnaires for obstructivesleep apnea Can J Anaesth “ 101007s126300109280x PMID Vasu TS Doghramji K Cavallazzi R Obstructive sleep apnea syndrome and postoperative complications clinical use of the STOPBANG questionnaire Arch Otolaryngol Head Neck Surg “ 101001archoto20101020 PMID Kumar S Mcelligott D Goyal A Baugh M Ionita RN Risk of Obstructive Sleep Apnea OSA in Hospitalized Patients Chest 1384779A Ong TH Raudha S FookChong S Lew N Hsu AA Aal H Simplifying STOPBANG use of a simplequestionnaire to screen for OSA in an Asian population Sleep Breath “œDrug Overdose Deaths Centers for Disease Control and Prevention Centers for Disease Control andPrevention June wwwcdcgovdrugoverdosedatastatedeathshtml Substance Abuse and Mental Health Services Administration Drug Abuse Warning Network National Estimates of DrugRelated Emergency Department Visits HHS Publication No SMA “PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management team DAWN Series D39 Rockville MD Substance Abuse and Mental Health Services Administration Schneiderhan JMD Clauw DMD Schwenk TMD Primary care of patients with chronic pain J Am MedAssoc “ Walker James M œChronic Opioid Use Is a Risk Factor for the Development of Central SleepApnea and Ataxic Breathing Journal of Clinical Sleep Medicine JCSM Official Publication of theAmerican Academy of Sleep Medicine American Academy of Sleep Medicine Aug wwwncbinlmnihgovpmcarticlesPMC1978331 PMID Webster LR Choi Y Desai H Webster L Grant BJ Sleepdisordered breathing and chronic opioid therapy Pain Med “ 101111j15264637200700343x PMID Onen SH Onen F Courpron P Dubray C How pain and analgesics disturb sleep Clin J Pain “ 10109701ajp000012975731856f7 PMID Cozowicz Crispiana œOpioids for Acute Pain Management in Patients with Obstructive SleepApnea Anesthesia Analgesia vol no pp “ 101213aneJaoude Philippe Lal Ashima Vermont Leaj Porhomayon Jahan Ali A ElSolh Pain Intensity and Opioid Utilization in Response to CPAP Therapy in Veterans with Obstructive Sleep Apnea on Chronic Opioid Treatment Journal of Clinical Sleep Medicine Barbor TF HigginsBiddle JC World Health anization Dept of Mental Health and SubstanceDependence Brief Intervention of Hazardous and Harmful Drinking A Manual for Use in Primary CareGeneva World Health anization Group WBIS A crossnational trial of brief interventions with heavy drinkers Am J Public Health “ 102105ajph867948 PMID Miller WR Rollinick S Motivational Interviewing Preparing People to Change Addictive Behavior NewYork and London Guilford Press Saunders B Wilkinson C Phillips M The impact of a brief motivational intervention with opiate usersattending a methadone program Addiction “ 101046j13600443199590341510x PMID Hester RK Miller WR Handbook of Alcoholism Treatment Approaches Vol Boston MA Allyn andBacon D™Agostino R Belanger A A Suggestion for Using Powerful and Informative Tests of Normality The American Statistician “ 1023072684359 Edwards A Note on the ˜Correction for Continuity™ in testing the significance of differencebetween populations Psychometrika B “ Young T Finn L Sleep disordered breathing and mortality eighteenyear followup of the Wisconsin sleep cohort Sleep Aug “ PMID Marshall NS Wong KK Sleep apnea as an independent risk factor for allcause mortality the Busselton Health Study Sleep Aug “ PMID Liang Y Turner B Assesing Risk for Drug Overdose in a National Cohort Role for Both Daily and TotalOpioid Dose J Pain Apr “ 101016jjpain201411007 PMID Heejin Kim MinSukim Treatment Outcomes and Compliance According to Obesity in Patientswith OSA European Archives of OtoRhinoLaryngology “ PelletierFleury N Rakotonanahary D Fleury B The age and other factors in the evaluation of compliance with nasal continuous positive airway pressure for obstructive sleep apnea syndrome A Cox™sproportional hazard analysis Sleep Med “ 101016s1389945700 PMID Anttalainen U Saaresranta T Kalleinen N Aittokllio J Vahlbergy T Polo O CPAP adherence and partial upper airway obstruction during sleep Sleep Breath “ 101007s1132500701025 PMID Sin DD Mayers I Man GC Pawluk L Longterm compliance rates to continuous positive airway pressure in obstructive sleep apnea a populationbased study Chest “ 101378chest1212430 PMID Ye Lichun Pien Grace W Ratcliffe Sara J Weaver Terri E Gender Differences in Obstructive SleepApnea and Treatment Response to Continuous Positive Airway Pressure J Clin Sleep Med “ PMID PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management team Mador M Henderson J Effect of Opioids on Sleep and Breathing in Chronic Pain Patients Journal ofClinical Sleep Medicine Aug “ 105664jcsm3952 PMIDPLOS ONE 101371journalpone0237359 August PLOS ONE 0c'

Thyroid_Cancer

Sequential Use of aYeastCEA Therapeutic CancerVaccine and AntiPDL1 Inhibitor inMetastatic Medullary Thyroid CancerJaydira Del Rivero Renee N Donahue Jennifer L Mart Ann W Gramza Marijo Bilusic Myrna Rauckhorst Lisa Cordes Maria J Merino William L Dahut Jeffrey Schlom James L Gulley and Ravi A Madan Genitourinary Malignancies Branch Center for Cancer Research National Cancer Institute Bethesda MD United States Developmental Therapeutics Branch Center for Cancer Research National Cancer Institute Bethesda MD United States Laboratory of Tumor Immunology and Biology Center for Cancer Research National Cancer Institute Bethesda MDUnited States Medstar Geetown Lombardi Comprehensive Cancer Center Geetown University Medical CenterWashington DC United States Laboratory of Pathology National Cancer Institute Bethesda MD United StatesMedullary thyroid cancer MTC accounts for ˆ¼ of all thyroid malignancies MTCderives from the neural crest and secretes calcitonin CTN and carcinoembryonic antigenCEA Unlike differentiated thyroid cancer MTC does not uptake iodine and I131RAI radioactive iodine treatment is ineffective Patients with metastatic disease arecandidates for FDAapproved agents with either vandetanib or cabozantinib howeveradverse effects limit their use There are ongoing trials exploring the role of less toxicimmunotherapies in patients with MTC We present a 61yearold male with the diagnosisof MTC and persistent local recurrence despite multiple surgeries He was startedon sunitinib but ultimately its use was limited by toxicity He then presented to theNational Cancer Institute NCI and was enrolled on a clinical trial with heatkilledyeastCEA vaccine NCT01856920 and his calcitonin doubling time improved in months He then came off vaccine for elective surgery After surgery his calcitoninwas rising and he enrolled on a phase I trial of avelumab a programmed deathligand PDL1inhibitor NCT01772004 Thereafter his calcitonin decreased on consecutive evaluations His tumor was subsequently found to express PDL1CEAspecific T cells were increased following vaccination and a number of potentialimmuneenhancing changes were noted in the peripheral immunome over the course ofsequential immunotherapy treatment Although calcitonin declines do not always directlycorrelate with clinical responses this response is noteworthy and highlights the potentialfor immunotherapy or sequential immunotherapy in metastatic or unresectable MTCKeywords medullary thyroid cancer CEA calcitonin immunotherapy PDL1 inhibitorINTRODUCTIONMedullary thyroid cancer MTC accounts for ˆ¼ of allis aneuroendocrine tumor deriving from the neural crestderived parafollicular or C cells of the thyroidgland About of MTC cases are sporadic and the remaining present as part of anautosomal dominant inherited disorder Activating mutations of the RET Rearranged duringthyroid malignancies ItEdited byEnzo LalliUMR7275 Institut de PharmacologieMolculaire et CellulaireIPMC FranceReviewed byMatthias KroissJulius Maximilian University ofW¼rzburg GermanyMouhammed Amir HabraUniversity of Texas MD AndersonCancer Center United StatesCorrespondenceJaydira Del RiverojaydiradelriveronihgovSpecialty sectionThis was submitted toCancer Endocrinologya section of the journalFrontiers in EndocrinologyReceived April Accepted June Published August CitationDel Rivero J Donahue RN Mart JLGramza AW Bilusic M Rauckhorst MCordes L Merino MJ Dahut WLSchlom J Gulley JL and Madan RA A Case Report of SequentialUse of a YeastCEA TherapeuticCancer Vaccine and AntiPDL1Inhibitor in Metastatic MedullaryThyroid CancerFront Endocrinol 103389fendo202000490Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cDel Rivero et alMTCTransfection protooncogene are characteristic with germlineactivating RET mutations seen in fMTC familial MTC andMEN multiple endocrine neoplasia 2aMEN2b “ MTCmost often produces both immunoreactive calcitonin CTNand carcinoembryonic antigen CEA which are used as tumormarkers The growth rate of MTC is estimated by usingRECIST v11 Response Evaluation Criteria in Solid Tumorshowever it can also be determined by measuring serum levelsof CTN and CEA over multiple time points to determinedoubling time which play an important role in the followupand management of MTC Calcitonin doubling times of yearsseem to be associated with a better longterm prognosis thanthose of months ltration on MTC Dendritic cellThe role of immunotherapy in MTC is not fully studiedHowever previous studies have identified evidence of TcellDCbasedimmunotherapy was also given in patients with solid tumorsincluding MTC and it was reported that vaccination withautologous tumorpulsed DCs generated from peripheral bloodwas safe and can induce tumorspecific cellular cytotoxicity Schott reported that subcutaneous injectionof calcitonin and CEA loaded DC vaccine in patients withmetastatic medullary thyroid cancer showed clinical benefitCalcitonin and CEA decreased in of patients and one of thesepatients had complete regression of detectable liver metastasisand reduction of pulmonary lesions A phase I study using theheatkilled yeastCEA vaccine GI6207 was performed at theNational Cancer Institute NCI A total of patients wereenrolled in a classic phase I design at dose levels One patientwith MTC had a significant ‚ammatory response at the sitesof her tumors and a substantial and sustained antigenspecificimmune response Furthermore the relatively low toxicity profileof therapeutic cancer vaccines could be advantageous comparedto approved tyrosine kinase inhibitors TKIs for some patientswith indolent recurrent or metastatic MTC Here we presenta case of a patient with recurrent MTC who was enrolled ona clinical trial with yeastbased vaccine targeting CEA Uponsurgical resection after vaccine his tumor was found to expressprogrammed deathligand PDL1 which may explain thepatient™s subsequent reponse to a PDL1 inhibitorCASE PRESENTATIONWe report a 61yearold male who initially presented with anenlarging anterior neck mass that was biopsied and found tobe consistent with the diagnosis of MTC no known somatic ermline mutation of the RET protooncogene Subsequentlyhe underwent a total thyroidectomy with bilateral neck lymphnode dissection He then had multiple local recurrences resultingin a total of five neck surgeries the last one occurring years after diagnosis Based on the elevated CTN levels andpersistent local recurrence he then started systemic treatmentwith oï¬label sunitinib years after diagnosis Whileon sunitinib his CTN levels nadired to pgml reference pgml down from pgml months after startingtreatment He continued for years and then stopped due toside eï¬ects His CTN levels after discontinuing sunitinib roseto pgmlOn followup imaging studies there was no evidence ofdistant metastases and he presented to the NCI with diseaseinvolving his thyroid bed and cervical nodes most of which werenot amenable to resection He then enrolled on a clinical trialwith yeastbased therapeutic cancer vaccine targeting CEA aphase study of GI6207 in patients with recurrent medullarythyroid cancer NCT01856920 During a 6monthprotocol“mandated surveillance he had a CTN of pgmLand CEA of ngmL reference “ ngmL that increasedto pgmL and CEA of ngmL CTN doubling timeof days During the subsequent 3month vaccine periodhis doubling time improved to days nadir CTN was pgmL and CEA ngmL He then chose to have electivesurgery to remove a neck lymph node and per protocol thevaccine was discontinuedApproximately months after surgery his calcitonin hadrisen to pgml and CEA ngmL and the patient wasenrolled on a phase I trial of avelumab a PDL1 inhibitor phase I label multipleascending dose trial to investigate the safetytolerability pharmacokinetics biological and clinical activity ofavelumab MSB0010718C a monoclonal antiPDL1 antibodyin subjects with metastatic or locally advanced solid tumorsNCT01772004 He then had five consecutive declines inhis calcitonin to pgml and CEA levels remained overallstable at ngmL while on the immune checkpoint inhibitoravelumab a decline not previously seen in his NCIclinical course A Response assessment by RECIST v11reported stable disease Figure These findings coincided with an immunerelated adverseevent asymptomatic rise in grade lipase that led to protocolmandated treatment discontinuation A subsequent analysis ofthe patient™s lymph node resected postvaccination revealed thatthe tumor was PDL1 positive B No baseline samplewas available for evaluation given that the patient was diagnosedover years prior to the latest surgery ImmuneAnalysisSufficient cryopreserved peripheral blood mononuclear cellsPBMCs were available from this patient to analyze CEAspecificCD4 and CD8 T cell responses before vaccination and aftersix and seven vaccinations with yeastCEA corresponding to and months respectively PBMCs were also examined daysfollowing one cycle administered every weeks for days ofavelumab Figure 3A This assay involves intracellular cytokinestaining ICS following a period of in vitro stimulation IVSwith overlapping 15mer peptide pools encoding the tumorassociated antigen CEA or the negative control pool HLA aspreviously described The patient did not have preexisting CEAspecific T cells but displayed a notable increase inCEAspecific T cells months following yeastCEA vaccinationfollowing subtraction of background and any value obtainedprior to vaccination there were CD4 cells producingIFNγ and CD4 cells producing TNF per — cellsplated at the start of the stimulation assay As visualized in thedot plots of Figure 3B the CD4 CEAspecific cells includedmultifunctional cells or cells producing cytokine Theincrease in CEAspecific T cells was not seen at the two later timepoints evaluatedFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cDel Rivero et alMTCFIGURE A Five consecutive declines in the patient™s calcitonin levels while on the immune checkpoint inhibitor a decline B Robustly positive PDL1staining after surgical resection of a neck lymph node after vaccine higher power on the rightFIGURE Cross sectional imaging studies with computed tomography of the neck A prior to PDL1 administration and B after a decrease in calcitoninshowing stable thyroid bed recurrenceFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cDel Rivero et alMTCFIGURE Induction of CEAspecific T cells and changes in peripheral immune cell subsets A Schema showing the timing of sequential immunotherapies andimmune assays B CEAspecific T cells were identified in PBMCs by intracellular cytokine staining following a period of in vitro stimulation with overlapping 15merpeptide pools encoding for the tumorassociated antigen CEA or the negative control peptide pool HLA Dot plots of IFNγ and TNF production by CD4 T cellsshowing induction of multifunctional CEAspecific T cells producing cytokine at months C“G PBMCs were assessed for the frequency of immune cellsubsets over the course of immunotherapy The most notable fluctuations were observed after initiation of avelumab indicated by black arrow The frequency overtime of Tregs C cDC D pDC E MDSC F and B cells G indicated as a percentage of total PBMCsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cDel Rivero et alMTCThe frequency of PBMC subsets was also evaluatedin this patient over his course of treatment at the NationalCancer Institute using 11color flow cytometry on cryopreservedPBMC as previously described Supplemental Table PBMCs were assayed prior to vaccination and monthsfollowing yeastCEA vaccine as well as at time points preand post and days avelumab Figure 3A Using change as a cutoï¬ the first fluctuation in immune cell subsetswas observed months following vaccination with yeastCEAand included an increase in regulatory Tcells Tregs an inhibitory immune subset compared to prevaccine levelsFigure 3C After the patient completed vaccine and underwentsurgery and prior to the initiation of avelumab the patienthad more Tregs Figure 3C and more conventionaldendritic cells cDC Figure 3D a subset that is involved inantigen presentation compared to prevaccine levels The mostdramatic fluctuations in immune subsets were noted at thetime point after weeks of avelumab and included decreasesin Tregs Figure 3C cDC Figure 3D and plasmacytoid DCpDC Figure 3E compared to preavelumab therapy levels pDCare tolerogenic DC exhibiting poor immunostimulatory abilityand their interaction with T cells often favors the generationof Tregs Increases in myeloid derived suppressor cellsMDSCs Figure 3F another immune suppressive subset andB cells Figure 3G were also noted after avelumab comparedto preavelumab levels There were no alterations in the CD4CD8 natural killer NK or NKT compartments noted at anytime point examinedDISCUSSIONFor many years doxorubicin was the only US Food and DrugAdministration FDA“approved treatment for patients withadvanced thyroid cancer however response rate in patients withMTC is up to with significant toxicity “ Recentlyin advanced MTC several TKIs such as axitinib cabozantinibgefitinib lenvatinib imatinib motesanib sorafenib pazopanibsunitinib and vandetanib have been studied in phase I IIand III clinical trials Vandetanib an oral inhibitor of VEGFRvascular endothelial growth factor receptor RET and EGFRepidermal growth factor receptor was approved by theFDA in April after a phase III trial demonstrated improvedmedian progressionfree survival PFS compared to placebohazard ratio CI “ and overall response rateof Cabozantinib an inhibitor of hepatocyte growthfactor receptor MET VEGFR2 and RET was approved bythe FDA in after a phase III trial demonstrated improvedmedian PFS of months relative to months in theplacebo group The impact of toxicity on patientswas clearly indicated and for cabozantinib of patientsrequired dose reductions and required dosing delays Therefore toxicity of FDAapproved TKI agents limits their usein patients with small volume asymptomatic or indolent disease Furthermore no clear data exist from these studies thateither agent impacts overall survival In addition RETspecificTKIs in development are Selpercatinib previously LOXO292and Blue667 with more specific RETtargeting activity Theseagents have demonstrated evidence of efficacy in early trialresults however further treatments are warranted withless toxicityEvidence for cellmediated immunity to tumorspecificantigens has been found in medullary thyroid cancer andearly studies suggested that MTCspecific T cells exist Emerging data suggest that the immune system may be relevantin the treatment of MTC “ Furthermoreimmunebased treatments have been studied Dentritic cell“basedimmunotherapy was given in patients with solid tumorsincluding MTC and it was reported that vaccination withautologous tumorpulsed DCs generated from peripheral bloodwas safe and can induce tumorspecific cellular cytotoxicity This case report may demonstrate the potential for therapeuticcancer vaccines to synergize with immune checkpoint inhibitionsequentially in MTC and that principle could be applied as well toother cancers that may have tumor microenvironments TMEsdevoid of baseline immune recognition The therapeutic cancervaccine in this trial was a heatkilled yeastbased vaccine designedto stimulate an immune response against CEA After a phase Itrial demonstrated safety transient injection site reaction wasthe most common adverse event and preliminary evidence ofimmunologic and clinical activity a phase II study was developedin MTC NCT01856920 The phase I study included apatient with MTC who had substantial ‚ammation at sitesof disease that followed months of the vaccine It isalso possible that the patient™s previous sunitinib is relevant inthis case report In a model using CEAtransgenic mice bearingCEA tumors continuous sunitinib followed by vaccine increasedintratumoralltration of antigenspecific T lymphocytesdecreased immunosuppressant Tregs and MDSCs reducedtumor volumes and increased survival The immunomodulatoryactivity of continuous sunitinib administration can create a moreimmunepermissive environment Despite the significant recent advances of antiPD1 andantiPDL1 therapy they still impact only a minority of patientswhose TMEs express those molecules at baseline One hypothesisis that sequential use of vaccine can drive immune cells to theTME resulting in an adaptive reaction by tumor cells potentiallyfrom the presence of cytokines produced by active immune cellsin the TME upregulating PDL1 and perhaps defining a rolefor antiPDL1PD1 therapies in patients who may not haveotherwise benefited from such immunotherapies Basedon this perspective combining or sequencing vaccines with antiPDL1PD1 therapies could broaden the clinical benefit for allpatients with immunologically œcold tumor microenvironmentsdevoid of reactive immune cells to enhance the clinical efficacyamong cancer patients with a variety of tumor types This casemay provide an example of how increasing peripheral Tcellactivation with vaccines can enable immune cells to then migrateto the TME and improve response rates to antiPDL1PD1therapies Indeed existing data with the FDAapprovedtherapeutic cancer vaccine for prostate cancer sipuleucelTindicate that vaccine did increase T cells in the TME aftertreatment Induction of CEAspecific T cells was noted in the peripheralblood of this patient following vaccination with yeastCEA butnot at later time points It is possible the CEAspecific cellshomed in on the TME inducing PDL1 expression subsequentlyFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cDel Rivero et alMTCseen on the tumor In addition fluctuations in the peripheralimmunome were noted in this patient over the course of therapywith yeastCEA vaccine and subsequent avelumab therapythese changes included both immunepotentiating and immunesuppressive alterations with the most notable fluctuationsoccurring after several administrations of avelumab The increasein suppressive elements may be a compensatory mechanisminduced to tamper down the immune activation induced by thediï¬erent immunotherapy treatments However as this patienthad metastatic diseaseit is unknown whether the changesin the peripheralimmunome were directly induced by thesequential immunotherapy regimens or potentially related todisease progressionAs with all case reports these presentations have limitationsthe fact that the patient did not have a biopsy at baseline prior tostarting the vaccine limits understanding of the baseline TMEThus it is unclear if the vaccine drove PDL1 expression orif it was preexisting in this patient Little data exist for thepresence of PDL1 expression on MTC tumor cells To furthercomplicate this case™s assessment the patient was previouslytreated with sunitinib which has been able to deplete Tregswhich alone or in combination with vaccine could have impactedthe PDL1 status of this patient Nonetheless data gleanedfrom using immunotherapy in such a rare disease are worthgreater examinationAlthough a decline in calcitonin does not directly correlatewith clinical responses in this case or in MTC in general themagnitude and consistency of the decline are noteworthy amidstdata that suggest the predictive value of calcitonin doubling timeand disease progression Also many patients with MTChave disease recurrence solely defined by serum tumor markersIn these patients the opportunity to impact calcitonin kineticswith immunotherapy may decrease the pace of the diseaseand delay progression to overt metastasis requiring systemictherapies TKIs that are associated with toxicity or ultimatelymetastatic diseaserelated morbidity Despite the eï¬ectivenessof TKIs in MTC opportunities for immunotherapy clinicaldevelopment may provide patients with additional treatmentoptions that are less toxic and could thus be used earlier in thedisease processETHICS STATEMENTWritten informed consent for publication of clinical detailsandor clinical images was obtained from the patientAUTHOR CONTRIBUTIONSJD RD and RM were responsible for study concept and designJD and RD acquired the data from the study and prepared themanuscript RD was responsible for the immune analysis andinterpretation RM reviewed the manuscript JM AG MB MRLC MM WD JS and JG read and approved the final versionof the manuscript All authors contributed to the andapproved the submitted versionFUNDINGThis work was supported by National Cancer Institute NationalInstitutes of Health Intramural Research Program This researchwas financially supported by Merck KGaA Darmstadt Germanyas part of an alliance between Merck KGaA and Pfizer given thatJAVELIN Solid Tumor is an alliancesponsored trialACKNOWLEDGMENTSThis work was selected for poster presentation at The EndocrineSociety 99th Annual Meeting Orlando FL in We arethankful for the support of the National Institutes of HealthClinical Center staï¬ including nurses clinical and researchfellows Merck KGaA Darmstadt Germany and Pfizer reviewedthe manuscriptfor medical accuracy only before journalsubmission The authors are fully responsible for the content ofthis manuscript and the views and opinions described in thepublication reflect solely those of the authorsSUPPLEMENTARY MATERIALfor this can be foundat httpswwwfrontiersins103389fendoThe Supplementary Materialonline202000490fullsupplementarymaterialREFERENCES Saad MF Ordonez NG Rashid RK Guido JJ Hill CS Jr Hickey RC Medullary carcinoma ofthe clinicalfeatures and prognostic factors in patients Medicine “ the thyroid A study of Kouvaraki MA Shapiro SE Perrier ND Cote GJ Gagel RF Hoï¬ AO RETprotooncogene a review and update of genotypephenotype correlationsin hereditary medullary thyroid cancer and associated endocrine tumorsThyroid “ 101089thy200515531 Del Rivero J Edgerly M Ward J Madan RA Balasubramaniam S Fojo T Phase III trial of vandetanib and bortezomib in adults with locallyadvanced or metastatic medullary thyroid cancer Oncologist “e4 101634theoncologist20180452 Saad MF Fritsche HA Jr Samaan NA Diagnostic and prognostic valuesof carcinoembryonic antigen in medullary carcinoma of the thyroid J ClinEndocrinol Metab “ 101210jcem585889 Saad MF Ordonez NG Guido JJ Samaan NA The prognostic value ofcalcitonin immunostaining in medullary carcinoma of the thyroid J ClinEndocrinol Metab “ 101210jcem595850 Brandi ML Gagel RF Angeli A Bilezikian JP BeckPeccoz P Bordi C et alGuidelines for diagnosis and therapy of MEN type and type J ClinEndocrinol Metab “ 101210jcem86128070 French JD Bible K Spitzweg C Haugen BR Ryder M Leveraging the immunesystem to treat advanced thyroid cancers Lancet Diabetes Endocrinol “ 101016S2213858716302777 Eng C Mulligan LM Healey CS Houghton C Frilling A Raue F et alHeterogeneous mutation of the RET protooncogene in subpopulations ofmedullary thyroid carcinoma Cancer Res “ Stift A Friedl J Dubsky P BachleitnerHofmann T Schueller G Zontsich T Dendritic cellbased vaccination in solid cancer J Clin Oncol “ 101200JCO200302135Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cDel Rivero et alMTC Schott M SeisslerJ Lettmann M Fouxon V Scherbaum WAFeldkamp JImmunotherapy for medullary thyroid carcinoma bydendritic cell vaccination J Clin Endocrinol Metab “ 101210jcem86107949 Bilusic M Heery CR Arlen PM Rauckhorst M Apelian D Tsang KY et alPhase I trial of a recombinant yeastCEA vaccine GI6207 in adults withmetastatic CEAexpressing carcinoma Cancer Immunol Immunother “ 101007s0026201315058 Ravaud A de la Fouchardiere C Caron P Doussau A Do Cao CAsselineau J A multicenter phase II study of sunitinib in patientswith locally advanced or metastatic diï¬erentiated anaplastic or medullarythyroid carcinomas mature data from the THYSU study Eur J Cancer “ 101016jejca201701029 Bastman JJ Serracino HS Zhu Y Koenig MR Mateescu V Sams SB et alTumorInfiltrating T cells and the PD1 checkpoint pathway in advanceddiï¬erentiated and anaplastic thyroid cancer J Clin Endocrinol Metab “ 101210jc20154227 Heery CR O™SullivanCoyne G Madan RA Cordes L Rajan A RauckhorstM Avelumab for metastatic or locally advanced previously treated solidtumours JAVELIN Solid Tumor a phase 1a multicohort doseescalationtrial Lancet Oncol “ 101016S1470204517302395 Del Rivero JGA Bilusic M Rauckhorsts M Cordes L Karzai F StraussJ Calcitonin response following sequential use of a yeastCEAtherapeutic cancer vaccine and avelumab a monoclonal antiPDL1 inhibitorin metastatic medullary thyroid cancer Poster 99th Annual Meeting of theEndocrine Society Orlando FL Heery CR Ibrahim NK Arlen PM Mohebtash M Murray JL Koenig K Docetaxel alone or in combination with a therapeutic cancer vaccinePANVAC in patients with metastatic breast cancer a randomized clinicaltrial JAMA Oncol “ 101001jamaoncol20152736 Heery CR Singh BH Rauckhorst M Marte JL Donahue RN Grenga I et alPhase I trial of a yeastbased therapeutic cancer vaccine GI6301 targetingthe transcription factor brachyury Cancer Immunol Res “ 10115823266066CIR150119 Donahue RN Lepone LM Grenga I Jochems C Fantini M Madan RA et alAnalyses of the peripheral immunome following multiple administrations ofavelumab a human IgG1 antiPDL1 monoclonal antibody J ImmunotherCancer 101186s404250170220y Lepone LM Donahue RN Grenga I Metenou S Richards J Heery CR Analyses of peripheral human immune cell subsets definingdiï¬erences with age and between healthy donors and cancer patients notdetected in analysis of standard immune cell types J Circ Biomark Matta BM Castellaneta A Thomson AW Tolerogenic plasmacytoid DC EurJ Immunol “ 101002eji201040839 Sculier J Thiriaux J Bureau G Lafitte J Recloux P Brohee D A phaseiistudy testing weekly platinum derivative combination chemotherapy as 2ndline treatment in patients with advanced smallcell lungcancer Int J Oncol “ 103892ijo62425 Nocera M Baudin E Pellegriti G Cailleux AF MechelanyCorone CSchlumberger M Treatment of advanced medullary thyroid cancer with analternating combination of doxorubicinstreptozocin and FUdacarbazineGroupe d™Etude des Tumeurs a Calcitonine GETC Br J Cancer “ 101054bjoc20001314 Wu LT Averbuch SD Ball DW de Bustros A Baylin SB McGuire WP IIITreatment of advanced medullary thyroid carcinoma with a combination ofcyclophosphamide vincristine and dacarbazine Cancer “ 1010021097014219940115732432aidcncr282073023130co2k Carlomagno F Vitagliano D Guida T Ciardiello F Tortora G Vecchio G ZD6474 an orally available inhibitor of KDR tyrosine kinase activityefficiently blocks oncogenic RET kinases Cancer Res “ Wells SA Jr Gosnell JE Gagel RF Moley J Pfister D Skinner M et alVandetanib for the treatment of patients with locally advanced or metastatichereditary medullary thyroid cancerJ Clin Oncol “ 101200JCO2009236604 Wells SA Jr Robinson BG Gagel RF Dralle H Fagin JA Santoro M et alVandetanib in patients with locally advanced or metastatic medullary thyroidcancer a randomized doubleblind phase III trial J Clin Oncol “ 101200JCO2011355040 Elisei R Schlumberger MJ Muller SP Schoï¬ski P Brose MS Shah MH et alCabozantinib in progressive medullary thyroid cancer J Clin Oncol “ 101200JCO2012484659 Viola D Cappagli V Elisei R Cabozantinib XL184 for the treatment oflocally advanced or metastatic progressive medullary thyroid cancer FutureOncol “ 102217fon13128 Drilon AE Subbiah V Oxnard GR Bauer TM Velcheti V Lakhani NJ et alA phase study of LOXO292 a potent and highly selective RET inhibitorin patients with RETaltered cancers J Clin Oncol 36Suppl 101200JCO20183615_suppl102 Ilanchezhian M Khan S Okafor C Glod J Del Rivero J Update on thetreatment of medullary thyroid carcinoma in patients with multiple endocrineneoplasia type Horm Metab Res 101055a11458479 [Epubahead of print] Hellstrom I Hellstrom KE Pierce GE Yang JP Cellular and humoralimmunity to diï¬erent types of human neoplasms Nature “ 1010382201352a0 Rocklin RE Gagel R Feldman Z Tashjian AH Jr Cellular immune responsesin familial medullary thyroid carcinoma N Engl J Med “ 101056NEJM197704142961502the thyroid Cellular Gee JM Williams MA Almoney R Sizemore G Medullary carcinomaimmune response to tumor antigen in aofheritable human cancer Cancer “ 01421975113651658AIDCNCR282036051930CO20 Muller S Poehnert D Muller JA Scheumann GW Koch M Luck RRegulatory T cells in peripheral blood lymph node and thyroid tissue inpatients with medullary thyroid carcinoma World J Surg “ 101007s0026801004846and Cressent M Pidoux E Cohen R Modigliani E Roth C Interleukinon ratmedullary thyroid carcinoma cells Eur J Cancer 31A2379“ interleukin4activitydisplaypotentantitumour Lausson S Fournes B Borrel C Milhaud G TreilhouLahille F Immuneresponse against medullary thyroid carcinoma MTC induced by parentalandor interleukin2secreting MTC cells in a rat model of human familialmedullary thyroid carcinoma Cancer Immunol Immunother “ 101007s002620050311 Farsaci B Higgins JP Hodge JW Consequence of dose scheduling of sunitinibon host immune response elements and vaccine combination therapy Int JCancer “ 101002ijc26219 Fu J Malm IJ Kadayakkara DK Levitsky H Pardoll D Kim YJ Preclinicalevidence that PD1 blockade cooperates with cancer vaccine TEGVAX toelicit regression of established tumors Cancer Res “ 10115800085472CAN132685 Antoni R Caroline RF Hodi S Wolchok JD Joshua AM Hwu W Association of response to programmed death receptor PD1blockade with pembrolizumab MK3475 with an interferon‚ammatoryimmune gene 33Suppl 101200jco20153315_suppl3001J Clin Oncolsignature Fong L Carroll P Weinberg V Chan S Lewis J Corman J Activatedlymphocyte recruitmentfollowingpreoperative sipuleucelT for localized prostate cancer J Natl Cancer Inst 106dju268 101093jncidju268into the tumor microenvironment Meijer JA le Cessie S van den Hout WB Kievit J Schoones JW Romijn JA Calcitonin and carcinoembryonic antigen doubling times as prognosticfactors in medullary thyroid carcinoma a structured metaanalysis ClinEndocrinol Oxf “ 101111j13652265200903666xConflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Del Rivero Donahue Mart Gramza Bilusic Rauckhorst CordesMerino Dahut Schlom Gulley and Madan This is an access distributedunder the terms of the Creative Commons Attribution License CC BY The usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this journal is cited in accordance with accepted academic practice No usedistribution or reproduction is permitted which does not comply with these termsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'

Thyroid_Cancer

Molecular Medicine The role of selenium metabolism andselenoproteins in cartilage homeostasisand arthropathiesDonghyun Kang Jeeyeon Lee Cuiyan Wu3 Xiong Guo3 Byeong Jae Lee24 JangSoo Chun5 andJinHong Kim AbstractAs an essential nutrient and trace element selenium is required for living anisms and its beneficial roles in humanhealth have been well recognized The role of selenium is mainly played through selenoproteins synthesized by theselenium metabolic system Selenoproteins have a wide range of cellular functions including regulation of seleniumtransport thyroid hormones immunity and redox homeostasis Selenium deficiency contributes to various diseasessuch as cardiovascular disease cancer liver disease and arthropathy”Kashin“Beck disease KBD and osteoarthritisOA A skeletal developmental disorder KBD has been reported in lowselenium areas of China North Korea and theSiberian region of Russia and can be alleviated by selenium supplementation OA the most common form of arthritisis a degenerative disease caused by an imbalance in matrix metabolism and is characterized by cartilage destructionOxidative stress serves as a major cause of the initiation of OA pathogenesis Selenium deficiency and dysregulation ofselenoproteins are associated with impairments to redox homeostasis in cartilage We review the recently exploredroles of selenium metabolism and selenoproteins in cartilage with an emphasis on two arthropathies KBD and OAMoreover we discuss the potential of therapeutic strategies targeting the biological functions of selenium andselenoproteins for OA treatmentIntroductionSelenium Se is an essential trace element in humans12Selenium is generally taken up from the diet through foodor other forms of external supplementation Dietaryselenium is obtained in the form of selenomethionineSeMet selenocysteine Sec selenite and selenate Significant health benefits have been attributed to seleniummetabolic systems that play major physiological roles inthyroid hormone metabolism immunity and antioxidantdefense23 Selenium is required for the production ofthyroid hormonemetabolizing enzymes and seleniumCorrespondence JinHong Kim jinhkimsnuackr1Center for RNA Research Institute for Basic Science Seoul South Korea2Department of Biological Sciences College of Natural Sciences Seoul NationalUniversity Seoul South KoreaFull list of author information is available at the end of the These authors contributed equally Donghyun Kang Jeeyeon Leesupplementation is thought to improve the function ofthyrocytes and immune cells4 Selenium supplementationdemonstrated immunostimulant effects such as enhancedproliferation of activated T cells activation of naturalkiller cells and tumor cytotoxicity mediated by cytotoxiclymphocytes56 In contrast selenium deficiency is associated with the occurrence virulence and disease progression of viral infections7Selenium inadequacy can lead to various types ofdiseases most notably cardiovascular disease8“ cancer13“ hepatopathy1617 and arthropathy Cardiovascular diseases are associated with systemic seleniumlevel with a higher risk at or μgL seleniumconcentration in the blood10 A type of endemic cardiomyopathy Keshan disease is linked to selenium deficiency811 Keshan disease occurs in lowselenium areasin Chinasodium seleniteand is prevented by The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Korean Society for Biochemistry and Molecular Biology 0cKang Experimental Molecular MedicinePage of studiesEpidemiologicalsupplementation12 Lowselenium status is correlatedwith a significantly increased risk of cancer incidenceand mortality13“haveprovided evidence on the cancerpreventing effects ofselenium18“ Selenium deficiency is also characterizedby elevated levels of oxidative stress markers in the liver21which significantly contribute to liver injury17 The oxidative stress caused by selenium deficiency further plays adetrimental role in joint development Selenium deficiency is the main cause of endemic Kashin“Beck diseaseKBD which is mainly reported in lowselenium areas ofChina North Korea and the Siberian region of RussiaMoreover there is a growing body of evidence suggestingthat the pathogenesis of osteoarthritis OA the mostcommon form of arthritis may be associated with selenium deficiency by resulting in oxidative stress22“However it is noteworthy that excessive selenium intakecan also cause selenosis2930 which accompanies adversesymptoms including fatigue diarrhea nausea increasedheart rate necrosis in liver and kidney and neurologicaldamage Chroniccompromisesimmune and reproductive systems in humanseventuallyselenosisOA is characterized by progressive loss of cartilageextracellular matrix ECM and pathological changes inother joint tissues such as subchondral bone sclerosisosteophyte formation and synovial ‚ammation31 Cartilage destruction is considered a hallmark of OA and is aresult of increased production of catabolic effectors32“and reduced matrix biosynthesis by chondrocytes36 OA isassociated with multiple etiologies involving systemicfactors such as age37 as well as local factors such asmechanical stress38 driven by weightbearing and jointinstability Both OAcausing factors have been found tocause oxidative stress in chondrocytes Oxidative stressresults from the abnormal production of reactive oxygenspecies ROS and the loss of cellular antioxidant capacityMany preclinical and clinical studies have indicated theaccumulation of oxidative burden in chondrocytesundergoing osteoarthritic changes3940 Emerging evidence suggests that oxidative stress is mechanisticallylinked to the initiation of osteoarthritic changes inchondrocytes through the acquisition of senescent phenotypes36 Therefore restoring redox homeostasis canserve as a rational therapeutic strategy to alleviate OAprogression Here we review the role of selenium metabolism in cartilage and bone and the significance ofmaintaining its homeostasis in the context of joint diseases such as KBD and OAOverview of the selenium metabolic systemThe selenium metabolic system and the biosynthesis ofselenoproteinsSelenium metabolism is a systemic process that includesandtransformationtransportationabsorptiontheOfficial journal of the Korean Society for Biochemistry and Molecular Biologyexcretion of selenium Fig Selenium is obtained inanic forms”SeMet and Sec”and inanic forms”selenite and selenate”from diet Selenium is taken up bythe liver that synthesizes and exports SELENOP whicheventually circulates through the bloodstream SELENOPwith multiple Sec residues41 transports selenium to othertissues and ans42 and the transported selenium isconverted to selenophosphate by intracellular seleniummetabolic pathways Selenium is excreted through exhalation and urine in the form of smallmolecule metabolites formed by sequential methylation4344Selenium plays biological roles predominantly in theform of selenoproteins synthesized by the seleniummetabolic system Ingested inanic selenium is firstreduced to hydrogen selenide H2Se via glutathioneGSH and thioredoxin TXN systems Selenide is furtherconverted to Sec amino acids for incorporation intospecific sites of selenoproteins such as the catalytic sites ofa selenoenzyme Mechanistically selenophosphate synthetase SEPHS2 catalyzes the production of selenophosphate through the reduction of hydrogen selenideThe subsequent reaction with phosphoseryltRNA PSertRNA[Ser]Sec yields SectRNA[Ser]Sec Sec amino acids areincorporated into polypeptidethrough themachinery utilizing the UGA codon Selenocysteineinsertion sequence binding protein SBP2 binds toselenocysteine insertion sequence SECIS element whichis located in the ²untranslated region ²UTR of selenoprotein mRNA and mediates the transfer of SectRNA[Ser]Sec to the Asite of ribosome which recognizesthe UGA codon as the Sec integration codon Collectivelythe selenoprotein translation machinery consists of SECISelement SBP2 Secspecific eukaryotic elongation factorEEFSEC and aminoacylated SectRNA[Ser]Sec therebyenabling UGA to be recognized as a Sec codon and utilized for translation into the growing polypeptidechainsSelenoproteinssome ofSelenoprotein is defined as a protein containing Secamino acid residue The biological functions of seleniumare mostly exerted through selenoprotein domains thatcontain Sec residues Twentyfive selenoprotein geneshave been identified in the human genome45 In mice atotal of selenoproteins have been characterized46 andtargeted deletion ofthese selenoproteinsdemonstrated their essential roles in developmental processes and in disease pathogenesis Selenoproteins can beclassified into subfamilies based on their cellular functionssuch as those implicated in antioxidation GPX1 GPX2GPX3 GPX4 redox regulation TXNRD1 TXNRD2TXNRD3 MSRB1 SELENOH SELENOM SELENOWthyroid hormone metabolism DIO1 DIO2 DIO3 selenium transport and storage SELENOP selenophosphatesynthesis SEPHS2 calcium metabolism SELENOK 0cKang Experimental Molecular MedicinePage of Fig Selenium metabolic system in mammals Selenium is absorbed from the diet undergoes several conversion steps and is incorporated intopolypeptide chains completing selenoprotein synthesis Dietary sources of selenium uptake exist in inanic form such as selenate and selenite andanic form such as Sec and SeMet Inanic forms are reduced by TXNRDTRX or GRXGSH systems and anic forms are cleaved by SCLYforming selenide Selenophosphate is synthesized from selenide by SEPHS2 and the subsequent reaction with PSertRNA[Ser]Sec mediated by SEPSECSyields SectRNA[Ser]Sec SectRNA[Ser]Sec is transferred to the Asite of ribosome mediated by SBP2 which binds to SECIS located in the ²UTR of aselenoprotein mRNA Finally the UGA codon is recognized as the Sec integration codon Abbreviations SeMet selenomethionine Secselenocysteine GRX glutathione reductase TRX thioredoxin TXNRD thioredoxin reductase GSH glutathione MGL methionine gammalyase SCLYselenocysteine lyase SEPHS2 selenophosphate synthetase SARS seryltRNA synthetase PSTK phosphoserylSeptRNA kinase SEPSECS SeptRNASectRNA synthase EEFSEC Secspecific eukaryotic elongation factor SBP2 SECIS binding protein SELENOT myogenesis SELENON protein foldingSELENOF SELENOI SELENOS and protein AMPylation SELENOO4748 The functions of other selenoproteins such as GPX6 and SELENOV still remain unclearGlutathione peroxidases GPXs such as GPX1 cytosolicGPX GPX2 gastrointestinal GPX and GPX4 phospholipid hydroperoxide GPX catalyze the decompositionof a great variety of peroxides thus protecting cellsagainst oxidative damage4950 Thioredoxin reductasesTXNRDs employ NADPH as an electron donor to revertoxidized TXN to a reduced dithiol the oxidation status ofwhich is critically implicated in regulating various cellbehaviors including proliferation and apoptosis51 Thephysiological significance of TXNRDs is further supported by the embryonic lethality of Txnrd1 or Txnrd2knockout mice5253 Deiodinases DIOs regulate thyroidhormone metabolism by catalyzing the conversion ofthyroid hormones from precursor thyroxine T4 to biologically active triiodothyronine T3 or inactive reverseT3 rT354 The expression levels of several selenoproteinsOfficial journal of the Korean Society for Biochemistry and Molecular Biologyare ‚uenced by the extent of selenium uptake Forexample seleniumdeficient animals and human cell linesexhibit reduced transcription of selenoproteins such asGPX1 DIOs SELENOI and SELENOW55“ A subset ofselenoproteins such as GPX1 and SELENOW is moresensitive to selenium supplementation or deficiency Thehierarchy of selenoprotein expression is more apparentwhen the intracellular level of selenium is limited1Seleniumresponsive genesgenesareseleniumcontainingSeleniumresponsivethe genes whoseexpression patterns are ‚uenced by supplementationwith selenium orcompoundsTreatment of a cancer cell line with methylseleninic acidin genes58 Theseinduced expression changesresponsive genes were closely associated with annotationsrelated to cell cycle regulation androgenresponsive genesand phase II detoxification pathway Selenium supplementation of macrophages diminished the expression oflipopolysaccharide LPSinduced pro‚ammatory genes 0cKang Experimental Molecular MedicinePage of such as cyclooxygenase2 COX2 and tumor necrosisfactorα TNFα59 suggesting that selenium has anti‚ammatory effects on the immune system The CTDdatabase httpctdbase reports the effect of environmental chemicals including selenium on gene expression profiles in various human tissuesThe role of selenium and selenoproteins incartilage development and KBDSelenium levels and its role in joint tissuesJoints are composed of various types of connective tissues including cartilage bone synovium meniscus andligament Among these tissues cartilage is the maincomponent that absorbs mechanical stress cushioningbones from impacting each other during various weightbearing activities In the human knee joint the seleniumconcentration in cartilage is approximately μgkg dryweight whereas the selenium concentrations in ligamentand meniscus are and μgkg dry weight respectively6061 The requirement of adequate physiologicalselenium levels for maintaining cartilage homeostasis hasbeen recognized Selenium deficiency retards the growthand development of cartilage and bone62“ Growthretardation was observed in rats after two generations ofselenium deficiency62 Mice fed a diet deficient in selenium resulted in fibrocartilage formation at the articularsurface ultimately showing degeneration of articularcartilage63 Selenium deficiency induced the expression ofthe chondrocyte hypertrophy marker gene type X collagenCOLX in articular cartilage64 The expression of parathyroid hormonerelated protein PTHrP which controlschondrocyte maturation during endochondral ossification was enhanced in both articular cartilage andhypertrophic growth plate following selenium deficiencyThese changes were in line with the phenotypic changesobserved in the cartilage of KBD patients64 However itshould be noted that growth retardation caused by selenium deficiency may also be associated with the deregulation of bone metabolism65 In a study by Cao et alselenium deficiency severely compromised bone microarchitecture as a result of increased bone resorption66Abnormalities in selenium metabolism and skeletaldevelopment diseasesSelenium deficiency is regarded as one of the initiatingfactors of KBD which is an endemic osteoarthropathycaused by the premature closure of epiphyseal plate andthe impaired skeletal development Skeletal deformities inhands fingers knees and elbows and in severe casesdwarfism and movement disorders are the symptoms ofKBD22 The KBD area roughly coincides with lowselenium areas including a geological belt extendingfrom northeast to southwest China North Korea andeastern Siberia22 A metaanalysis showed that seleniumOfficial journal of the Korean Society for Biochemistry and Molecular Biologylevels in the water soil cereal and corn in KBD endemicregions were lower than they were in nonendemicregions supporting the fact that the level of selenium intissue is predominantly affected by dietary intake23 In linewith this finding selenium levels in the whole bloodserum hair and urine of KBD patients were markedlylower than those of healthy controls24Selenoprotein gene polymorphisms are associated withincreased susceptibility to KBD There were significantdifferences in the allelic frequency of GPX1 Pro198Leurs1050450 between the KBD and control group67 Inaddition the mRNA level of GPX1 and enzyme activity oftotal GPX in blood were lower in the KBD group thanthey were in the control group67 Haplotypes of TCCTTC and TTT of rs1050450 rs3811699 and rs1800668in GPX1 gene also had a significant link to KBD68 Asinglenucleotide polymorphism SNP in the promoterregion of SELENOS rs28665122 ˆ’105G A was relatedto the increased risk of KBD and upregulation of PI3KAktsignaling in patients with KBD69 In this study tertbutylhydroperoxide tBHPtreatmentinduced chondrocyteapoptosis was mitigated by selenium supplementation viasodium selenitetreatment which suppressed thePI3KAkt pathway The minor Aallele of SELENOFrs5859 was associated with a significantly higher incidenceof KBD70The animals fed a seleniumdeficient diet recapitulatedsome of the pathological manifestations of KBD stronglysupporting the notion that selenium deficiency is criticallyassociated with the development of this endemic arthropathy Selenium deficiency impaired bone and cartilagegrowth with the exhibition of premature chondrocytehypertrophy as evidenced by an increased expression ofCOLX compatible with the phenotypes in KBD cartilage64The lowselenium condition in combination with threemycotoxins deoxynivalenol DON nivalenol NIV and T yielded procatabolic changes and hypertrophic phenotype of chondrocytes as evidenced by the loss of aggrecanand type II collagen COLII and the increase in COLX andmatrix metalloproteinases MMPs expressionrespectively71 In contrast selenium supplementation partiallyalleviated these mycotoxininduced damages in chondrocytes71 In rats dietary selenium deficiency over twogenerations caused the onset of physiological seleniuminsufficiency72 In this condition pathological changes inthe epiphyseal plate were observed with the decreasedexpression of COLII and GPX1 in the chondrocytes suggesting a possible association of reduced chondrocyte anabolism and antioxidant capacity with the epiphyseal platelesions observed in KBD72 The relevance ofimpairedselenium metabolism to the onset of KBD was furthervalidated using a mouse genetic deletion model Targeteddeletion of SectRNA[Ser]Sec Trsp gene in osteochondroprogenitor cells from embryonic stage caused the 0cKang Experimental Molecular MedicinePage of depletion of selenoproteins in skeletal systems causinggrowth retardation abnormalities in the epiphyseal growthplate delayed endochondral ossificationand chondronecrosis which recapitulated the major pathologicalfeatures of KBD73As a prophylactic treatment selenium supplementationswere given to children living in a KBD area The supplemented group showed elevated physiological seleniumlevels in their hair samples and exhibited a substantiallylower prevalence of KBD74 A metaanalysis including fiverandomized controlled trials RCTs and ten prospectivenonRCTs statistically demonstrated the benefits of selenium supplementation in preventing KBD in children75Selenium metabolism and OAPhysiological significance of oxidative stress inchondrocytesOA is the most common form of arthritis and is primarilycharacterized by the loss of cartilagespecific ECM and otherpathological changes in joints including subchondral bonesclerosis osteophyte formation and synovial ‚ammation31Articular cartilage is composed of abundant proteoglycans inwhich sulfated glycosaminoglycan chains such as chondroitinsulfates are bound to a core protein such as aggrecan Loss ofcartilage matrix during OA progression is a combined resultof increased catabolic process in cartilage and reduced anabolic activity of chondrocytes The molecularlevel understanding of OA pathogenesis has led to the identification ofmajor catabolic enzymes ADAMTS576 MMP377 andMMP1378 which mediate the degradation of cartilagematrix Pro‚ammatory cytokines drive the expression ofthese catabolic factors in chondrocytes through the activationof transcription factors such as HIF2α32 and NFκB79Abnormalities in various metabolic pathways such as glucose80 or amino acid metabolic system81 in chondrocyteshave been implicated in activating catabolic cascades inosteoarthritic cartilage82 Moreover increased cellular uptakeof Zn2 through the upregulation of zinc transporter ZIP8activates metalregulatory transcription factor1 MTF1which in turn induces the expression of matrixdegradingenzymes in chondrocytes3383 Regulation of catabolism bythefurthershowed the association of metabolic abnormalities with thecatabolic process of OA34cholesterol“CH25H“CYP7B1“RORαaxisMeanwhile the upstream regulatory mechanism eliciting an imbalance in OA matrix homeostasis needs furtherinvestigation OAcausing factorssuch as age andmechanical stress lead to excessive oxidative stress inchondrocytes3738 Consistently clinical and preclinicalOA studies indicated a cumulative oxidative burden inosteoarthritic chondrocytes3940 Emerging evidence suggests that oxidative stress plays a significant role in OAdevelopment and the disease progression can be mitigatedby counteracting oxidative stress3684“In generalOfficial journal of the Korean Society for Biochemistry and Molecular Biologyoxidative stress results from the abnormal production ofROS and the loss of cellular antioxidant capacity Synovialfluid from patients with latestage OA who were undergoing knee joint replacement had a lower level of oxidoreductases than that from healthy controls87 In partthe increase in oxidative stress is attributable to mitochondrial dysfunction in OA chondrocytes8889 OAchondrocytes displayed reduced mitochondrial DNAcontent mitochondrial dysfunction and diminishedexpression of NRF2 which regulates the transcription ofoxidoreductase genes89 Similarly chondrocytes fromaged individuals exhibited increased ROS burden andmitochondrial and genomic DNA damage90“ Therefore the proper maintenance of redox homeostasis canpotentially serve as a rational therapeutic strategy toprotect against OA progressionPotential roles of selenium metabolism in OAThe protective effect of selenium in OA has beenexplored in a large number of epidemiological and geneticstudies Table The concentration of selenium in serumwas significantly lower in OA patients than that of normalcontrols25 Similarly the results from a populationbasedcohort study demonstrated the linkage between lowselenium levels in toenails with OAassociated pain anddisease severity2627 Several studies have indicated thatcartilage matrix homeostasis is impaired in seleniumdeficiency Lowselenium status diminished COLIIexpression level regulated by SOX9 which is known as amaster regulator required for maintaining cartilage matrixIn fact SOX9 was destabilized by thehomeostasisdownregulation ofseleniumresponsive PRMT5 thatsustains SOX9 stability via methylation93 In anotherstudy rats fed a seleniumdeficient diet exhibited lowsulfotransferase activity which resulted in diminishedforcontents ofmechanicalcartilagematrix28 In contrast selenium supplementation ameliorated the spontaneous degeneration of articular cartilagein STR1 N mice by increasing the expression of GPXs94In cultured chondrocytes pretreatment with SeMetmarkedly inhibited nitric oxide NO and prostaglandinE2 PGE2 production in response to pro‚ammatorycytokine IL1β95 Expression of SBP2 a factor recognizingSECIS element had a positive correlation with GPX1 andGPX4 expression and antioxidant capacity in chondrocytes96 Oxidation resistance mediated by SBP2 wasdiminished in response to IL1β treatment in vitro and indamaged regions of cartilage in OA patients96 Downregulation of selenoprotein mRNAs including GPX397GPX1 and GPX49698 and Selenop99 was observed inhuman and mouse OA chondrocytessulfated glycosaminoglycan essentialstressabsorbingpropertyofGenetic factors such as SNPs in selenoproteins wereidentified to be risk factors for OA development A GAG 0cKang Experimental Molecular MedicinePage of Table List of selenoproteins associated with the pathogenesis of arthropathies KBD and OAGeneGPX1GPX3GPX4DIO2DIO3SELENOFSELENOPSELENOSFunctionExpression in OASNPAntioxidantReduction of hydrogen peroxide and anic peroxidesDownregulatedPlasma antioxidantDetoxification of lipid hydroperoxidesMetabolism of lipidsActivation of hormonesDeiodination of T4 to T3Inactivation of hormonesConversion of T4 to rT3Protein foldingStorage and transport of SeAntioxidant propertiesProtein foldingERassociated protein degradationDownregulatedDownregulatedUpregulatedDownregulatedrs1050450 KBDrs3811699 KBDrs1800668 KBDrs225014 OArs12885300 OArs945006 OArs5859 KBDrs28665122 KBDRef“haplotype in SELENOS gene was significantly associatedwith increased levels of‚ammatory factors in OApatient plasma100 SNPs in DIO2 which converts precursor thyroid hormone T4 to its active form T3 were alsorelated to genetic susceptibility to OA developmentLevels of DIO2 mRNA and protein were markedly upregulated in OA cartilage101 A common DIO2 haplotypecomposed of the minor Callele of SNP rs225014 and thecommon Callele of SNP rs12885300 was significantlyassociated with advanced hip OA as indicated by a higherodds ratio101“ Locus rs225014 which confers risk toOA was associated with the differential methylation ofCpG located in the upstream region of DIO2 gene andwas correlated with upregulated DIO2 expression inOA104 Meanwhile DIO3 depletes the resources that canbe utilized for the production of active thyroid moleculesby catalyzing the conversion of T4 and T3 into inactivemetabolites The minor Gallele of the DIO3 variantrs945006 was associated with a protective effect againstOA development105However a few aspects regarding the relationshipbetween selenium and OA remain controversial Firstseveral studies indicate that there are no differences inselenium levels between OA and normal tissues Theselenium concentrations in synovial fluid and plasma of OA patients were not significantly different from thoseof healthy controls106 Similarly no significant difference in selenium concentration was noted between sixdogs with posttraumatic OA and six control dogs107Second the beneficial effect of selenium supplementationin alleviating OA symptoms has been debated The resultsfrom a controlled doubleblind trial of patientsOfficial journal of the Korean Society for Biochemistry and Molecular Biologyrevealed that the supplementation of a formulation containing selenium with vitamins A C and E SeACE didnot have any remarkable curative effect compared to aplacebo108 In a study with an independent cohort theprevalence of radiographic knee OA was not significantlyassociated with dietary selenium intake109Nonethelessit is apparent that selenium deficiencydysregulation of selenoproteins and genetic variations inselenoprotein genes serve as potential risk factors for OAThe vital role of selenium metabolism in maintainingcartilage homeostasis is expected considering its criticalinvolvementin regulating cellular processes such aschondrogenic differentiation of progenitor cells maintenance of redox homeostasis and DNA damage repair inchondrocytes which are covered in the next sectionIntracellular roles of selenium metabolism andselenoproteins in cartilageChondrogenic differentiation programs of progenitor cellsSelenium exerts various beneficial effects to promoteproliferation and differentiation of chondrogenic progenitorcells110111 Selenium supplementation stimulated the proliferation of ATDC5 chondrogenic cells even under serumdeprivation by inducing cyclin D1 expression110 Deficiencyof SELENOO interfered with the chondrogenic differentiation of ATDC5 cells by suppressing the expression ofchondrogenic genes SOX9 COLII and aggrecan anddecreasing the activity of alkaline phosphatase112 Knockdown of Gpx1 reduced the chondrogenic differentiation ofATDC5 cells by modulating intracellular GSHoxidizedGSH GSSG ratio113 Selenop was differentially upregulatedduring the chondrogenic differentiation of micromass 0cKang Experimental Molecular MedicinePage of culture of mesenchymal cells isolated from mouse limbbuds114 In line with the effects of selenium metabolism andselenoproteins in chondrogenic progenitor cells observedin vitro deficient uptake of selenium severely affectedchondrogenic differentiation of mesenchymal lineage cellsin mice64andOsteochondroprogenitorspecific deletion of Trsp genesignificantly impaired chondrogenic programs causingabnormalities in bone and cartilage development in mice73endochondralossificationthusAntioxidant defense and redox homeostasisfunction ofattributed to theThe protective effects of selenium on cartilage are primarilyantioxidantdefense115“ The metabolism and survival of chondrogenic progenitors and chondrocytes are greatly compromised by ROS including free radicals peroxides andsuperoxide anions118“ Recent studies strongly supportthe notion that mitochondrial dysfunction and oxidativestress are the main drivers of OA pathogenesis37Although ROS play essential roles in the maintenance ofbasal cellular activities such as chondrocyte proliferationand matrix remodeling in cartilage excessive oxidativestress causes detrimental events such as cellular senescence36121 dedifferentiation122 and apoptosis123 ROScause oxidative damage to various cellular componentsand disrupt the balance between ECM catabolism andanabolism119 ROS suppress mitochondrial oxidativephosphorylation and ATP production which are essentially required to sustain cartilage matrix synthesis124 Inaddition ROS induce matrix degeneration through theupregulation of matrixdegrading enzyme expressionwhile this effecttreatment123125 The detrimental effects of ROS on cartilagehomeostasis can be effectively alleviated by augmentingcellular antioxidant activity under stress conditions andseveral attempts have been made to treat OA by targetingthe regulators involved in oxidative stress production incartilage84“is abolished by antioxidantThe protective role of selenium metabolism is thoughtto be exerted through the neutralization of ROS viaantioxidant activities of selenoproteins including GPXsand TXNRDs Bone marrow stromal cells cultured inmedium supplemented with low selenite concentrationexhibited ROS accumulation along with the reducedexpression of GPXs TXNRDs and other seleniumindependentinmicronuclei generation which is an indication of chromosome damage126 Both GPX1 expression and activitywere substantially lower in mice fed a seleniumdeficientdiet than those in mice fed a normal dietleading todecreased trabecular number reduced femoral trabecularvolumetotal bone volume ratio and trabecular separation66 The rats exposed to a seleniumdeficient diet withT2 toxin showed increased lipid peroxidation level andoxidoreductaseenzymesresultingOfficial journal of the Korean Society for Biochemistry and Molecular Biologydecreased antioxidant GPX activity in their serum andcartilage127 A seleniumdeficient dietinduced theexpression of miR1385p which in turn suppressed theexpression of SELENOM that has antioxidant functionand caused mitochondrial dysfunction and apoptosis ofchondrocytes128 Lead Pbinduced oxidative stress andtoxicity reduced the expression of selenoprotein mRNAsand the effect was mitigated by selenium supplementation129 In summary the antioxidant properties of selenoproteins showed therapeutic potential by counteractingthe accumulation of damage induced by oxidative stress incartilageDNA damage repairIt is well known that DNA damage pathways play substantial roles in the progression of arthropathies119 Theexpression of genes related to DNA damage was changedin the cartilage of KBD patients130131 Chronic DNAdamage induces the initiation of apoptosis or cellularsenescence in chondrocytes36132133 Selenium has apotential to reduce DNA damage and increase DNArepair capacity134 In part the beneficial effect of seleniumon genomic stability is associated with the antioxidationeffect of selenoproteins such as GPXs and TXNRDswhich remove ROS before they cause DNA damage134Cancer cells supplemented with selenium nM sodiumselenite or μM SeMet showed elevated levels of GPX1and TXNRD1 enzyme activity effectively protectingagainst DNA strand breaks induced by ultraviolet A orH2O2induced oxidative stress135 SeMet reduced theextent of DNA damage and enhanced DNA repair capacity by inducing repair complex formation in DNAdamaged cells through U

Thyroid_Cancer

Genomic characterization of malignant progressionin neoplastic pancreatic cystsMicha«l No«et alIntraductal papillary mucinous neoplasms IPMNs and mucinous cystic neoplasms MCNsare noninvasive neoplasms that are often observed in association with invasive pancreaticcancers but their origins and evolutionary relationships are poorly understood In this studywe analyze samples from IPMNs MCNs and small associated invasive carcinomas from patients using whole exome or targeted sequencing Using evolutionary analyses weestablish that both IPMNs and MCNs are direct precursors to pancreatic cancer Mutations inSMAD4 and TGFBR2 are frequently restricted to invasive carcinoma while RNF43 alterationsare largely in noninvasive lesions Genomic analyses suggest an average window of overthree years between the development of highgrade dysplasia and pancreatic cancer Takentogether these data establish noninvasive IPMNs and MCNs as origins of invasive pancreatic canceridentifying potential drivers of invasion highlighting the complex clonaldynamics prior to malignant transformation and providing opportunities for early detectionand interventionA list of authors and their affiliations appears at the end of the paperNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178Pancreatic cancer is a deadly disease with a dismal prognosisthat is predicted to soon be the second leading cause ofcancer death in the United States1 However like otherepithelial malignancies pancreatic cancer arises from noninvasiveprecancerous lesions that are curable if detected and treated earlyenough Although the majority of pancreatic cancers are believedto originate in microscopic precancerous lesions pancreaticintraepithelial neoplasia or PanIN a significant minority arise inassociation with larger cystic neoplasms that can be detectedusing currently available imaging technologies2 These neoplasmswhich includeintraductal papillary mucinous neoplasmsIPMNs and mucinous cystic neoplasms MCNs are frequentlydiagnosed incidentally on abdominalidentifying acohort of atrisk patients with an important opportunity forprevention of invasive pancreatic cancer2 However preventionmust be balanced with potential overtreatment of lowrisk lesionsas pancreatic resection carries significant morbidity and evenoccasional mortality3 There is a critical need to understand themolecular alterations that are associated with the development ofinvasive cancer as these represent potential biomarkers to identify cysts at high risk for progression to carcinoma and thusrequiring clinical interventionimagingAlthough genomic analyses have been performed on hundredsof invasive pancreatic cancers relatively few noninvasive neoplasms have been analyzed comprehensively Whole exome andtargeted sequencing of small cohorts of IPMNs and MCNs haverevealed driver genes characteristic of each type of cystic neoplasm4“ while targeted analyses in larger cohorts have confirmed the prevalence of specific driver gene mutations thatcorrelate with grade of dysplasia or histological subtype7 Thesestudies have confirmed that hotspot mutations in the oncogenesKRAS and GNAS occur in lowgrade lesions while mutations inother driver genesincluding CDKN2A TP53 RNF43 andSMAD4 occur with increasing prevalence in lesions with highgrade dysplasia or associated invasive carcinoma8 Targeted nextgeneration sequencing has been used to analyze pancreatic drivergenes in different regions of IPMNs revealing a surprising degreeof intratumoral genetic heterogeneity even with respect to wellcharacterized driver gene mutations9“ However the aboveanalyses were based on studies of either single regions from eachneoplasm or a limited number of genes from multiple regionsand did not provide an analysis of the evolutionary relationshipbetween different regions of pancreatic cysts and associatedcancers These limitations highlight the need for comprehensivegenomic analysis of these cysts and associated invasive cancers tounderstand the molecular alterations that underlie the transitionto invasive carcinomaIn this study we perform whole exome sequencing of IPMNsand MCNs and their associated invasive carcinomas Importantlywe focus our study on small invasive carcinomas cm inorder to more precisely analyze the genetic alterations that occurat malignanttransformation in pancreatic tumorigenesis Inaddition in a subset of our samples we perform deep targetednext generation sequencing on a larger set of additional tissuesamples in order to assess mutated loci through entire neoplasmsincluding areas of lowgrade dysplasia highgrade dysplasia andinvasive carcinoma These analyses reveal important features ofpancreatic tumorigenesisincluding evolutionary relationshipsbetween different regions within cystic neoplasms as well asmolecular alterations that may drive the transition from a noninvasive precursor lesion to invasive cancerResultsOverall approach In order to dissect the molecular relationshipsbetween noninvasive dysplastic lesions and invasive pancreaticcancers we performed whole exome sequencing of neoplastictissue samples from patients with small invasive carcinomas cm associated with neoplastic pancreatic cysts including patients with IPMNs and patients with MCNs Supplementary Data Whole exome sequencing was performed onone sample from the noninvasive component with highgradedysplasia and one sample from the invasive cancer in each caseand for three cases an additional noninvasive sample with lowgrade dysplasia was also analyzed by whole exome sequencingMatched normalsamples were analyzed by whole exomesequencing in each case to exclude germline variants and toidentify somatic mutations Whole exome sequencing was performed with an average total coverage of — distinct coverageof — generating TB of sequencing data SupplementaryData In addition to whole exome analyses we performed targetednext generation sequencing of microdissected tissue samplesfrom seven of the above cases six IPMNs and one MCN Forthese targeted analyses we performed laser capture microdissection to isolate neoplastic cells from every available tissue block ofthe noninvasive cyst and cancer specimens Separate sampleswere microdissected based on grade of dysplasia cell morphollocation This resulted in “ogy architecture and spatialadditional samples per case The targeted panel analyses includedall mutated loci identified in the whole exome sequencing of theseseven cases as well as the entire coding regions of wellcharacterized pancreatic driver genes Supplementary Data The targeted sequencing had an average coverage of —distinct coverage of — Supplementary Data We developed an integrated mutation calling pipeline torigorously assess mutations in all sample types in our analyses inorder to confidently identify even subclonal alterations in sampleswith low neoplastic purity see œMethods Fig Supplementary Data In addition we utilized both on target and off targetreads to examine focal copy number changes as well as loss ofheterozygosity throughout the genome Fig SupplementaryData and From our whole exome sequencing analyses weidentified an average of somatic mutations in samples fromnoninvasive components range “ and an average of somatic mutations in invasive carcinoma samples range“Fig 1a Supplementary Data An average of somatic mutations were shared between the noninvasive andinvasive components while somatic mutations were unique tosamples from noninvasive components and somatic mutationswere unique to samples from invasive cancer Fig 1a We alsoidentified an average of five shared copy number alterationsbetween noninvasive and invasive components as well as anaverage of one copy number alteration unique to samples frominvasive cancer A similar mean proportion of somatic mutationsand copy number alterations were unique to invasive samples for somatic mutations for copy number alterationsAnalysis of our combined whole exome and targeted sequencing data provided multiple insights into IPMN and MCNtumorigenesis In every analyzed case there were multiple sharedmutations between the noninvasive and invasive componentsThese included both driver and passenger mutations indicatingthat they shared a common phylogenetic ancestor Fig 1a b Inaddition accumulation of unique mutations in both noninvasiveand invasive components demonstrated independent evolutionafter the divergence of the subclone that gave rise to the invasiveFig Supplementary Figs S1“S18 Analysis ofcanceradditional adjacent lowgrade or highgrade samples from thesame lesions revealed a subset of shared mutations suggestingthat these dysplastic lesions preceded the development of theinvasive carcinoma Fig Supplementary Figs S1 S2 S3 S5and S16 Evolutionary analyses showed a branched phylogeny inNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178asnoitatuMCancerization onlyDuctal cancer onlyMucinous cancer onlySharedIPMN onlyMCN onlyPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMbKRASTP53CDKN2ASMAD4RNF43GLI3GNASMUC16PTPRTTGFBR2ATMCNTN5PIK3CAALKAPCRYR2STK11CTNNB1ERBB4EGFRSF3B1NOTCH1KEAP1ERBB2MYCRETTSC2PrecursorSharedCancerFig Somatic mutations identified in matched noninvasive and invasive cancer samples a In each patient sample multiple mutations were sharedbetween the noninvasive and invasive cancer samples gray In addition some mutations were limited to the noninvasive bluegreen while others werelimited to the cancer redpink Darker colors indicate alterations that were likely restricted to one component but where sequencing coverage in thesecond component was limited The proportions of shared and distinct mutations varied between different lesions b Somatic mutations in the mostfrequently mutated genes are categorized as shared between noninvasive and cancer gray limited to noninvasive blue or limited to cancer redMutations in some genes such as KRAS were always shared while others were enriched in samples from noninvasive RNF43 or cancer SMAD4MutationsMTP1MTP2MTP3MTP4MTP5MTP6MTP7MTP8MTP9MTP11MTP13MTP14MTP18MTP19MTP23MTP24MTP26MTP30chr1chr2chr3chr4chr5chr6chr7chr8chr9chr10chr11chr12chr13chr14chr15chr16chr17chr18chr19chr20chr21chr22Copy number log2ˆ’ˆ’ˆ’SamplesLG IPMNHG IPMNLG MCNHG MCNDuctal cancerMucinous cancerCancerizationFig Copy number alterations identified in matched noninvasive and invasive cancer samples Chromosomal gains red and losses blue are shownfor each chromosome in each patient with noninvasive samples on the left and cancer samples on the rightNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178MTP19HEbefore LCMafter LCMMTP8KRASG12DKRASG12RKRASQ61Hˆ’1225380275_TGKRASQ61Hˆ’1225380275_TAKRASG12VTMEM56ˆ’RWDD3c5662TC [SP]KCNA5A451AC17orf98T142MDUSP27T652TADAMTS12P429LCOL12A1R933HSCAF8G961VTMEM246A372AMCM10E613KPNLIPRP1T465TC13orf35L33LSMAD6L179LGRIN2AS397SAP1G1T798TTP53E171FCHO1c22471GC [SP]LRFN1S632SLTBP4P243LLOHchr17001ˆ’ABRF645SALPK1R873RLOHchrX275ˆ’DELchr9218ˆ’2377CDKN2ABSND861NSUPT6HI104TPLEKHF1D258DLOHchr221716ˆ’LTBRR425GACOT11R306LOR6N1Y120YENPP5Y341YITGB3C562BTKP116TSLC1A7G465DFGF3R104QZBED4P1100LSIM1R192CPHC1S627CMS4A1T41TLOHchr712725ˆ’SH2D5R199WRNPEPL1G413VGAL3ST2P85PJADE2N352SHHLA1A97VTMEM141Q61QBPTFR296HKLHL22R603RGALEQ261LANKS4BQ368RKRI1S326SOR8I2C240CLRTM2G319VLOHchr97123ˆ’LAMB3R887LSPTAN1I1484FTP53R175HTissue sourceLG IPMNHG IPMNMUCINOUS CANCERDUCTAL CANCERCANCERIZATIONSequencingTargeted SeqWhole Exome SeqDriver MutationHotspotInactivating_OtherDELLOH µm µmMPT19 LG IPMN µm µm µmMPT19 HG IPMN µm_________ µm µm µmMPT19 ductal cancer µm µm µmMPT19 cancerization T2 T1 µm µmMPT8 LG IPMN µm µm µmMPT8 HG IPMN µm__ µm µm µmMPT8 mucinous cancer T1 T2 TTC39AN83ILRRC8DI741MOBSCNR2837QRGS7A195AOR14A2A156ACCDC13S534SEGFLAMG553VITGA1V27LBDP1N622NKIAA0319V824FRIMS1R211QPRDM13A303VIFNGR1M1PHACTR2A348PGPR85F208FGNED83HHTR7T240MGRM5T946KKRASG12DUNC13CN497SBNIP2D58DCCDC33D354NCLDN9P187PNOTUMR308CPIEZO2R2407QC19orf24G52AMUC16P9201QCCNE1R85WGNASR201HPHF21BA116TTNS1R894QNDUFAF6L320ISLC15A1E26KCLUHR433PRR23AA151TBRD3R313HUSP34R3136HRHOAK27ESIM1A654TNRG1A540VVRK3S129NCCDC166A84TST3GAL4V165MTSPAN17P63PMAD1L1S327SC17orf47E142GCHERPL47ITRPC5E418DZFP37R18WGNASD464NMED12E1497KBAG2T93TRNF43R371CACNG2V255MMAGEB18A63VBRINP3A748APDE1BE401EQRICH2R160RRNF43P370fsINTS1G527VMYOM3G152SPRRT3P407STGFBR2R553HPDZD2I810IFAM120BR646CEPHB4V682MNOBOXR302CCEP164R897QMAB21L1T171MZCCHC14T80MCD68L295LGAS2L2S728LRUNDC3AE49EPHBI122VDNMT1N109NZNF865A721ACASS4S376SKCNJ6R322SLC5A4L390PLRRC8CR355HNPR1R439HPPP1CBV263LBIRC6E4424VIL1Q468QMOGAT3cˆ’2359GAUSP20A675TMEX3BS256YMUC13T30AZBTB10R456QSLC26A3V54IC9orf84E8DPPP1R36R303CPLEKHA6Q171SSH3P639PDSCAML1G1252DSLCO1C1R639KENDOVA2VCOL9A1A680TMC2RF228LBPIFB2A427SLOHchr812546ˆ’DELCDKN2ARNF43P660fsRNF43C471fsRNF43E39fsRNF43M1_G4delRNF43S321RNF43P231LFig Somatic mutations identified in MTP19 and MTP8 in targeted and whole exome sequencing We show mutations identified in each sampleincluding lowgrade IPMN light blue highgrade IPMN dark blue ductal cancer red mucinous cancer pink and cancerization purple The type ofsequencing analysis targeted or whole exome performed for each sample is indicated in a track on the bottom Representative images of neoplastic tissuestained by hematoxylin and eosin as well as isolated regions before and after laser capture microdissection are shown for MTP19 and MTP8NATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178MTP2MTP8MTP3MTP19MTP1MTP5MTP24LG IPMNHG IPMNLG MCNHG MCNDuctal cancerMucinous cancerCancerizationMutationsFig Evolutionary reconstruction of samples analyzed by whole exome and targeted sequencing In all cases noninvasive samples bluegreenprecede invasive samples redpink in the evolutionary history In MTP5 different invasive cancer samples are placed in different regions of thephylogeny highlighting multiple independent invasion events in this lesion In MTP19 a sample of cancerization purple has descended from invasivecancer sampleseach case with multiple clonally related but distinct dysplasticsamples from each neoplasm Fig Importantly removal ofdriver gene mutations from these analyses did not significantlyalter the resulting phylogenies indicating that the evolutionaryrelationships are supported by mutations in addition to driveralterations which might be shared by chance Thus the inferredevolutionary relationships between IPMNMCNs and cancersamples are robust as the probability of sharing a nonhotspotmutation due to chance alone is vanishingly small in million while the mean number of shared point mutations was the vast majority of which did not occur in hotspotsAnother potentialexplanation forshared mutationsthis sample impurityinnoninvasive and invasive samples is the presence of a smallnumber of cancer cells contamination in IPMNMCN samplesOur detailed pathological characterization and macro or microdissection minimized the risk ofInaddition variant allele frequencies VAFs of shared mutationsin IPMNMCN and cancer samples can also help to evaluate thelikelihood of such contamination In mutations shared betweenthe IPMNMCN and cancer the mean VAF in the noninvasivesamples was with only of shared mutations having aVAF below in the noninvasive samples These high VAFsindicate that the shared mutations were not the result of a smallnumber of cancer cells contaminating the noninvasive samplesOverall these data provide evolutionary evidence that IPMNs andMCNs were precursors to invasive pancreatic cancer with lowgrade regions usually preceding highgrade regions and ultimatelyresulting in invasive carcinomaDriver genes of IPMNMCN tumorigenesis Through wholeexome and targeted sequencing analyses of IPMNsMCNs andassociated invasive carcinomas we confirmed the high prevalenceof mutations in previously identified pancreatic driver genesincluding mutations of KRAS of cases GNAS CDKN2A TP53 SMAD4 and TGFBR2 Fig 1b Somatic mutations were also identified in RNF43 which has been previously highlighted for its role as a driver inmucinproducing pancreatic cysts4 Somatic mutations wereobserved at low prevalence in key positions in the PI3K PIK3CATSC2 and WNT APC CTNNA2 CTNNB1 signaling pathwaysas well as in STK11 Fig 1b Supplementary Data Alteration ofthese genes and pathways has been previously reported in afraction of IPMNs471314 The two MCNs analyzed were similarto the IPMNs in the cohort with hotspot mutations in KRAShomozygous deletion of CDKN2A and inactivating mutations inRNF43 among others but as expected these MCNs did not haveGNAS alterations Supplementary Figs S3 S76In addition to driver genes previously reported in IPMNs ourdata provide an opportunity to discover novel drivers of IPMNtumorigenesis We identified somatic mutations in the DNAdamage response gene ATM in of lesions including onenonsense mutation Fig 1b Supplementary Data In additionwe identified alterations in Hedgehog pathway member GLI3 in of cases Fig 1b Supplementary Data We alsoidentified somatic mutations in a previously described hotspot inSF3B1 which encodes a protein critical for RNA splicing Fig 1bSupplementary Data Amplifications of the wellcharacterizeddriver genes ERBB2 and MYC were each observed in a single caseand have not been previously reported in IPMNs SupplementaryData Other altered genes with a previously unknown role inIPMN tumorigenesis include MUC16 four cases PTPRT fourcases and CNTN5 three cases Fig 1b Intriguingly in onecase an STK11 mutation was found in combination with biallelicloss”theATM lossand cancerspecific biallelic KEAP1NATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178combination ofreported in lung cancers Supplementary Fig S315these three mutations has previously beenAlthough KRAS mutations occur in the majority of IPMNs andare thought to initiate tumorigenesis in these lesions two IPMNslacked mutations in this gene One case contained a hotspotmutation in codon of GNAS another potential initiator ofIPMN tumorigenesis as well as alterations in TP53 and RNF43Supplementary Fig S15 The other case lacked mutations in anyof the frequently altered pancreatic driver genes but containedhotspot mutations in both CTNNB1 S45P and SF3B1 H662QSupplementary Fig S10 These cases highlight alternativepathways of initiation and progression in IPMNs lacking KRASmutationsOrder of genetic alterations in IPMNMCN tumorigenesis Ourmultiregion sequencing approach of IPMNsMCNs and associated invasive carcinomas provided insights into the order ofspecific genetic alterations in pancreatic tumorigenesis In ofthe cases at least one somatic mutation in the initiating drivergenes KRAS and GNAS was shared between the noninvasivecomponent and associated invasive cancer with the remainingcase lacking mutations in these genes Somatic mutations in TP53and CDKN2A were also shared in the noninvasive componentand associated invasive cancer in the majority of cases In contrast SMAD4 had alterations confined to the invasive carcinomain three cases and was shared between noninvasive and invasivesamples in four cases Fig 1b The majority of SMAD4 alterations in all sample types were biallelic including in noninvasive samples and in invasive samples SupplementaryData Alteration of TGFBR2 which functions in the samesignaling pathway as SMAD4 was also restricted to the cancer inone case Fig 1b The other genes with mutations restricted tothe invasive cancer CDKN2A CNTN5 PIK3CA KEAP1 andRET only had this pattern in a single sample Fig 1bOur study also identified driver mutations in subclones ofnoninvasive neoplasms that diverged from and were not presentin invasive cancer These included hotspots mutations in wellcharacterized oncogenes and inactivating mutations in tumorsuppressor geneseg PIK3CA pE545K CTNNB1 pS45FSMAD4 pE33fs and multiple inactivating RNF43 mutations inpatient MTP3 Supplementary Fig S3 Mutations in RNF43were a particularly striking finding in these cases as somenoninvasive components contained several different RNF43mutations each limited to a small number of sections and noneinvolving the invasive cancer Fig Supplementary Fig S3 Inaddition to heterogeneity in RNF43 in early lesions we alsoidentified two cases with multiple mutationsin KRAS inprecursor lesions of which only one was present in the invasivecancer For example in MTP19 KRAS pG12V was present in themajority of IPMN samples as well as all the invasive cancersamples but there were an additional four other KRAS mutationsall occurring in hotspot positions that were present in a smallnumber of sectionsin lowgrade IPMN samples Fig Intriguingly these three lowgrade IPMN regions shared nomutations with the invasive cancertheyrepresented genetically independent clonessuggesting thatNotably while there were often many differences in thesomatic point mutations identified in the matched noninvasiveand invasive samples the copy number profiles were quite similarbetween IPMNMCNs and invasive cancers Fig Supplementary Data and the proportion of copy number alterationsunique to cancer samples was similar to that observed forsomatic mutations While homozygous deletion of some genesoccurred in the invasive cancer but notthe noninvasivecomponent such as CDKN2A in MTP8 Fig analyses ofchromosomal gains and losses through assessment of allelicimbalance revealed that an average of of the genome wassimilar in copy number in matched noninvasive and invasivesamples Fig Supplementary Data Insights into pancreatic neoplasia revealed by sequencing Thesamples analyzed by targeted sequencing were characterizedmorphologically and meticulously isolated using laser capturemicrodissection Even with this process we identified samples intwo cases that were characterized morphologically as IPMNs butthrough genomic and evolutionary analyses were determined tobe identical to or descendants of the associated invasive cancersFor example in MTP19 some of the samples originally identifiedmorphologically as noninvasive IPMN and T1 sharedall the mutations present in the invasive cancer sample T2 andcontained additional mutations suggesting that these samplesdescended from the cancer Figs Evolutionary analysessuggested that some of the morphologically identified IPMNsamples in this case as well as MTP1 actually representedintraductal spread of invasive carcinoma also referred to ascancerization of the ducts In these cases after invading thestroma the carcinoma invaded back into and colonized the cystsuch that it was morphologically indistinguishable from IPMNwith highgrade dysplasiaIn one case MTP5 we also identified an interesting pattern ofmultifocal invasion of the carcinoma In this case we analyzedfive different samples from invasive cancer”three samples wereisolated from a mucinous carcinoma and two samples wereisolated from a ductal carcinoma Based on evolutionary analysesof the patterns of shared and distinct mutations in the cancersand IPMNs we conclude thatthere were multiple separateinvasion events in this lesion as represented by the mutationsshared between the invasive cancers and noninvasive componentsas well as those that were unique to the specific invasive cancersFig Supplementary Fig S5As our study represents the largest cohort of comprehensivelysequenced IPMNsMCNs we also analyzed mutational signaturesin our dataset Intriguingly our data contrast somewhat with themutational signatures previously reported in pancreatic ductaladenocarcinoma PDAC1617 Like PDAC the most prominentmutational signature was associated with age Signature 1Awhich was identified in almost every case SupplementaryFig S19 However we also identified signatures associated withAPOBEC enzymes four cases smoking three cases andmismatch repair deficiency cases Although smoking isconsidered a risk factor for pancreatic cancer until now themutational signature associated with smoking has not beenreported in pancreatic neoplasia18Evolutionary timeline of highgrade IPMN to PDAC To estimate the time between the development of highgrade IPMN andPDAC we evaluated Bayesian hierarchical models for the numberof acquired mutations under a range of possible mutation ratesThese models estimate the time interval between a founder cell ofa PDAC and the ancestral precursor cell in the associated highgrade IPMN assuming that mutation rates and cell division timesare constantseeœMethods We performed this analysis on the paired WES datafrom of our cases Supplementary Fig S20 We excludedMTP19 because our evolutionary analyses demonstrated intraductal spread of invasive carcinoma and as such we lacked WESdata from an IPMN sample in this case In the analyzed casesthe average median time to progression from IPMN to PDAC was years but the models showed a bimodal distribution Thismedian time was nearly years for patients but nearly yearsthis period of developmentthroughoutNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178for patients with more than acquired mutations highlightingpotential variability in progression time between patients Forexample in patient MTP1 most models suggested an average of years between the development of the IPMN and the PDAC CI “ years In contrast for patient MTP2 with additional mutations acquired in the PDACthe transitionappears to have been slower with an average estimate of years CI “ years from the Bayesian models Overall theseanalyses suggest that for most patients there is a significantwindow of time between development of highgrade dysplasiaand pancreatic cancer providing an opportunity for surveillanceand interventionDiscussionThis study representsthe largest dataset of whole exomesequencing of IPMNs and MCNs to date Importantly our dataestablished that both IPMNs and MCNs are direct precursors ofinvasive pancreatic cancer Fig This conclusion has beenpreviously suggested by the morphological relationship betweenthe noninvasive neoplasms and invasive cancer on traditionalhistologic sections as well as shared driver gene mutations intargeted sequencing studies679“ However the presence ofmany shared driver and passenger mutations clearly demonstrated the common origin of IPMNsMCNs and invasive pancreatic cancers in our study and evolutionary analyses revealedthat dysplastic lesions precede invasive cancers Evolutionaryanalyses suggested that highgrade noninvasive lesions occur over years before invasive carcinoma providing a window ofopportunity for early detection and interventionIn this study we identified somatic mutations in driver genesthat had not been previously implicated in IPMNsMCNs Forexample we identified alterations in the DNA damage responsegene ATM in of the analyzed cases Germline mutations inATM have been recently reported in patients that developedIPMNs highlighting the potential importance of this gene inIPMN risk19 In addition mucinous colloid carcinomas aresignificantly more common than typical ductal carcinomas inpatients with germline ATM mutations further highlighting thelink between mutations in this gene and IPMNs20 Although theATM gene is large potentially increasing the likelihood of passenger or artifactual mutations even larger genes such as TTNhad a lower mutation prevalence suggesting that at least some ofthe alterations identified in ATM are likely to be bona fidesomatic mutations Somatic mutations in GLI3 which encodes acomponent of the Hedgehog signaling pathway were identified in of cases Somatic mutations in GLI3 were recently reportedin a distinct morphological variant of pancreatic carcinomaundifferentiated carcinoma with osteoclastlike giant cells aswell as at a low prevalence in sporadic PDAC suggesting that theimportance of GLI3 mutations and its signaling pathway inpancreatic tumorigenesis may extend beyond IPMNsMCNs21“The hotspot mutations in SF3B1 which encodes a protein criticalfor RNA splicing are also potential drivers in the IPMN pathwayHowever somatic mutations in this gene have been reported in avariety of other neoplasms including hematologic malignanciesand uveal melanoma24“We highlight somatic alteration of the SMAD4 pathway as aputative driver of progression to invasive cancer as mutations inSMAD4 or TGFBR2 occurred only in invasive cancer samples in of the cases analyzed SMAD4 was the only gene with cancerspecific mutations in more than one case highlighting thepotentially unique role this gene plays in pancreatic carcinogenesis This role has been previously suggested by next generationsequencing of highgrade PanINs showing an absence of SMAD4mutations in precancerous lesions as well as cancerspecificSMAD4 mutationsreported in a paired PanINcarcinomaanalyses2728 Loss of SMAD4 expression limited to invasivecarcinomas has been reported in MCN and IPMNassociatedinvasive cancers and targeted sequencing of a small number ofIPMNs and matched cancers identified a single case with aSMAD4 mutation occurring only in the cancer72829 In our datathere were also four cases where mutations in SMAD4 wereshared between noninvasive and invasive cancer samples and twowhere SMAD4 mutations were limited to the noninvasive component Although our wholeexome approach could not detect alltypes of SMAD4 alterations such as rearrangements or epigeneticchanges the majority of SMAD4 mutations observed in bothnoninvasive and invasive components affected both alleles Takentogether this suggests that the role of SMAD4 mutations may notbe universal and may depend on other factors including cellintrinsic such as somatic mutations in other driver genes andcell extrinsic such as stromal and immune microenvironmentmechanismsAlthough some of our cases had SMAD4 mutations limited tothe invasive cancer most of the IPMNMCNassociated cancerslacked driver gene alterations that were associated with invasivedisease suggesting that malignant progression is not universallydriven by point mutations Previous studies have specificallydemonstrated the importance of copy number alterations andchromosomal rearrangements in pancreatic tumorigenesis30 Wedid not identify large differences in the copy number profilesbetween noninvasive components and associated invasive cancers suggesting that global genomic instability may be importantas an early feature of tumorigenesis but is not likely to drivemalignant transformation in many casesOur study also revealed prevalent genetic heterogeneity indriver gene mutations in early lesions demonstrating morecomplex processes than previously suggested by traditional lineartumorigenesis models Similar to our recently reported polyclonalorigin of IPMNs12 we identified multiple independent clonesinitiated by distinct KRAS mutations in two cases in the currentstudy In addition our study identified multiple distinct inactivating mutations in RNF43 limited to unique tumor subclones apattern previously observed by our group and not shared by othergenes in our whole exome sequencing analyses1231 In most casesRNF43 mutations were enriched in noninvasive components andabsent from the associated invasive cancers More generally weobserved multiple instances of clear driver mutations includinghotspot mutations in oncogenes as well as inactivating mutationsin tumor suppressor genes that were limited to the noninvasivecomponents and not present in the associated invasive cancerThus these mutations occur and clonally expand in the IPMN orMCN but are not present in the subclone that subsequentlyinvades Such independent evolution of premalignant lesions hasbeen observed in other an sites and does not diminish theconclusion of our evolutionary analyses that IPMNsMCNs aretheseprecursors ofobservations suggest unique selective processes at different timepoints in tumorigenesis such that mutations selected in theprecancerous lesion are not selected for or are even selectedagainst in the invasive cancerinvasive pancreatic cancer32“ RatherIn addition to these observations about clonal evolution innoninvasive lesions our data also provide genetic evidence formultiple underappreciated processes in pancreatic neoplasiaFirst we provide genetic evidence for intraductal spread ofinvasive carci

Thyroid_Cancer

"Immigrant statusfamily relationsACP contemplationACP discussionburial planninga b s t r a c tObjectives To examine how immigrant status and family relationships are associated with advance careplanning ACP engagement and endoflife EOL preference in burial planning among older ChineseAmericans the largest subgroup of Asian AmericansDesign Crosssectional surveySetting Communities in Honolulu Hawai™iParticipants Participants were older Chinese Americans aged years and olderMeasures Measures included ACP contemplation ACP discussion and EOL preference in burial planningimmigrant status family cohesion family conflict demographic information and health statusResults Results show that in comparison to foreignborn Chinese Americans USborn Chinese Americanswere more likely to have ACP contemplation [odds ratio OR confidence interval CI ] ACP discussion OR CI and preferences for burial plans at the end of life OR CI Family conflict increased the possibility of having ACP contemplation OR CI ACP discussion OR CI and EOL preference in burial planning OR CI whereas family cohesion was not associated with these study outcomesConclusions and Implications This study suggests that ACP should be adapted to be more culturallyappropriate especially in a time of coronavirus and xenophobia such as framing ACP as a tool to helpfamilies reduce stress while fulfilling filial obligations in order to ensure equitable access to ACP“ AMDA e The Society for PostAcute and LongTerm Care MedicineAdvance care planning ACP is a process of understanding andcommunicating individuals™ values goals and preferences regardingendoflife EOL care12 Contemplation of individuals™ EOL wishes anddiscussions with families can be as important as discussions withphysicians and completion of an advance directive in guiding care34ACP is a social process built on relationships and alleviation ofburden on others a means to prepare for death and a measure toexercise the ethical principle of patient autonomy5 Burial planningcan ensure individuals™ wishes are executed and relieve the burden ofloved ones to determine what the deceased would have wantedduring the time of grief In this sense burial planning is an importantThis study was supported by a research grant from the Rory Meyers College ofNursing at New York UniversityThe authors declare no conflicts of interest Address correspondence to Bei Wu PhD Rory Meyers College of Nursing NewYork University First Avenue Room New York NY USAEmail address beiwunyuedu B Wu101016jjamda20200604015258610“ AMDA e The Society for PostAcute and LongTerm Care Medicineelement of ACP6 Therefore it makes sense to examine ACP contemplation ACP discussion with family and EOL preference in burialplanning togetherACP can improve quality of EOL care for individuals including lessinhospital death and increased hospice use7 Despite the benefits ofACP the participation rate of ACP remains low especially among olderadults of racial and ethnic minorities Studies found that in the UnitedStates Blacks and Hispanics are less likely to have an EOL discussion adurable power of attorney and an advance directive than their Whitecounterparts89 but there is a lack of knowledge on ACP engagementamong Chinese Americans the largest subgroup of Asian Americansand the fastestgrowing minority group in the USA10Compared with nativeborn Chinese AmericansforeignbornChinese Americans may face more cultural and logistical challengesin ACP engagement because of their limited English proficiencygreater cultural burden in discussing death and dying and acceptingindividual autonomy and lack of ACP knowledge1112 In addition theeffectiveness of ACP may rely on the involvement knowledge and 0cY Pei JAMDA xxx 1e4cooperation of family members13 however because of the lack of richand comprehensive measures of family relationships in previousresearch on ACP few studies have examined the extent to whichfamily relationships ‚uence individuals™ ACP engagement To fill thisknowledge gap this study aimed to examine how immigrant statusand family relationships are associated with ACP contemplation ACPdiscussion with family and preference in burial planning among olderChinese AmericansMethodsDataData were derived from a survey conducted in Honolulu Hawai™iwhere approximately of the total population is composed ofChinese Americans and of the adult population are immigrants14We used snowball sampling and convenience sampling to identify andrecruit key informants from local Chinese groups social anizationsbusinesses and faithbased agencies based on their capacity ofaccessing Chinese communities and their willingness to assist inrecruiting Chinese older adults in the community We collaboratedwith key community leaders This is a common and effective strategyto recruit respondents from minority populations15 as random sampling is challenging because of the unfeasibility of constructing acompleted sampling frame cultural appropriatenesstime andexpense16 The inclusion criteria for the survey participants includedHonolulu residents aged years and older who selfidentified asChinese The detailed recruitment and data collection methods werereported in previous studies17 The participants provided informedconsent prior to the data collection This study was approved by theinstitutional review board at the university with which the secondauthor was affiliated A total of participants were recruited fromJanuary to September MeasuresDependent variables ACP engagement and EOL preference in burialplanningACP engagement includes ACP contemplation and ACP discussionACP contemplation and ACP discussion was assessed by asking respondents if they previously had thought about their endoflifecare plan with family and had discussed the plan with familyrespectivelyEOL preference in burial planning was measured by a hypothesizedquestion Respondents were asked whether formulating a burial planwas one of the most important things for them to consider if theywere diagnosed with a terminal illness and only had months to liveamong several other options Other mentioned options includedhaving religious beliefssupport alleviating pain reducing care andfinancial burden on family and extending their lifeIndependent variablesImmigrant status was measured by asking respondents whetherthey were US or foreignbornFamily relationships were measured by reliable and valid existingscalesdfamily cohesion and family conflict The index of familycohesion was assessed by asking respondents whether familymembers like to spend free time with each other family membersfeel very close to each other and family togetherness is veryimportant18Family conflict was measured using the 5item Family CulturalConflict scale which assesses cultural and intergenerational conflictperceived by respondents in their family19CovariatesSociodemographic variables included gender age marital statuseducation financial strain living arrangement and social activityparticipation Health need factors included selfrated health comorbidity a continuous variable that examines the existence of at least chronic conditions including heart diseases stroke cancer diabeteshypertension high cholesterol thyroid disease arthritis liverrelateddiseases and others disabilities in activities of daily living andpsychological distress Psychological distress was assessed by theKessler Psychological Distress Scale K1020AnalysisFirst we summarized the sample characteristics Then we usedlogistic regression models and calculated odds ratios ORs to testwhether immigrant status family cohesion and family conflict wereassociated with ACP engagement and EOL preferences All the analyses were conducted using Stata version The missing rates for ACP contemplation ACP discussion and EOLpreferences in burial planning were and respectively Toreduce sampling errors and attain more stable analytical results weconducted multiple imputations MIs for each model All thedependent variables were imputed and the imputed values wereretained in the analysis We used imputed data sets as there werehigh levels of missingness on the dependent variables21 For sensitivity analysis a dependent variable was imputed and imputed valueswere deleted for analysis MID The MID method produced ORs thatwere almost identical to those in the model where the imputed valueswere retainedResultsTable summarizes sample characteristics It shows that less thanhalf of the participants had ACP contemplation and ACP discussion Only had EOL preference in burial planning inthe hypothesized situationTable shows ORs with confidence intervals CIs from logisticregressions The USborn Chinese Americans were more likely to haveACP contemplation OR CI ACP discussion OR CI and preference in burial planning OR CI than the foreignborn Higher levels of familyconflict were associated with higher likelihood of ACP contemplationOR CI ACP discussion OR CI and preference in burial planning OR CI whereasfamily cohesion was not significantly related to these outcomesDiscussionThis study aimed to examine the roles of immigrant status andfamily relationships in the associations between ACP engagement andgiving EOL preferences to burial planning among older ChineseAmericans The USborn Chinese Americans were more likely to haveACP contemplation and ACP discussion than the foreignborn Thismay be because the foreignborn Chinese Americans have lower socioeconomic status less English proficiency lower levels of acculturation and less knowledge about ACP and the US healthcare systemthan their USborn counterparts111222 In addition these individuallevel differences may be mixed with other systemlevel barrierswithin the US healthcare system to worsen the disparities in ACPengagement23 For example Chinese American immigrants may havea stronger belief that family and society are held in higher regard thanindividuals and attribute a higher value to collectivism of family andsociety rather than patient autonomy in EOL decision making12Moreover because traditional Chinese culture expects children tocarry the role of protecting their parents™ health safety and generalwellbeing many Chinese children may construe this responsibility as 0cTable Characteristics of the Study Sample of Chinese Americans N ¼ or Mean SDCodingY Pei JAMDA xxx 1e4ACP engagementACP contemplationACP discussionEOL preferences in burial planningUSbornFamily RelationshipsFamily cohesionFamily conflictFemaleAgeMarriedEducationFinancial strainLiving aloneParticipation in social activitiesSelfrated health as excellentgoodNumber of chronic diseaseADL disabilityPsychological distressADL activities of daily living never and don™t want tonever but want toreluctant to yes never and don™t want tonever but want toreluctant to yes no yes no yes least cohesivee12 most cohesive least cultural conflicte10 most cultural conflict male female unmarried married not at alle3 a great deal no yes no yes no yes no help needed needs help least distressfule5 most distressfulmaking every effort to prolong their older parents™ life which maysometimes be in opposition to their parents™ own wishes24 Thesepotential factors surrounding older Chinese immigrants may helpexplain this population™s lack of engagement in ACP Healthcare providers in turn should pay closer attention to these factors in order tothoroughly evaluate patients™ EOL wishes It is noted that the USbornChinese Americans were far more likely to have preferences in burialplanning than the foreignborn The finding is consistent with a previous study in that decisions such as EOL care and funeral and burialpreplanning are impacted by similar factors25 Indeed EOL care decision making and burial planning are integrated processes at theend of life26 and burial plan is included in some advance directivedocuments in practice Future studies on ACP need to consider burialplanningSecond family cohesion was not associated with ACP contemplation ACP discussion and EOL preference in burial planning whereasfamily conflict increased the possibility of ACP contemplation ACPdiscussion and EOL preferences in burial planning The finding isinconsistent with previous study conducted among White olderadults revealing that the positive family relationship encourageswhereas problematic family relationship hinders ACP engagement27The inconsistency is likely due to the fact that Chinese Americansvalue family in the process of EOL decision making2829 The lack ofassociation between family cohesion and ACP engagement may bebecause older adults with higher levels of family cohesion have tobalance between the potential benefit and harm of ACP engagementOn the hand older Chinese Americans may have positive attitudesabout ACP engagement and believe that ACP engagement is importantand necessary because it allows them to witness their loved ones™death and dying experience12 On the other hand closeknit familialrelationships may make both older Chinese Americans and theirfamilies feel more uncomfortable to start a conversation on EOL carebecause discussions about death and dying are often considered ataboo in Chinese culture30 In this sense strong family ties may havelimited impact on ACP engagement An explanation for the significantrelationship between family conflict and ACP engagement could bethat higher levels of family conflict may indicate a greater need for ACPengagement This is because the members in these families are lesslikely to know about the EOL care preferences of older adults and betrusted in the EOL decision making13 These findings suggest thatculture may play an important role in the complex association between family relationships and ACP engagementSeveral limitations of the study deserve mentioning First thecrosssectional data from a small region limit our ability to generalizefindings to older Chinese Americans living in other parts of the UnitedStates as well as to make causalinferences Second the ACPengagement in our study only included ACP contemplation ACP discussion and preference in burial planning Future studies need toinclude more ACP options such as the completion of living wills oradvance directives and the selection of a durable power of attorneyfor health care to understand more about ACP engagement in ChineseAmerican families Third ACP knowledge is an important confoundingvariable for both immigrant status and ACP engagement Futurestudies on ACP engagement need to consider this variableConclusions and ImplicationsDespite these limitations this study sheds light on how immigrantstatus and family relationships shape ACP engagement among olderChinese AmericansIt is found that immigrant status decreaseswhereas family conflict increases the likelihood of having ACPcontemplation ACP discussion and preference in burial planningTable Factors Associated With Advance Care Planning Engagement Results of Logistic Regression N ¼ USborn ref ¼ foreignbornFamily relationshipsFamily cohesionFamily conflictACP ContemplationOR CI ACP DiscussionOR CI EOL Preferences inBurial Planning OR CI All models adjusted for age gender marital status education financial strain living alone social activity participation selfrated health number of chronic disease activitiesof daily living and psychological distress 0cY Pei JAMDA xxx 1e4Health care providers may consider patients™ immigrant status andfamily relationships to better serve ethnically diverse populationsGiven that cultural factors play an important role in ACP engagementACP should be adapted to be more culturally appropriate amongChinese Americans especially in a time of coronavirus and xenophobia such as framing ACP as a tool to help families reduce stresswhile fulfilling filial obligations in order to ensure equitable access toACPAcknowledgmentWe thank Katherine Wang for her editorial assistance We wouldalso like to thank the research team at the University of Hawaii fortheir data collectionReferences Rietjens JAC Sudore RL Connolly M Definition and recommendations foradvance care planning An international consensus supported by the EuropeanAssociation for Palliative Care Lancet Oncol 201718e543ee551 Sudore RL Lum HD You JJ Defining advance care planning for adults Aconsensus definition from a multidisciplinary Delphi panel J Pain SymptomManage 201753821e832e821 Sudore RL Schickedanz AD Landefeld CS Engagement in multiple steps ofthe advance care planning process A descriptive study of diverse older adultsJ Am Geriatr Soc 2008561006e1013 Sudore RL Knight SJ McMahan RD A novel website to prepare diverseolder adults for decision making and advance care planning A pilot studyJ Pain Symptom Manage 201447674e686 Dahlin C Giansiracusa D Communication in palliative care In Ferrell BCoyle N editors Textbook of Palliative Nursing New York NY Oxford University Press p 67e96 KataokaYahiro MR Conde FA Wong RS Advance care planning amongAsian Americans and Native Hawaiians receiving haemodialysis Int J PalliatNurs 20101632e40 Bischoff KE Sudore R Miao Y Advance care planning and the quality ofendoflife care in older adults J Am Geriatr Soc 201361209e214 Kale MS Ornstein KA Smith CB Kelley AS Endoflife discussions with olderadults J Am Geriatr Soc 2016641962e1967 Harrison KL Adrion ER Ritchie CS Low completion and disparities inadvance care planning activities among older Medicare beneficiaries JAMAIntern Med 20161761872e1875 Pew Research Center Key facts about Asian Americans a diverse and growingpopulation Available at wwwpewresearchfacttank20170908keyfactsaboutasianamericans Accessed November Gao X Sun F Ko E Knowledge of advance directive and perceptions ofendoflife care in ChineseAmerican elders The role of acculturation PalliatSupport Care 2015131677e1684 Lee MC Byon HD Hinderer K Alexander C Beliefs in advance care planningamong Chinese Americans Similarities and differences between the youngerand older generations Asian Pac Isl Nurs J 2017283e90 Parks SM Winter L Santana AJ Family factors in endoflife decisionJ Palliat Med making Family conflict and proxy relationship179e184 Tong M Sentell T Insights in public health Challenges investigating healthoutcomes in Chinese Americans using populationbased survey data Hawaii JMed Public Health Ibrahim S Sidani S Strategies to recruit minority persons A systematic reviewJ Immigr Minor Health 20145882e888 Spring M Westermeyer J Halcon L Sampling in difficult to access refugeeand immigrant communities J Nerv Ment Dis 2003191813e819 Zhang W Liu S Zhang K Wu B Neighborhood social cohesion resilience andpsychological wellbeing among Chinese older adults in Hawai™i Gerontologist201960229e238 Rivera FI Guarnaccia PJ MulvaneyDay N Family cohesion and its relationship to psychological distress among Latino groups Hisp J Behav Sci 30357e378 Alegria M Vila D Woo M Cultural relevance and equivalence in theNLAAS instrument Integrating etic and emic in the development of crosscultural measures for a psychiatric epidemiology and services study of LatinosInt J Methods Psychiatr Res 200413270e288 Kessler RC Andrews G Colpe LJ Short screening scales to monitor population prevalences and trends in nonspecific psychological distress PsycholMed 200232959e976 Johnson DR Young R Toward best practices in analyzing datasets withmissing data Comparisons and recommendations J Marriage Fam 926e945 Carr D The social stratification of older adults™ preparations for endoflifehealth care J Health Soc Behav 201253297e312 Shen MJ Prigerson HG Tergas AI Maciejewski PK Impact of immigrant statuson aggressive medical care counter to patients™ values near death amongadvanced cancer patients J Palliat Med 20192234e40 Bowman K Singer P Chinese seniors™ perspectives on endoflife decisions SocSci Med 200153455e464 Kelly CM Masters JL DeViney S Endoflife planning activities An integratedprocess Death Stud 201337529e551 Tanaka M Takahashi M Kawashima D Endoflife activities amongin Japan Omega Westport communitydwelling older adults Carr D Moorman SM Boerner K Endoflife planning in a family context Doesrelationship quality affect whether and with whom older adults planJ Gerontol B Psychol Sci Soc Sci 201368586e592 Hinderer KA Lee MC Chinese Americans™ attitudes toward advance directivesAn assessment of outcomes based on a nursingled intervention Appl Nurs Res20194991e96 YonashiroCho J Cote S Enguidanos S Knowledge about and perceptions ofadvance care planning and communication of ChineseAmerican older adultsJ Am Geriatr Soc 2016641884e1889 Chi HL Cataldo J Ho EY Rehm RS Can we talk about it now Recognizing theoptimal time to initiate endoflife care discussions with older ChineseAmericans and their families J Transcult Nurs 201829532e539 0c"

Thyroid_Cancer

annotated fluorescence image dataset for training nuclear segmentation methods \u2009œ‰ Eva Bozsaky19 Fikret Rifatbegovic Florian Kromp Magdalena ambros1 Maria Berneder1210 Tamara Weiss1 Daria Lazic1 Wolfgang D¶rr410 Allan Hanbury Sabine taschnerMandl Lukas Fischer Klaus Beiske7 Peter F ambros Inge M ambros12 \u2009œ‰Fullyautomated nuclear image segmentation is the prerequisite to ensure statistically significant quantitative analyses of tissue preparationsapplied in digital pathology or quantitative microscopy The design of segmentation methods that work independently of the tissue type or preparation is complex due to variations in nuclear morphology staining intensity cell density and nuclei aggregations Machine learningbased segmentation methods can overcome these challenges however high quality expertannotated images are required for training Currently the limited number of annotated fluorescence image datasets publicly available do not cover a broad range of tissues and preparations We present a comprehensive annotated dataset including tightly aggregated nuclei of multiple tissues for the training of machine learningbased nuclear segmentation algorithms The proposed dataset covers sample preparation methods frequently used in quantitative immunofluorescence microscopy We demonstrate the heterogeneity of the dataset with respect to multiple parameters such as magnification modality signaltonoise ratio and diagnosis Based on a suggested split into training and test sets and additional singlenuclei expert annotations machine learningbased image segmentation methods can be trained and evaluatedBackground SummaryBioimage analysis is of increasing importance in multiple domains including digital pathology computational pathology systems pathology or quantitative microscopy1“ The field is currently rapidly expanding mainly facilitated by technological advances of imaging modalities in terms of resolution throughput or automated sample handling Moreover publicly available databases and platforms allow the access to image datasets and annotations enabling the research community to develop sophisticated algorithms for complex bioimage analysisDigital pathology relies on tissue sections as a basis for diagnosing disease type and grade or stage78 Moreover the accurate quantification of subcellular information including nuclear features at the single cell level is critical for the characterization of tumor heterogeneity plasticity response to therapeutic intervention910 and others Several approaches to visualise subcellular compartments such as nuclear morphology andor biological markers in tissues or cell preparations are well established Hematoxylin and eosin HE histological stainings immunohistochemical IHC and immunofluorescence IF stainings Whereas HE and IHC stainings and visualisation by bright field microscopy are standard procedures in routine diagnostic laboratories and pathology departments IF is more often employed in research settings than in routine diagnostics Prominent examples recently proved 1Tumor biology group Children™s Cancer Research Institute Zimmermannplatz Vienna Austria 2Labdia Labordiagnostik GmbH Zimmermannplatz Vienna Austria 3Software Competence Center Hagenberg GmbH SCCH Softwarepark Hagenberg Austria 4ATRABApplied and Translational Radiobiology Department of Radiation Oncology Medical University of Vienna Whringer G¼rtel Vienna Austria 5Institute of Information Systems Engineering TU Wien Favoritenstrasse Vienna Austria 6Complexity Science Hub Josefstdter StraŸe Vienna Austria 7Department of Pathology Oslo University Hospital Ullernchaussen N0379 Oslo Norway 8Department of Pediatrics Medical University of Vienna Whringer G¼rtel Vienna Austria 9These authors contributed equally Florian Kromp Eva Bozsaky 10Deceased Maria Berneder Wolfgang D¶rr œ‰email floriankrompccriat sabinetaschnerccriatScientific Data 101038s4159702000608wwwwnaturecomscientificdata 0cthe feasibility and power of IFbased quantitative analysis for eg detection of blood circulating or bone marrow disseminated tumor cells for minimal residual disease MRD diagnostics or the detection of genetic alterations by fluorescence in situ hybridization FISH11“ Although the digitization of fluorescence stainings is more challenging and time consuming when compared to the digitization of HE or IHC stainings up to or more cellular or subcellular markers can be visualized in multiplex assays1415 This depicts a beneficial gain of information on individual cells and cell compartmentsA prerequisite for any reliable quantitative imagebased analysis however is the accurate segmentation of nuclei in fluorescence images5616 In order to reach sufficient power for statistical analysis fully automated segmentation algorithms are preferred While tissues and cell preparations presenting with well separated nuclei can be segmented based on simple intensity thresholds densely packed tissues or cell aggregations require more sophisticated algorithms for nuclear segmentation Segmentation algorithms focusing on the separation of objects instances are called instance segmentation algorithms or instance aware segmentation algorithms Although designed to split aggregating nuclei they frequently fail to separate each nucleus in cases of tightly clustered nuclei17 Remaining aggregations of nuclei lead in the worst case to their exclusion from the downstream analysis potentially causing a biologically significant biasTo overcome these drawbacks novel deep learningbased image segmentation approaches are currently developed in many labs worldwide They promise to solve most segmentation problems as long as highquality expertannotated datasets are available to train the systems However there are only a limited number of annotated nuclear image datasets publicly available While annotated datasets outlining nuclei in HE or IHC images can be obtained a comprehensive dataset including IF images of tissue sections of diverse origin and annotated nuclei is currently not available to the best of our knowledge Apart from the tedious process of annotation this may be due to the fact that it is challenging to decide whether an aggregation consists of one or multiple nuclei This is because in epifluoresecence microscopy images nuclei often appear blurry and within cell aggregations their borders are frequently not definableIn summary the time consuming and challenging process of annotation hampered the generation and publication of annotated fluorescence image datasets including tightly aggregated and overlapping nucleiWe hereby present an expertannotated comprehensive dataset18 that can be used to train machine learningbased nuclear segmentation algorithms The presence and annotation of tightly aggregating and partially overlapping nuclei enable the algorithms to learn how to solve the task of accurate instance aware nuclear segmentation The dataset consists of annotated IF images of different biological tissues and cells of pathological and nonpathological origin covering the main preparation types used in imagingbased biomedical research settings Schwann cell stromarich tissue from a ganglioneuroblastoma cryosections a Wilms tumor tissue cryosection a neuroblastoma tumor tissue cryosection bone marrow cytospin preparations infiltrated with neuroblastoma cells neuroblastoma tumor touch imprints cells of two neuroblastoma cell lines CLBMa STANB10 cytospinned on microscopy glass slides and cells of a normal human keratinocyte cell line HaCaT cytospinned or grown on microscopy glass slides The characteristics of neuroblastoma and the Schwann cell stromarich ganglioneuroblastoma tumors have been previously described by Shimada in detail19 Multiple modalities Zeiss Axioplan II Zeiss and Leica laser scanning microscopes LSM were used for image acquisition while using different magnifications 10x 20x 40x 63x objectives Nuclei in IF images were annotated by trained biologists carefully curated by an experienced disease expert and finally reviewed and curated by an external disease expert and pathologist The final annotated dataset forming the ground truth dataset was split into a training set and a test set to be used for machine learningbased image segmentation architectures The training set consists of images with similar characteristics while the test set partially consists of images with varying image characteristics To enable a detailed assessment of the generalizability of trained segmentation algorithms with respect to image parameters such as the magnification or the signaltonoise ratio the images of the test set were divided into classes based on common image characteristics In addition to the expertannotated ground truth randomly selected nuclei from images of each class were marked on the raw images and presented to two independent experts subject to annotation These annotations further called singlenuclei annotations serve to validate the quality of the annotated dataset by comparing the ground truth annotations to the singlenuclei expert annotations and to set a baseline for machine learningbased image segmentation architecturesIn conclusion the proposed expertannotated dataset presents a heterogeneous realworld dataset consisting of fluorescence images of nuclei of commonly used tissue preparations showing varying imaging conditions sampled using different magnifications and modalities The dataset can be used to train and evaluate machine learningbased nuclear image segmentation architectures thereby challenging their ability to segment each instance of partially highly agglomerated nucleiMethodsPatient samples Tumor n and bone marrow n samples of stage M neuroblastoma patients the Schwann cell stromarich part of a patient with a ganglioneuroblastoma tumor and one wilms tumor patient were obtained from the Children™s Cancer Research Institute CCRI biobank EK18532016 within the scope of ongoing research projects In addition two patientderived neuroblastoma cell lines CLBMa STANB10 were used Written informed consent has been obtained from patients or patient representatives Ethical approval for IF staining and imaging was obtained from the ethics commission of the Medical University of Vienna EK12162018 All authors confirm that we have complied with all relevant ethical regulationsPreparation and IFstaining of tumor tissue cryosections The freshfrozen tumor tissues of one ganglioneuroblastoma patient one neuroblastoma patient and one Wilms tumor patient were embedded into tissuetekOCT and µm thick cryosections were prepared Sections were mounted on Histobond glass slides Scientific Data 101038s4159702000608wwwwnaturecomscientificdatawwwnaturecomscientificdata 0cMarienfeld fixed in formaledhyde and stained with 46diamino2phenylindole DAPI a blue fluorescent dye conventionally used for staining of nuclei for cellular imaging techniques Finally slides were covered with Vectashield and coverslips were sealed on the slides with rubber cementPreparation and IFstaining of HaCaT human skin keratinocyte cell line The HaCaT cell line a spontaneously transformed human epithelial cell line from adult skin20 was cultivated either in culture flasks or on microscopy glass slides Cell cultures were irradiated and Gy harvested cytospinned airdried and IF stained Cells grown and irradiated on the glass slides were directly subjected to IF staining Cells were fixed in formaldehyde for minutes at °C and were permeabilized with sodium dodecyl sulfate SDS in PBS for minutes Slides were mounted with antifade solution Vectashield containing DAPI and coverslips were sealed on the slides with rubber cementPreparation and IFstaining of tumor touch imprints and bone marrow cytospin preparations Touch imprints were prepared from fresh primary tumors of stage M neuroblastoma patients as previously described21 Mononuclear cells were isolated from bone marrow aspirates of stage M neuroblastoma patients by density gradient centrifugation and cytospinned as described22 After fixation in formaldehyde for minutes cells were treated according to the Telomere PNA FISH Kit Cy3 protocol Dako mounted with Vectashield containing DAPI covered and sealedPreparation and IFstaining of neuroblastoma cell line cytospin preparations STANB10 and CLBMa are cell lines derived from neuroblastoma tumor tissue of patients with stage M disease Preparation and drugtreatment were conducted as described23 Briefly cells were cultured in the absence or presence of nM topotecan a chemotherapeutic drug for weeks detached and cytospinned to microscopy glass slides Preparations were airdried fixed in formaldehyde immuno and DAPI stained covered and sealedFluorescence imaging Samples were imaged using an AxioplanII microscope from Zeiss equipped with a Maerzhaeuser slide scanning stage and a Metasystems Coolcube camera using the Metafer software system V386 from Metasystems an AxioplanII microscope from Zeiss equipped with a Zeiss AxioCam Mrm using the Metasystems ISIS Software for microscopy image acquisition an LSM microscope from Zeiss equipped with an Argonlaser nm a photomultiplier tube PMT detector “ nm and a motorized Piezo Zstage using the Zeiss Zen software package and a SP8X from Leica equipped with a Diode Laser and a PMT detector “ nm For the presented dataset we digitized the DAPI staining pattern representing nuclear DNA Additional immunofluorescence or FISH stainings were in part available An automatic illumination time was set as measured by pixel saturation Metasystems Metafer and ISIS or defined manually Zeiss and Leica LSMs Objectives used were a Zeiss PlanApochromat — objective Zeiss Axioplan II numerical aperture air a Zeiss — PlanApochromat Zeiss LSM numerical aperture oil a Zeiss — PlanApochromat Leica SP8X nuermical aperture oil a Zeiss PlanNeofluar — objective Zeiss Axioplan II numerical aperture and a Zeiss PlanApochromat — objective Zeiss Axioplan II Zeiss LSM and Leica SP8X numerical aperture oil Representative field of views FOVs were selected according to the following quality criteria sharpness intact nuclei and a sufficient number of nucleiGround truth annotation Nuclei image annotation was performed by students and expert biologists trained by a disease expert To accelerate the time consuming process of image annotation a machine learningbased framework MLF was utilized supporting the process of annotation by learning characteristics of annotation in multiple steps24 The MLF annotations result in a coarse annotation of nuclear contours and have to be refined to serve as ground truth annotation Therefore annotated images were exported as support vector graphic SVG files and imported into Adobe Illustrator AI CS6 AI enables the visualization of annotated nuclei as polygons overlaid on the raw nuclear image and provides tools to refine the contours of each nucleus An expert biologist and disease expert carefully curated all images by refining polygonal contours and by removing polygons or adding them if missing Finally an expert pathologist was consulted to revise all image annotations and annotations were curated according to the pathologist™s suggestions In cases where decision finding was difficult a majority vote including all experts™ suggestions was considered and annotations were corrected accordingly Images were exported and converted to Tagged Image File Format TIFF files serving as nuclear masks in the ground truth dataset A sample workflow is illustrated in Fig a0Dataset split As the dataset is intended to be used to train and evaluate machine learningbased image segmentation methods we created a dataset split into training set and test set The training set consists of multiple images of ganglioneuroblastoma tissue sections normal cells HaCaT as cytospin preparations or grown on slide and neuroblastoma tumor touch imprints and bone marrow preparations For each of these types of preparation multiple images using the same magnification 20x or 63x imaged with the same modality Zeiss Axioplan II and the Metasystems Metafer Software and showing a good signaltonoise ratio were included The test set consists of additional images of these preparation types and moreover includes images of different preparation types eg neuroblastoma cell line preparations Wilms tumor and neuroblastoma tumor tissue sections imaged with different modalities Zeiss and Leica confocal LSM Zeiss and Metafer ISIS software and different signaltonoise ratiosScientific Data 101038s4159702000608wwwwnaturecomscientificdatawwwnaturecomscientificdata 0cacS1S2S3bdFig Workflow for ground truth image annotation a Raw image visualizing HaCaT cytospinned nuclei b A machine learning framework was used to annotate the raw image learning from user interaction within three consecutive steps S1 foreground extraction S2 connected component classification red nonusable objects blue nuclei aggregations green single nuclei and S3 splitting of aggregated objects into single nuclei resulting in an annotation mask c Zoomin of the SVGfile showing the nuclear image overlaid with polygons representing each annotated nucleus Polygons were modified by expert biologists to fit effective nuclear borders Challenging decisions on how to annotate nuclei mainly occurring due to aggregated or overlapped nuclei were presented to an expert pathologist and corrected to obtain the final ground truth d The curated SVGfile was transformed into a labeled nuclear maskTo enable an objective comparison of image segmentation architectures to the ground truth annotations with respect to varying imaging conditions we classified each image of the test set into one of classes according to criteria such as sample preparation diagnosis modality and signaltonoise ratio The details are presented in Table a0 The recommended dataset split into training set and test set and the test set classes can be downloaded along with the datasetSinglenuclei annotation To set a baseline for machine learningbased image segmentation methods and to validate the proposed dataset nuclei were randomly sampled from the ground truth annotations for each of the classes marked on the raw images and presented to two independent experts for image annotation Annotation was carried out by a biology expert with longstanding experience in nuclear image annotation further called annotation expert and a biologist with experience in cell morphology and microscopy further called expert biologist Nuclei were annotated using SVGfiles and Adobe illustrator The singlenuclei annotations described as singlecell annotations within the dataset can be downloaded along with the datasetAnnotation criteria The annotation of nuclei in tissue sections or tumor touch imprints is challenging and may not be unambiguous due to outoffocus light or nuclei damaged nuclei or nuclei presenting with modified morphology due to the slide preparation procedure We defined the following criteria to annotate nuclear images¢\t Only intact nuclei are annotated even if the nuclear intensity is low in comparison to all other nuclei present¢\t Nuclei have to be in focus¢\t¢\t Nuclear borders have to be annotated as exact as resolution and blurring allows forIf parts of a nucleus are out of focus only the part of the nucleus being in focus is annotatedScientific Data 101038s4159702000608wwwwnaturecomscientificdatawwwnaturecomscientificdata 0cAcronymGNBIGNBIINBINBIINBIIINBIVNCINCIINCIIITSDescriptionganglioneuroblastoma tissue sectionsganglioneuroblastoma tissue sections with a low signaltonoise rationeuroblastoma bone marrow cytospin preparationsneuroblastoma cell line preparations imaged with different magnificationsneuroblastoma cell line preparations imaged with LSM modalitiesneuroblastoma tumor touch imprintsnormal cells cytospin preparationsnormal cells cytospin preparations with low signaltonoise rationormal cells grown on slideother tissue sections neuroblastoma WilmsTable Test set split into classes to evaluate the generalizability of machine learningbased image segmentation methods with respect to varying imaging conditions¢\t Nuclei are not annotated if their morphology was heavily changed due to the preparation procedure¢\t Nuclei from dividing cells are annotated as one nucleus unless clear borders can be distinguished between the resulting new nucleiData recordsThe dataset presented here is hosted in the BioStudies database under accession number SBSST265 identifiersbiostudiesSBSST26518 It consists of fluorescence images of immuno and DAPI stained samples containing nuclei in total The images are derived from one Schwann cell stromarich tissue from a ganglioneuroblastoma cryosection images2773 nuclei seven neuroblastoma NB patients images931 nuclei one Wilms patient image102 nuclei two NB cell lines CLBMa STANB10 images1785 nuclei and a human keratinocyte cell line HaCaT images2222 nuclei In addition the dataset is heterogenous in aspects such as type of preparation imaging modality magnification signaltonoise ratio and other technical aspects A summary of the dataset composition and relevant parameters eg diagnosis magnification signaltonoise ratio and modality with respect to the type of preparation is presented in Fig a0 Detailed information for each image is provided along with the dataset The signaltonoise ratio was calculated as follows We used the binarized ground truth annotation masks to calculate the meanforeground nuclear and meanbackground signal First we calculated the mean intensity of all raw image pixels covered by the masks™ foreground region resulting in the meanforeground signal By applying a morphological dilation on the binary foreground region using a diskshaped structuring element of size pixels and by inverting the resulting mask mean intensity values of raw image pixels covered by the inverted mask were calculated resulting in the meanbackground signal Dilation was applied to exclude pixels neighboring nuclei from the calculation as they do not represent the background signal but present increased intensity values due to blurred nucleiEach image of the dataset is provided in TIFFformat In addition we provide two types of annotations annotated labeled masks in TIFFformat and SVGfiles describing the exact position of each nucleus as a polygon The SVGfiles reference a nuclear image stored in Joint Photographic Experts Group JPEGformat and provide all annotated objects within an additional layerX Y ˆXTechnical ValidationTo validate the proposed dataset and to set a baseline for machinelearning based image segmentation methods we compared the singlenuclei annotations to the ground truth annotation We calculated the Dice coefficient Y of each nucleus comparing the ground truth annotation X and the corresponding expert annotaDCtion Y Finally we computed the mean Dice coefficient for each of the classes by calculating the mean value of the Dice coefficients of all annotations of a class The overall Dice coefficient was computed by calculating the mean value of all annotations of all classes The results are presented in Table a0The overall Dice coefficients of achieved by the expert biologist and achieved by the annotation expert show that the concordance in annotations of nuclear contours on the pixel level is high between expert annotations and the ground truth A Dice score close to the optimal score cannot be achieved due to the nature of fluorescence images eg blurriness resulting in fuzzy nuclear borders and highly overlapping nuclei with partially invisible borders Varying Dice coefficients between classes are due to different imaging conditions For example images in the GNBII class were more blurry and present lower signaltonoise ratios than images of eg the NCI to NCIII classes thus exact nuclear contours cannot be determined Dice scores achieved by the annotation expert were higher than the scores achieved by the expert biologist in eight out of ten classes This was an expected result as the task of image annotation differs from image acquisition and visual biological interpretation Thus the annotation expert™s experience in image annotation was of benefit to achieve higher coefficients The presented scores and the released singlenuclei ground truth can be further used to benchmark the accuracy of machine learningbased image segmentation architectures in comparison to the baseline set by human annotatorsScientific Data 101038s4159702000608wwwwnaturecomscientificdatawwwnaturecomscientificdata 0c GNBNBDiagnosisNormal HaCaT TWTissue Cell line grown oCell line c y tp i nse ll li n e c ytospinCTU toucharrowe mnoB Signal to noise x worarBone mTU touch Cell line c y te ll li n e c ytospin sop i nCell line gro x xx Tissue Cell line grown worarBone mTU touchMaterial PreparationCell line grownTissue sectionCTU toucharrowe mnoBZeissLeicawnTissue sectionSISIModality Metafe rFig Heterogeneity of the proposed dataset with respect to the type of preparation GNB ganglioneuroblastoma NB neuroblastoma TU touch tumor touch imprint Tissue tissue sectionAnnotatorBiol expAnnot expGNBIGNBII NBINBIINBIIINBIVNCINCIINCIIITSOverallTable Mean Dice coefficient between randomly selected nuclei of the manual annotations and the ground truth annotations with respect to the human annotator Annot exp annotation expert Biol exp expert biologist GNBI ganglioneuroblastoma tissue sections GNBII ganglioneuroblastoma tissue sections with a low signaltonoise ratio NBI neuroblastoma bone marrow cytospin preparations NBII neuroblastoma cell line preparations with different magnifications NBIII neuroblastoma cell line preparations with different magnifications and imaged with LSM modalities NBIV neuroblastoma tumor touch imprints NCI normal cells HaCaT cytospin preparations NCII normal cells HaCaT with low signaltonoise ratio NCIII normal cells HaCaT grown on slide TS other tissue sections neuroblastoma Wilms tumor Bold values set the baseline for machine learningbased image segmentation methodsCode availabilityWe provide code to transform predicted annotation masks in TIFFformat to SVGfiles for curation by experts as well as the transformation from SVGfiles to TIFFfiles The contour sampling rate when transforming mask objects to SVGdescriptions can be set in accordance to the size of predicted nuclei Therefore new nuclei image annotation datasets can easily be created utilizing the proposed framework and a tool to modify SVGobjects such as Adobe Illustrator The code is written in python and is publicly available under githubcomperlfloccriNuclearSegmentationPipelineScientific Data 101038s4159702000608wwwwnaturecomscientificdatawwwnaturecomscientificdata 0cReceived August Accepted July Published xx xx xxxxreferences Irshad H Veillard A Roux L Racoceanu D Methods for Nuclei Detection Segmentation and Classification in Digital Histopathology A Review Current Status and Future Potential IEEE Rev Biomed Eng “ Meijering E Carpenter A E Peng H Hamprecht F A OlivoMarin J C Imagining the future of bioimage analysis Nat Ching T Opportunities and obstacles for deep learning in biology and medicine J R Soc Interface Blom S Systems pathology by multiplexed immunohistochemistry and wholeslide digital image analysis Sci Rep “ Biotechnol “ Waters J C Accuracy and precision in quantitative fluorescence microscopy J Cell Biol “ Ronneberger O Spatial quantitative analysis of fluorescently labeled nuclearstructures Problems methods pitfalls Chromosome Res “ unfavorable groups Cancer “ Ambros I M Morphologic features of neuroblastoma Schwannian stromapoor tumors in clinically favorable and Gurcan M N Histopathological Image Analysis A Review IEEE Rev Biomed Eng “ Saltz J Spatial anization and Molecular Correlation of TumorInfiltrating Lymphocytes Using Deep Learning on Pathology Liu J Molecular Mapping of Tumor Heterogeneity on Clinical Tissue Specimens with Multiplexed Quantum Dots ACS Nano Images Cell Rep “ “ Ambros P F Mehes G Ambros I M Ladenstein R Disseminated tumor cells in the bone marrow Chances and consequences of microscopical detection methods Cancer Lett “ Narath R L¶rch T Rudas M Ambros P F Automatic Quantification of Gene Amplification in Clinical Samples by IQFISH Cytometry B Clin Cytom “ Mhes G Luegmayr A Ambros I M Ladenstein R Ambros P F Combined automatic immunological and molecular cytogenetic analysis allows exact identification and quantification of tumor cells in the bone marrow Clin Cancer Res “ Schubert W Analyzing proteome topology and function by automated multidimensional fluorescence microscopy Nat Ostalecki C Multiepitope tissue analysis reveals SPPL3mediated ADAM10 activation as a key step in the transformation of Jung C Kim C Impact of the accuracy of automatic segmentation of cell nuclei clusters on classification of thyroid follicular Biotechnol “ melanocytes Sci Signal lesions Cytometry Part A “ Xing F Yang L Robust NucleusCell Detection and Segmentation in Digital Pathology and Microscopy Images A Comprehensive Review IEEE Rev Biomed Eng “ Kromp F An annotated fluorescence image dataset for training nuclear segmentation methods BioStudies Database identifiersbiostudiesSBSST265 Shimada system Cancer “ Shimada H Ambros I M Dehner L P Hata J Joshi V V The International Neuroblastoma Pathology Classification the Boukamp P Normal Keratinization in a Spontaneously Immortalized J Cell Biol “ Brunner C Tumor touch imprints as source for whole genome analysis of neuroblastoma tumors Plos One Mhes G Detection of disseminated tumor cells in neuroblastoma Log improvement in sensitivity by automatic immunofluorescence plus FISH AIPF analysis compared with classical bone marrow cytology Am J Pathol “ TaschnerMandl S Metronomic topotecan impedes tumor growth of MYCNamplified neuroblastoma cells in vitro and in vivo by therapy induced senescence Oncotarget “ Kromp F Machine learning framework incorporating expert knowledge in tissue image annotation IEEE ICPR “ AcknowledgementsThis work was facilitated by an EraSME grant project TisQuant under the grant no and by a COIN grant project VISIOMICS under the grant no both grants kindly provided by the Austrian Research Promotion Agency FFG and the St Anna Kinderkrebsforschung Partial funding was further provided by BMK BMDW and the Province of Upper Austria in the frame of the COMET Programme managed by FFGAuthor contributionsF Kromp A Hanbury S TaschnerMandl and PF Ambros conceived the study M Berneder E Bozsaky T Weiss S TaschnerMandl and M Ambros prepared samples for the dataset W Doerr provided essential material F Kromp E Bozsaky IM Ambros M Ambros PF Ambros T Weiss and S TaschnerMandl acquired all images of the annotated dataset F Kromp T Weiss E Bozsaky F Rifatbegovic IM Ambros and K Beiske participated in annotation or curation of the ground truth dataset or the singlenuclei annotations F Kromp and L Fischer performed the statistical analysis of the singlenuclei annotations F Kromp S TaschnerMandl PF Ambros IM Ambros D Lazic and L Fischer interpreted the data F Kromp and E Bozsaky wrote the manuscript with input from all authors S TaschnerMandl F Rifatbegovic A Hanbury PF Ambros IM Ambros D Lazic and L Fischer revised the manuscriptCompeting interestsThe authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to FK or STMReprints and permissions information is available at wwwnaturecomreprintsScientific Data 101038s4159702000608wwwwnaturecomscientificdatawwwnaturecomscientificdata 0cPublisher™s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsOpen Access This article is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate if changes were made The images or other third party material in this article are included in the article™s Creative Commons license unless indicated otherwise in a credit line to the material If material is not included in the article™s Creative

Thyroid_Cancer

Neck Tissues A a0Systematic ReviewJerome a0R a0Lechien1234 Stphane a0Hans13 a0· Maria a0R a0Barillari18 a0· Giovanni a0Cammaroto19 a0· Graldine a0Descamps12 a0· Julien a0Hsieh110 a0· Luigi a0Vaira111 a0· Giacomo a0De a0Riu111 a0· Leigh a0Sowerby112 a0· Isabelle a0Gengler113 a0· Justin a0Michel15 a0· Sven a0Saussez124 a0· Thomas a0Radulesco15 a0· Christian a0Calvo‘Henriquez16 a0· Carlos a0M a0Chiesa‘Estomba17 a0· Received July Accepted August Springer ScienceBusiness Media LLC part of Springer Nature AbstractTo review the data regarding the expression of angiotensin converting enzyme2 ACE2 and transmembrane protease serine2 TMPRSS2 in head and neck tissue Scopus Cochrane Library Medrxiv Google Scholar and PubMEDMEDLINE were searched by four independent investigators for studies investigating ACE2 or TMPRSS2 expressions in head and neck tissues The following outcomes were considered sample origin animal versus human detection method anatomical location and cell types PRISMA checklist and modified population intervention comparison outcome timing and setting PICOTS framework were used to perform the review Of the identified studies met our inclusion criteria Thirteen studies were conducted during the severe acute respiratory syndrome a0coronavirus2 SARSCoV2 pandemic ACE2 and TMPRSS2 were expressed in oral pharyngeal sinusonasal human mucosa The following cell types expressed ACE2 basal apical goblet minor salivary and endothelial cells TMPRSS2 was found in goblet and apical respiratory cells ACE2 and TMPRSS2 were found in the olfactory region especially in sustentacular nonneural and neural stem cells Animal studies suggested that ACE2 expression may vary regarding age There was an important heterogeneity between studies in the methods used to detect ACE2 and TMPRSS2 leading to a potential identification bias The SARSCoV2 receptors ACE2 and TMPRSS2 are both expressed in many head and neck tissues enabling the viral entry into the host anismKeywords ACE2 a0· TMPRSS2 a0· SARSCoV2 a0· COVID a0· Coronavirus a0· Head NeckIntroductionThe renin angiotensin aldosterone system is one of the most important systems regulating the homeostasis of cardiovascular and pulmonary function this involves many molecules including angiotensin converting enzyme2 ACE2 [] ACE2 is also known to be the functional receptor of some coronavirus species as initially discovered in during the severe acute respiratory syndrome coronavirus SARSCoV epidemic [] The current pandemic of coronavirus disease Jerome R Lechien and Thomas Radulesco have contributed equally to the paper and are joint as cofirst authorsJustin Michel and Sven Saussez equally contributed to the paper and are cosenior authorsjeromelechienumonsacbe Jerome R Lechien Extended author information available on the last page of the COVID19 has brought to light the importance of ACE2 regarding development of infection viral spread and the development of the clinical COVID19 [] At the same time another SARSCoV2 receptor has been identified the transmembrane protease serine2 TMPRSS2 []ACE2 and TMPRSS2 tissue expressions are particularly important to identify viral entry pathways and to better understand the anrelated clinical presentation of the disease [ ] Further evaluation of ACE2 and TMPRSS2 expression in ear nose and throat mucosa is warranted to shed light on the pathophysiology of disease in the head and neck [“]The aim of this systematic review is to summarize the current data about the expression of ACE2 and TMPRSS2 in head neck tissueVol01234567891 0c MethodsThe review was conducted regarding the Preferred Reporting Items for a Systematic Review and Metaanalysis PRISMA checklist [] A modified population intervention comparison outcome timing and setting PICOTS framework was used to structure the review process [] For this review the PICOTS structure was kept but adapted to experimentalbasic research studies on human and animal tissuesStudiesAnimal and human experimental published studies in Englishlanguage peerreviewed journals were considered Preprint studies were also considered in light of the current pandemic and the significant wealth of knowledge derived over the last few months All studies where investigators assessed ACE2 or TMPRSS2 expressions in head neck tissues through immunochemistry IHC in a0situ hybridization Western Blot RNA sequencing RNAseq or reverse transcription polymerase chain reaction RTPCR were evaluatedParticipants and a0Inclusion CriteriaThe papers had to include either human or animal subjects The authors extracted substantial information about the sample characteristics including species involved and ACE2 and TMPRSS2 identification methodOutcomesThe primary outcome studied was tissue expression of ACE2 and TMPRSS2 The anatomical location the types of cells that expressed both receptors were recorded Particular attention was paid to the method used to detect ACE2TMPRSS2 in tissues Additional useful information such as viral impact on the functioning of the tissuecell that expressed the receptor or interindividual differences were also collectedIntervention and a0ComparisonBecause the aim of the study was to investigate the tissue ACE2 and TMPRSS2 expression we did not consider potential intervention on patient or animal modelsHead and Neck PathologyTiming and a0SettingWe included the studies where the receptor analysis was made on normal subjects andor infected patientsSearch StrategyThe PubMedMEDLINE Google Scholar Medrxiv Scopus and Cochrane search was conducted by independent authors JRL TR CCH GD CMCE to identify papers published between January and April The authors screened publications with database s and available full texts referring to the condition The following keywords were used for the search strategy ˜ACE2² ˜TMPRSS2² ˜COVID19² ˜COVID™ ˜SARS™ ˜coronav™ ˜coronavirus™ ˜salivary™ ˜gland™ ˜Receptor™ ˜Head™ ˜Neck™ ˜Nasal™ ˜ear nose throat ENT™ ˜Tissue™ and ˜Cell™ The authors investigated papers for number of samplesindividuals study type design inclusion criteria and ACE2TMPRSS2 detection outcomesResultsThe electronic search identified papers of which met our inclusion criteria Table a0 [“] A total of studies investigated the expression of ACE2 and TMPRSS2 in human head and neck tissues while five papers focused on mouse and two on monkey samples respectively Table a0 One study focused on ACE2 genetic analysis without reporting sitespecific anatomical expression [] The flow chart of the study process is available in Fig a0 Five studies were preprint [ “ ]Tissue Expression in a0HumanACE2 ExpressionACE2 was assessed in studies [“] The expression of ACE2 was found in all mucosa of the respiratory upper tract including trachea [ ] sinus and nasal cavities [ ] Among the respiratory mucosa ACE2 was expressed in several types of cells including epithelial goblet and endothelial cells [ ] One study reported that ACE2 was expressed on ciliated epithelial cells and not on nonciliated goblet cells [] Butowt et a0al compared the intensity of expression of ACE2 in the upper and lower respiratory tract [] They found that nasal epithelial cells had lower levels of ACE2 expression compared with epithelial cells of the lower respiratory tract [] Among the nasal region two studies investigated the ACE2 expression in the 0cHead and Neck Pathology Table Studies reporting ACE2 or TMPRSS2 head and neck expressionAuthorsDesignVaarala Mixed [] StudyHamming HumanSamples MethodsHuman TMPRSS2MouseHuman ACE2RNAseqACE2 TMPRSS2 expressionSalivary glandsTMPRSS2 humanOral Nasal Nasopharyngeal Epithelium endotheliumACE2 human all mucosa [] StudyIHCHumanJia [] StudyHuman ACE2Tracheal Epithelium endotheliumIHC biotinylation ACE2 humanLiuExperimenal Monkey ACE2 [] StudyIHCNaso Oro Hypopharyngeal Tracheal EpitheliumACE2 MonkeyVirion in SalivaBilinska Animal [] StudyBrannMixed [] StudyButowtMixed [] StudyMouse ACE2 TMPRSS2 Olfactory EpitheliumRNAseq RTPCRACE2 sustentacular cellsIn situ hybridization TMPRSS2 sustentacular cellsWB IHCMouse ACE2 TMPRSS2 Olfactory EpitheliumHuman RNAseqACE2 TMPRSS2 Mouse nonneuronal cellsACE2 TMPRSS2 Human glial and neuronal stem cellsMouse ACE2 TMPRSS2 Olfactory EpitheliumHuman RNAseqACE2 Mouse Human non neuronal cellsCaoHumanHuman ACE2 gene [] StudyGenetic AnalyzisChenHuman [] StudyHikmetHuman [] StudyHuman ACE2RNAseqHuman ACE2IHCLeeHumanHuman ACE2TMPRSS2 Mouse Human neuronal nonneuronal cellsRespiratory EpitheliumACE2 TMPRSS2 Human Lower Airway NasalNo localization providedSalivary glandsACE2 humanNasopharyngeal EpitheliumACE2 human no expression in nasopharynxTracheal Nasal Sinusal EpitheliumFindings TMPRSS2 is expressed in human salivary gland tissues ACE2 was found in endothelial arteries veins and epithelial cells of nasal rhinopharyngeal and oral mucosa Precisely the epithelium expression concerned the basal layer cells ACE2 was more expressed on the apical than the basolateral surface of polarized airway epithelia ACE2 is expressed in salivary gland ducts of the pharyngeal glands ACE2 was expressed in epithelial cells lamina propria respiratory tract Virus was found in saliva of infected monkeys ACE2 TMPRSS2 are expressed in sustentacular cells of the olfactory epithelium but notmuch less in most olfactory receptor neurons Expression of the entry proteins increases in animals of old age In human ACE2 TMPRSS2 were not identified in purified olfactory neurons ACE2 was identified in glial cells olfactory stem cells Nasal epithelial cells have lower levels of ACE2 TMPRSS2 compared with epithelial cells of the lower respiratory tract ACE2 has nonneuronal expression in olfactory epithelium The expression of ACE2 TMPRSS2 mouse were increased in elderly mouse unique expression quantitative trait loci variants were found for ACE2 The genotypes of ACE2 gene polymorphism may be characterized by higher expression levels of ACE2 in East Asian population There would be different susceptibility or response to SARSCoV2 in different populations ACE2 is expressed in human granular cells of salivary glands There was no ACE2 expression in nasopharyngeal cells ACE2 is expressed in ciliated epithelial cells cilia anelle 0c Table continuedAuthorsDesign [] StudyHumanLiHikmetHuman [] Study [] StudySungnak Human [] StudySamples MethodsIHCACE2 TMPRSS2 expressionACE2 humanHuman ACE2Human ACE2IHCRNAseqThyroidNasopharyngeal EpitheliumACE2 human no expression in nasopharynxACE2 humanHuman ACE2 TMPRSS2 Airway Nasal epitheliumRNAseqACE2 humanTMPRSS2 human subset of ACE2 cellsXuHumanHuman ACE2 TMPRSS2 Oral Epithelium [] StudyRNAseqHumanXu [] StudyWuHuman [] StudyHuman ACE2RNAseqHuman ACE2RNAseqACE2 humanACE2 humanOral T cells B cells fibroblastsACE2 humanMinor salivary glandsACE2 humanNasal Oral EpitheliumHead and Neck PathologyFindings2There was no ACE2 expression in the nonciliated goblet cells ACE2 expression is influenced by patient demographics clinical characteristics comorbidities or medication use The use of ACE inhibitor drugs did not increase ACE2 protein expression ACE2 is expressed by thyroid cells There was no ACE2 expression in nasopharyngeal cells ACE2 was expressed in airway epithelial cells ACE2 is more expressed in nasal epithelial cells compared with other respiratory cells goblet ciliated cells TMPRSS2 is only expressed in a subset of ACE2 cells ACE2 is expressed in the oral cavity epithelial cells ACE2 expression was higher in tongue than buccal and gingival tissues ACE2 is expressed in minor salivary glands ACE is expressed in nasal epithelial cells The was a higher virus concentration in the nasalswab comparing with throatswab which is attributed to ACE2expression in nasal epithelial cells ACE2 TMPRSS2 are coexpressed in nasal goblet secretory cellsMixedZiegler [] StudyACE2 Angiotensin Converting Enzyme2 IHC Immunohistochemistry RTPCR reverse transcription polymerase chain reaction SARSCoV2 severe acute respiratory syndrome a0coronavirus2 TMPRSS2 transmembrane protease serine2 WB Western BlottingHuman ACE2 TMPRSS2 Sinusal Nasal goblet epithelial cellsMonkey RNAseqACE2 TMPRSS2 Humanmucosa of the olfactory region including olfactory bulb [ ] The ACE2 receptor was identified in sustentacularnonneuronal cells of the olfactory tissues Moreover ACE2 was found in a low proportion of neuronal stem cells in the olfactory bulb [ ] The expression of ACE2 in olfactory neurons nonstem cells remains uncertain because Butowt et a0al and Brann et a0al observed that ACE2 has only nonneuronal expression pattern in olfactory epithelium [ ]Five studies investigated ACE2 expression in oral and pharyngeal regions including oral and hypo oro and nasopharyngeal spaces [ “] The study that explored ACE2 expression in human nasopharynx [] did not exhibit significant ACE2 immunostaining in nasopharyngeal cells [] ACE2 receptor was identified in oral endothelial [] epithelial [ ] and salivary [] cells Xu et a0al found that ACE2 was also expressed in T and B cells as well as fibroblasts of the oral cavity [] Moreover ACE2 was expressed in major salivary gland tissues [] and thyroid tissue [] In many publications authors reported the type of cells goblet versus epithelial versus stem cells that expressed ACE2 or TMPRSS2 Table a0 Interestingly Xu et a0al almost as much ACE2 expression in the thyroid as in the lungs []The genetic analysis of Cao et a0al reported that there are unique expression quantitative trait loci variants in the East Asian population supporting a gene polymorphism and tissuerelated differences between individuals [] 0cHead and Neck Pathology Fig Flow chart ACE2 Angiotensin Converting Enzyme2 TMPRSS2 transmembrane protease serine2TMPRSS2 ExpressionTMPRSS2 expression was investigated in studies [ ] Similarly to ACE2 TMPRSS2 was identified in nasal [ ] and respiratory mucosa cells [] including both epithelial and goblet cells with higher expression in lower airway compared with upper airway [] Moreover TMPRSS2 receptor was identified in sustentacular and neuronal olfactory cells [ ] but not in olfactory neurons [] TMPRSS2 was also identified in salivary major gland tissue []ACE2 TMPRSS2 Tissue Expression in a0Mouse and a0MonkeySix studies used animal models to assess ACE2 or TMPRSS2 expressions in head and neck tissues [ “ ] The mouse studies of Butowt et a0al and Bilinska et a0al revealed that elderly mice had higher expression of both ACE2 and TMPRSS2 in nasal mucosa compared with younger mice [ ] In olfactory tissue ACE2 was identified in sustentacularnonneuronal and neural stem cells of mice [“] Liu et a0al analyzed ACE2 expression in monkeys [] 0c Head and Neck PathologyTable Summary of Cell Expression of ACE2 and TMPRSS2AuthorsBilinska []Brann []SamplesMouseHuman MouseTissueOlfactoryOlfactoryButowt []Human MouseNasalOlfactoryChen []Hamming []Hikmet []Jia []Lee []Li []Liu []Sungnak []Vaarala []Xu []Xu []Wu []Ziegler []HumanHumanHumanHumanHumanHumanMonkeyHumanMajor Salivary GlandOral Nasal NasopharyngealNasopharyngealTracheal Nasal SinusalThyroidPharyngealTrachealTracheal NasalHuman MouseHumanMajor Salivary GlandOralHumanHumanHuman Mouse MonkeyMinor Salivary GlandNasalOralNasal SinusalCell typesSustentatorialSustentatorialNeuronalStem NeuronalEpithelialSustentatorialNeuronalGranularBasal layerEndothelialEpithelialApical EpithelialEndothelialApical EpithelialGobletUnspecifiedMinor salivary ductalBasal layerGobletApical EpithelialUnspecifiedApical EpithelialFibroblastT and BcellsUnspecifiedUnspecifiedBasal layerGobletACE2ˆ’ˆ’ˆ’NATMPRSS2ˆ’ˆ’NANANANANANANANANANANANANANANANANAACE2 Angiotensin Converting Enzyme2 NA not available TMPRSS2 transmembrane protease serine2reporting a higher ACE2 expression in tracheal naso oro and hypopharyngeal tissues as well as in the salivary ducts of the pharyngeal gland and consequently in saliva In this study the cell expression was mainly localized in the lamina propria In the same vein Vaarala et a0al reported TMPRSS2 expression in mouse salivary tissues []Cell Detection MethodsThe following methods have been used for detecting ACE2 and TMPRSS2 in cells of human and animal tissue RNAseq N IHC N RTPCR N in a0situ hybridization ISH N and WB N Different detection approaches were used in studies [ ] One study reported specific genetic analysis [] There were significant differences between studies regarding methods used While Ziegler et a0al and Sungnak et a0al detected ACE2 by RNAseq in goblet cells Lee et a0al did not find any immunohistochemical labeling [ ] However the results reported in sustentacular cells agree in the same direction whatever the technique used whether by RNAseq or by ISH and immunocytochemistry [“] The discrepancies are rather observed between studies having performed immunohistochemistry Indeed using two different antibodies Hikmet did not find ACE2 expression in nasopharynx epithelium whereas others demonstrated the staining of the apical surface of epithelia and ciliated epithelial cells [ ] Interestingly all the studies which carried out RNAseq found an expression of ACE2 or TMPRSS2 at the epithelial level which implies that the technique used could generate biases between the studies [ “ “] 0cHead and Neck Pathology DiscussionThe presentation of COVID19 infection may be in several clinical forms ranging from anosmia in isolation to severe multiple an failure and death The mechanisms underlying the COVID19 polymorphism are still unknown To infect tissues SARSCoV2 needs to entry into the cells which is allowed through ACE2 and TMPRSS2 receptors [] The identification of virus receptor expression in the tissues makes particularly sense to better understand the clinical expression of the disease This systematic review sheds light on many pointsFirst ACE2 and TMPRSS2 receptors are expressed in epithelial and nonepithelial cells throughout the head and neck The head and neck expression may support the otolaryngological clinical picture of the disease which was recently found in European and North American COVID patients [ ] By entering the body via the epithelial cells of the upper aerodigestive tract mucosa the SARSCoV2 virus leads to an inflammatory reaction and the development of otolaryngological symptoms Nasal entrance of the virus through high ACE2 expression was supported in the study of Wu et a0al who found a higher virus concentration in nasal swabs compared with throat swabs []The olfactory cleft is a nasal region that has drawn the attention of many researchers over the past few weeks Indeed recent data supported that more than of COVID19 patients developed subjective olfactory dysfunction especially when patients suffered from mildtomoderate forms of the disease [ ] Because ACE2 and TMPRSS2 are both expressed in the nasal mucosa of the olfactory cleft entrance into the olfactory bulb seems plausible Once in the bulb according to some human studies [ ] the virus could infect cells that express ACE2 or TMPRSS2 namely glial and neuronal stem cells Fig a0Integrating the molecular clinical and radiological characteristics of SARSCOV2 olfactory loss may shed light about its pathophysiological process Taking into account that the loss is often temporary SARSCOV2 may primarily infect the sustentacular cells supporting the olfactory sensory neurons This infection may cause rapid disruption of the olfactory epithelium structure and function with a possible inflammatory response inducing sudden onset smell loss This inflammation is observed in a minority of patient with congested olfactory cleft who underwent CT scan [ “]ACE2 and TMPRSS2 are also found in horizontal basal olfactory stem cells located in the basal layer They are less exposed to the external environment thus less likely to be infected in first line the loss would have been more Fig Epithelium of Olfactory Cleft The figure summarizes the olfactory cleft epithelium 0c Head and Neck Pathologyprogressive However once infected they might slow down recovery time because horizontal basal cells give rise to many cell type in the olfactory epithelium They may also contribute to virus spread to the olfactory bulb vascular pericytes Magnetic resonance imaging MRI studies of the olfactory bulb [ ] in which ACE2 are only expressed in vascular pericytes but not in neurons may show inflammatory signs suggesting that the infection process can extend more centrally and promote inflammatory response [] Inflammatory causes are often quickly reversible for example after a oneweek trial of high dose of corticosteroids or simply days after the resolution of the viral infection suggesting that the olfactory neurons and bulbs are still somewhat intact This seems to be the case for a majority of patients In contrast with more sustained destruction of neuronal olfactory structures the recovery time is much longer and may take “ a0years given the slow neuroregeneration process [ ]In this systematic review we found that three studies reported high ACE2 expression in major or minor salivary gland human tissues [ ] These data corroborate the literature findings that reported a salivary pattern of SARSCoV2 and related parotitis [ ] Moreover the virus spread into the salivary gland tissues allows us a better understanding the mechanisms underlying salivary transmission Interestingly in Liu et a0al observed that monkeys infected by SARSCoV had a salivary viral spread which was associated with a salivary virion excretion [] These data support the need to conduct future studies investigating the presence of SARSCoV2 in the saliva of infected human and to corroborate the saliva findings with the ACE2 salivary gland expressionThe head and neck expression of ACE2 and TMPRSS2 and the related otolaryngological symptom pattern seems obvious but could vary across individuals and populations In support of this Lee et a0al observed that ACE2 expression is influenced by patient demographics clinical characteristics comorbidities and medication [] As reported in the genetic analysis of Cao et a0al there would be different susceptibilities or responses to SARSCoV2 in different populations [] The polymorphism of ACE2 and TMPRSS2 expression could explain the clinical differences between individuals Indeed many physicians reported in clinical and epidemiological studies different clinical presentation of the disease [ ] which could be associated with virus mutations [ ] The virus mutations and the related impact on receptor binding and infectivity is another point that has to be considered in future studies Otherwise according to the Bgee database https bgee expression of ACE2 and TMPRSS2 evaluated in murine models may increase with age These findings have to be confirmed in humans but could explain more severe clinical pictures in the elderlyThe present study has several limitations First the heterogeneity between studies about the detection method may lead to detection bias as some approaches are more sensitive than others Some studies interrogate gene expression at mRNA level and others at protein levels both types of analysis having their advantages and limitations Compared to transcriptomic analyzes immunohistochemistry brings additional important spatial information in tissue samples but recently Sorokin et a0al demonstrated high and statistically significant correlations between the RNA sequencing and immunohistochemical measurements [] Interestingly they highlighted the complementarity of both techniques for measuring cancer biomarkers in FFPE samples However differences observed across IHC studies suggest the involvement of many parameters The antibody specificity is a big challenge to ensure reproducibility of antibodybased studies and given the high homology between ACE1 and ACE2 cautions must be taken regarding antibody selection Besides a report from the International Working Group for Antibody Validation IWGAV proposed five scientific approaches to validate antibody specificity [] then such strategies must be considered in future investigations to confirm the published observations In addition it seems essential to enlarge and diversify patient cohorts and to combine transcriptomic and proteomic strategies as well as colocalize different markers of SARSCoV2 such as ACE2 and TMPRSS2 to provide an accurate representation of ACE2 expression through all head and neck areas of the whole populationSecond the majority of studies that were conducted during the SARSCoV2 pandemic did not consider many demographic and clinical factors such as the age of patients from who the tissues were extracted or the use of ACE inhibitor medications among others Third some otolaryngological regions remain uninvestigated such as the vocal folds The investigation of these remaining regions may shed further light on some recently reported unusual clinical phenomena such as severe dysphonia []ConclusionACE2 and TMPRSS2 are both expressed in head and neck tissues which may explain the otolaryngological clinical pattern of the disease and the entry of SARSCoV2 into the host anism Future studies considering demographical and clinical characteristics of patients from who the tissues are extracted are needed to better understand the cell entry mechanisms of SARSCoV2Author contributions JR TR SS JM design acquisition of data data analysis interpretation drafting final approval and accountability 0cHead and Neck Pathology for the work final approval of the version to be published agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved CCH CMCE MRB IG design data analysis interpretation revising the manuscript for important intellectual content final approval of the version to be published final approval and accountability for the work final approval of the version to be published agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved LS SH GC GD JH LV GR design acquisition of data data analysis interpretation drafting some parts of the manuscript final approval of the version to be published final approval and accountability for the work final approval of the version to be published agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolvedCompliance with Ethical Standards Conflict of interest The authors declare that they have no conflict of interestReferences Crackower MA Sarao R Oudit GY et a0al Angiotensinconverting enzyme is an essential regulator of heart function Nature “ Li W Moore MJ Vasilieva N Sui J Wong SK Berne MA Somasundaran M Sullivan JL Luzuriaga K Greenough TC Choe H Farzan M Angiotensinconverting enzyme is a functional receptor for the SARS coronavirus Nature “ Wang Z Xu X scRNAseq profiling of human testes reveals the presence of the ACE2 receptor a target for SARSCoV2 infection in spermatogonia leydig and sertoli cells Cells https doi103390cells Shang J Wan Y Luo C Ye G Geng Q Auerbach A Li F Cell entry mechanisms of SARSCoV2 Proc Natl Acad Sci USA https doi101073pnas20031 Vaira LA Hopkins C Salzano G et a0al Olfactory and gustatory function impairment in COVID19 patients An Italian objective multicenterstudy Head Neck https doi101002hed26269 Lechien JR ChiesaEstomba CM Place S Van Laethem Y Cabaraux P Mat Q Huet K Plzak J Horoi M Hans S Barillari MR Cammaroto G Fakhry N Martiny D Ayad T Jouffe L Hopkins C Saussez S COVID19 task force of YOIFOS Clinical and epidemiological characteristics of European patients with mildtomoderate Coronavirus Disease J Intern Med https doi101111joim13089 Lechien JR ChiesaEstomba CM De Siati DR et a0al Olfactory and gustatory dysfunctions as a clinical presentation of mildtomoderate forms of the coronavirus disease COVID19 a multicenter European study Eur Arch Otorhinolaryngol https doi101007s0040 McInnes MDF Moher D Thombs BD et a0al Preferred reporting items for a systematic review and metaanalysis of diagnostic test accuracy studies the PRISMADTA statement JAMA “ https doi101001jama201719163 Thompson M Tiwari A Fu R Moe E Buckley DI A Framework To Facilitate the Use of Systematic Reviews and MetaAnalyses in the Design of Primary Research Studies Rockville Agency for Healthcare Research and Quality US Vaarala MH Porvari KS Kellokumpu S Kyll¶nen AP Vihko PT Expression of transmembrane serine protease TMPRSS2 in mouse and human tissues J Pathol “ https doi10100210969896200099999999 Hamming I Timens W Bulthuis ML Lely AT Navis G van Goor H Tissue distribution of ACE2 protein the functional receptor for SARS coronavirus A first step in understanding SARS pathogenesis J Pathol “ https doi101002path1570 Jia HP Look DC Shi L et a0al ACE2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia J Virol “ https doi101128JVI792314614 Liu L Wei Q Alvarez X et a0al Epithelial cells lining salivary gland ducts are early target cells of severe acute respiratory syndrome coronavirus infection in the upper respiratory tracts of rhesus macaques J Virol “ https doi101128JVI02292 Bilinska K Jakubowska P Von Bartheld CS Butowt R Expression of the SARSCoV2 entry proteins ACE2 and TMPRSS2 in Cells of the olfactory epithelium identification of cell types and trends with age ACS Chem Neurosci https doi101021acsch emneu ro0c002 Brann DH Tsukahara T Weinreb C et a0al Nonneuronal expression of SARSCoV2 entry genes in the olfactory system suggests mechanisms underlying COVID19associated anosmia Preprint https doi1011012020032500908 Butowt R Bilinska K SARSCoV2 olfaction brain infection and the urgent need for clinical samples allowing earlier virus detection ACS Chem Neurosci “ https doi101021acsch emneu ro0c001 Cao Y Li L Feng Z Wan S Huang P Sun X Wen F Huang X Ning G Wang W Comparative genetic analysis of the novel Coronavirus 2019nCoVSARSCoV2 receptor ACE2 in different populations Cell Discov https doi101038s4142 Chen R Wang K Yu J et a0al The spatial and celltype distribution of SARSCoV2 receptor ACE2 in human and mouse brain Preprint https doi1011012020040703065 Hikmet F Mar L Uhln M Lindskog C The protein expression profile of ACE2 in human tissues bioRxiv https doi1011012020033101604 Lee IT Nakayama T Wu CT et a0al Robust ACE2 protein expression localizes to the motile cilia of the respiratory tract epithelia and is not increased by ACE inhibitors or angiotensin receptor blockers Preprint https doi1011012020050820092 Li MY Li L Zhang Y Wang XS Expression of the SARSCoV2 cell receptor gene ACE2 in a wide variety of human tissues Infect Dis Poverty https doi101186s4024 x Sungnak W Huang N Bcavin C et a0al SARSCoV2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes Nat Med “ https doi101038s4159 Xu H Zhong L Deng J Peng J Dan H Zeng X Li T Chen Q High expression of ACE2 receptor of 2019nCoV on the epithelial cells of oral mucosa Int J Oral Sci https doi101038s4136 80200074x Xu J Li Y Gan F Du Y Yao Y Salivary glands potential reservoirs for COVID19 asymptomatic infection J Dent Res https doi10117700220 Wu C Zheng S Chen Y Zheng M Singlecell RNA expression profiling of ACE2 the putative receptor of Wuhan 2019nCoV in the nasal tissue https doi1011012020021120022 0c Head and Neck Pathology Ziegler CGK Allon SJ Nyquist SK et a0al SARSCoV2 receptor ACE2 is an interferonstimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues Cell 2020S0092““ https doi101016jcell202004035 Kaye R Chang CWD Kazahaya K Brereton J Denneny JC III COVID19 anosmia reporting tool initial findings Otolaryngol Head Neck Surg https doi10117701945 Lechien JR ChiesaEstomba CM Hans S Barillari MR Jouffe L Saussez S Loss of smell and taste in European patients with mild to moderate COVID19 Ann Intern Med https doi107326M202428 Vaira LA Salzano G Deiana G De Riu G Anosmia and ageusia common findings in COVID19 patients Laryngoscope https doi101002lary28692 Spinato G Fabbris C Polesel J Cazzador D Borsetto D

Thyroid_Cancer

Breast cancer BC is the most common malignant tumour in women worldwide and one of the most common fataltumours in women DeltaNotchlike epidermal growth factor EGFrelated receptor DNER is a transmembraneprotein involved in the development of tumours The role and potential mechanism of DNER inepithelial“mesenchymal transition EMT and apoptosis in BC are not fully understood We find that DNER isoverexpressed in BC tissue especially triplenegative breast cancer TNBC tissue and related to the survival of BC andTNBC patients In addition DNER regulates cell EMT to enhance the proliferation and metastasis of BC cells via theWntcatenin pathway in vitro and in vivo Moreover the expression levels of catenin and DNER in BD tissue arepositively correlated The simultaneously high expression of DNER and catenin contributes to poor prognosis in BCpatients Finally DNER protects BC cells from epirubicininduced growth inhibition and apoptosis via the Wntcatenin pathway In these results suggest that DNER induces EMT and prevents apoptosis by the Wntcatenin pathway ultimately promoting the malignant progression of BC In our study demonstrates thatDNER functions as an oncogene and potentially valuable therapeutic target for BCIntroductionBreast cancer BC is the most common malignanttumour in women worldwide and one of the most common fatal tumours in women12 BC treatments can beused to improve patient outcome3 However tumourrecurrence and metastasis and chemotherapeutic resistance are the most common causes of cancer treatmentfailure Therefore the need to screen and identify keyregulatory factors in the process of tumour recurrenceand metastasis for the treatment of BC is urgentCorrespondence Si Sun karensisi126com or Shengrong Sun sun137sinacom1Department of Breast and Thyroid Surgery Renmin Hospital of WuhanUniversity Wuhan Hubei China2Department of Pathophysiology Wuhan University School of Basic MedicalSciences Wuhan Hubei ChinaFull list of author information is available at the end of the These authors contributed equally Zhong Wang Zhiyu LiEdited by S TaitTumour EMT is a multifactorial and complex event inwhich epithelial properties and the ability to adhere toadjacent cells are lost and mesenchymal and stem cellphenotypes are eventually obtained4“ EMT a crucialregulatory mechanism by which tumours acquire invasiveand metastatic abilities and the ability to resist apoptosisplays an irreplaceable role in the development of malignant tumours8“ Recent studies upon activation of theclassical Wntcatenin pathway catenin enters andaccumulates in the nucleus which induces the transcription and translation of downstream target genes thusaccelerating EMT10 Therefore maintaining cateninactivity is important for the Wntcatenin pathway andtumour progressionDNER a neuronspecific transmembrane protein foundin a variety of peripheral cells11“ is a member of theatypical Notch ligand family and binds to Notch1 receptor1115 DNER is expressed at abnormally high levels in The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of various cancer tissues16 and promotes the proliferationmigration and invasion of cancer cells1617 but has aninhibitory effect on cell proliferation in glioma14 Nevertheless the precise function and underlying molecularmechanisms of EMT and chemosensitivity in BC areunclearIn this study we have revealed the previously unrecognized role of DNER in cancer progression EMT andthe apoptosis of BC cells Furthermore we investigatedthe expression of DNER and its relationship with survivalin BC and TNBC patients In addition we have providedevidence for the correlation between DNER and cateninand the prognostic value of the highlevel expression ofDNER and catenin in BC patients Finally the crucial roleof catenin in DNERinduced EMT and the inhibitoryeffect of DNER on apoptosis have been revealed Takentogether our results elucidate the potential functions andmechanism of DNER in EMT and apoptosis in BC cellsand provide a new therapeutic pathway for the recurrence metastasis and chemotherapy resistance of BCMaterials and methodsEthics statementTwo groups of the same human tissue specimens wereacquired from patients of Renmin Hospital of WuhanUniversity who were diagnosed with BC from to One group of specimens was promptly stored atˆ’ °C for western blotting and PCR analysis The othergroup of specimens was fixed in formalin and paraffinizedfor immunohistochemistry IHC All patients did notreceive chemotherapy radiotherapy or immunotherapyThis research was approved by the Ethics Committee ofRenmin Hospital of Wuhan University and informedconsent was obtained from all patientsCell culture and reagentsHuman BC cell lines MCF7 and MDAMB468 cellswere obtained from American Type Culture Collectionand incubated by their corresponding recommendedmethod All celllines were mycoplasmafree by morphological examination and verified for their authenticities by STR profiling Epirubicin was purchased fromPfizer Pharmaceutical Co Ltd Wuxi China and dissolved in physiological saline CHIR catenininhibitor and XAV939 catenin agonist were purchased from Selleck Shanghai China and dissolvedin DMSO “ “ and The stainingintensity was evaluated as follows no staining weak staining moderate staining and strongstaining The final protein staining score was the percentage score multiplied by the intensity score finalprotein staining scores were divided into three categoriesas follows negative “ low expression and “ high expressionsiRNA and plasmid transfectionscrambleDNER siRNA ²GCUUUGCCAGUCCAAGAUUTTsiRNA ²UUCUCCGAACGUGUandCACGUTT were synthesized from GenePharma CoShanghai China FLAGDNER and FLAGNC werepurchased from GeneChem Co Shanghai China Whencells in a sixwell plate had grown to the appropriatedensity siRNA and plasmids were transiently transfectedwith Lipofectamine3000 Invitrogen USA and RNAiMAX Invitrogen USA respectively according to themanufacturer™s instructions After h of transfection thecells were used for subsequent experimentsqRTPCRTotal RNA from tissue specimens and cell samples wasextracted by using TRIzol Invitrogen USA according tothe protocol and then reverse transcribed to cDNA usinga TransScript FirstStand cDNA Synthesis Kit TaKaRaJapan qRTPCR was implemented by using SYBR GreenMastermix TaKaRa Japan with an ABI 7900HT RealTime PCR system USA The primer sequences areshown in Supplemental Table Cell Counting Kit CCK8 assayAfter a series of interventions equal numbers of BCcells were plated into 96well plates and cultured for days Ten microlitres of CCK8 CK04 Dojindo Japansolution was added to each well and the cells wereincubated at °C for h The absorbance was determined at nmWound healing assayAfter intervention the cells were seeded into sixwellplates When the cell density exceeded the cells werewashed twice with PBS and scratches were made with ayellow plastic pipette tip Cells were cultured in serumfree medium for h and photographed under amicroscopeImmunohistochemical stainingInvasion assayIHC staining was performed as previously described18The results of IHC staining were evaluated by two independent pathologists and scored according to the percentage of positive tumour cells and staining intensityThe percentage of positive cells was scored as follows After a series of treatments × cells in serumfreemedium were plated in the upper chambers of a Transwell apparatus with Matrigel Corning NY USA Medium in the bottom chambers containing FBS servedas an attractant After h of incubation cells that passedOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of through the chamber membrane were fixed with precooled formaldehyde and stained with crystal violetC0121 Beyotime The cells were counted and photographed under a microscopeWestern blottingThe prepared tissue and cell samples were separated byprotein SDSPAGE and transferred to a nitrocelluloseNC membrane The membrane was blocked in skimmilk powder for h at room temperature and immunoblotted with primary antibody at °C overnight Afterincubation with secondary antibody at room temperaturefor h protein expression was detected with corresponding protein development instrument and quantifiedby ImageJ software W S Rasband Image J NIH Theantibodies used are listed in Supplementary Table Nuclear and cytoplasmic protein extractionNuclear and Cytoplasmic Extraction Reagent P0027was purchased Beyotime Biotechnology The nuclear andcytoplasmic proteins were extracted according to theinstructions and then used for subsequent experimentsFlow cytometry to detect apoptosisA FITC Annexin V Apoptosis Detection Kit I BDPharmingen USA was used to detect cell apoptosis The cellswere seeded in sixwell plates After a series of interventionscells were processed following the manufacturer™s protocolFig DNER is upregulated in BC tissues and correlated with poor prognosis in BC and TNBC patients a The expression levels of DNER inluminal A and TNBC tumour tissues compared with adjacent tissue by IHC magnification × b The mRNA levels of DNER in luminal A and TNBCtumour tissues compared with adjacent tissue c The DNER protein expression in BC tissues and adjacent tissues by western blotting d TheKaplan“Meier analysis showed the RFS of BC and TNBC patients with DNER high expression or DNER low expression e The staining of DNER Ecadherin and Ncadherin in BC tissue by IHC magnification × f Correlation analyses of protein expression levels between Ecadherin Ncadherinand DNER p p vs the control groupOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of and the cell fluorescence was measured with a FACScan flowcytometer FACScan Becton DickinsonTable Clinicopathological associations of DNERexpression in breast cancerAnimal experimentsTo acquire MDAMB468 cells with DNER stablyknocked down and MCF7 cells stably overexpressingDNER cells were transfected with DNER knockdown andoverexpression lentivirus GeneChem Shanghai Chinaand then selected with puromycin When the transfectionefficiency approached the DNER protein level wasdetected with western blotting All experimental procedures were conducted according to the Regulations ofExperimental Animal Administration issued by the Animal Committee of Wuhan University The mice wererandomly divided into two groups A total of × stable cells in μl PBS were subcutaneously inoculatedinto the right iliac fossa of to 5weekold femaleathymic nude mice BALBc After a certain period ofintervention the mice were sacrificed by anaesthesia andxenografts were removed for weighing and photographing The expression of relative proteins was detected bywestern blotting and IHCFor mammaryfatpad tumour assays we establishedMDAMB231 cells with DNER stably knocked downThe mice were randomly divided into two groups × stable cells were resuspended in a mixture of PBS andMatrigel and then injected into the fourth mammaryfat pad on the same side of nude mice To observe lungmetastasis tumours were excised by surgical operationwhen they reached about mm3 Ten days after theoperation the mice were sacrificed by anaesthesia and thenumber of metastatic tumours per lung were determinedThe entire lung tissues were fixed with formalin andsectioned for haematoxylin and eosin HE staining todetermine the presence of lung metastasis The entirelung tissues were fixed with formalin and sectionedfor haematoxylin and eosin HE staining to determinethe presence of lung metastasisImmunofluorescenceImmunofluorescence staining was performed as previously described19 In brief after corresponding treatments the cells fixed with paraformaldehyde wereperforated by TritonX for min and blockedwith BSA for h Next the cells were incubated withcatenin dilution overnight at °C and thenincubated for min with 488conjugated antibodyInvitrogen A11034 Finally the slides were stained withDAPI for min The images of sample were analyzed bylaser confocal microscopy Zeiss LSM Statistical analysisStatisticalSPSS software SPSS Inc Chicago IL and GraphPadanalyses were performed usingOfficial journal of the Cell Death Differentiation AssociationVariablesLowN HighN P valueAge at diagnosis years‰¤GradeWellModeratelyPoorlyTumour size cm‰¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveERNegativePositivePRNegativePositiveHER2NegativePositiveKi67 ‰¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically significant P ER oestrogen receptor PR progesterone receptor HER2 human epithelial growthfactor receptor2Prism GraphPad Software La Jolla CA USA All datawere analyzed with at least three independent experiments and are presented as the mean ± SD A survivalcurve was prepared by Kaplan“Meier analysis and thelogrank test was used to compare survival differencesbetween groups Pearson™s correlation method was used 0cWang Cell Death and Disease Page of Table Clinicopathological associations of DNERexpression in triple negative breast cancerVariablesLowN HighN P valueAge at diagnosis years‰¤GradeWellModeratelyPoorlyTumour size cm‰¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveKi67 ‰¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically significant P to analyze the correlation between DNER and cateninA chisquare test was used to analyze associationsbetween DNER expression levels and clinical characteristics Oneway ANOVA was used to compare differencesin three or more groups Differences in which p were considered statistically significantResultsDNER is upregulated in BC tissues and correlated withpoor prognosis in BC and TNBC patientsTo determine the role of DNER in development of BCwe first measured the expression levels of DNER in BCtissue and matched adjacent normal breast tissue by IHCThe expression level of DNER in BC tissue was markedlyhighertheexpression in TNBC was higher than that in luminal A BCFig 1a We also detected the expression of DNER in BCtissue by PCR the results of which were consistent withthose of IHC experiments Fig 1b To further verifytissue moreoverthan thatin adjacentOfficial journal of the Cell Death Differentiation AssociationDNER expression in BC we utilized western blotting todetect DNER protein expression in BC and adjacent tissues As expected compared with DNER expression inadjacent tissues DNER expression in BC tissues wassignificantly elevated Fig 1c Furthermore the highestDNER expression level was found in TNBC tissue Theclinicopathological characteristics with different expression of DNER in all BC and TNBC patients were shown inTables and Kaplan“Meier analysis of RFS showed thatthe group expressing high levels of DNER had a worseprognosis than the group expressing low levels of DNERThe results of survival analysis of TNBC patients were thesame as that of BC patients and TNBC patients had ashorter RFS than BC patients Fig 1d Next to verifywhether the poor prognosis of BC patients caused byDNER is related to EMT we detected the correlationbetween DNER and EMTrelated markers The resultsshowed that DNER expression was negatively correlatedwith the expression of Ecadherin while positively correlated with Ncadherin expression Fig 1e f In addition we found that high expression of mesenchymalmarkers was significantly associated with high expressionof DNER in BC through the TCGA database httpgepiacancerpkucn Although the negativecorrelationbetween Ecadherin and DNER in TCGA database wasnot significant it also presented a negative trend Supplementary Fig 2A The results therefore suggested thatDNER is highly expressed in BC and that elevated DNERprotein expression contributes to the progression of BCespecially TNBCDNER increases the biological functions of BC cells in vitroTo evaluate the effect of DNER on BC cell proliferationmigration and invasion we used siRNA to suppressDNER expression in both MCF7 and MDAMB468cells Compared with DNER expression in the control andscramble siRNA groups DNER was silenced by almost and in MCF7 and MDAMB468 cells transfected with siRNA respectively Fig 2a b As shown inFig 2c DNER knockdown visibly downregulated thegrowth rate of BC cells by CCK8 assay Next a woundhealing assay was used to evaluate cell migration capacityCompared with wound closure in the scramble siRNAgroup DNER knockdown significantly inhibited woundclosure after h in BC cells Fig 2d In addition theTranswell assay revealed that DNER knockdown clearlyreduced BC cell invasion Fig 2e These results suggestthat DNER acts as a cancerpromoting gene in BC cellsTo further confirm the role of DNER in BC progressionDNER was overexpressed by transfection with the FLAGDNER plasmid for h As shown in Supplementary Fig1A DNER was successfully overexpressed in the two BCcell lines In striking contrast with the effects of DNERknockdown the ability of cell proliferation migration and 0cWang Cell Death and Disease Page of Fig DNER knockdown inhibits cell proliferation and metastasis of BC cells a b The knockdown efficiency of DNER in MCF7 and MDAMB cells c Cell growth was measured by CCK8 assay after DNER knockdown in two BC cell lines d Wound healing assay was used to determine themigratory ability of BC cells with DNER knockdown e The invasion capacity of BC cells with knockdown of DNER was confirmed by Transwell assayDown Quantitative analysis of invasion ratio was shown The values are the mean ± SD from three independent experiments nsp p p p p vs the control groupinvasion was markedly enhanced after DNER overexpression Supplementary Fig 1B“E Taken togetherthese results indicated that DNER plays a crucial role inBC growth and metastatic potentialDNER induces EMT in BC cellsTumour cell EMT promotes the malignant progressionand metastasis of tumour cells10 We next examinedwhether DNER has a regulatory effect on BC cell EMTTo assess this function we detected EMTrelated proteinexpression by western blotting DNER knockdown significantly upregulated epitheliallike marker Ecadherinexpression and downregulated mesenchymal marker Ncadherin Vimentin Snail expression Fig 3a b Conversely overexpression of DNER dramatically shown theopposite effect Fig 3c d These results indicate thatDNER drives EMT in BC cells To provide further evidence of this effect of DNER on EMT we suppressedDNER expression and then transfected cells with theFLAGDNER plasmid to restore the DNER protein levelwe then determined whether DNER overexpression couldreverse changes in the expression of EMTrelated proteins As shown in Fig 3e f DNER knockdown alone hadan inhibitory effect on EMT whereas DNER knockdownand FLAGDNER transfection suppressed the effect ofDNER knockdown on Ecadherin and partially restoredthe expression of Ncadherin Vimentin and Snail Theseresults suggest that DNER plays a pivotal role in inducingEMT in BC cellsOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER induces EMT in BC cells a b EMTrelated proteins Ecadherin Ncadherin Vimentin and Snail were detected by western blotting inDNER knockdown cells Right quantitative analysis of the optical density ratio of Ecadherin Ncadherin Vimentin and Snail compared with actinare shown c d EMTrelated protein levels were measured by western blotting after DNER overexpression in BC cells Right quantitative analysis ofthe optical density ratio of Ecadherin Ncadherin Vimentin and Snail compared with actin are shown e f DNER was overexpressed in DNERknockdown cells and then western blotting detected the expression of EMTrelated proteins The values are the mean ± SD from three independentexperiments p p p vs the corresponding groupDNER activates the Wntcatenin signalling pathway andis positively correlated with cateninPrevious reports have shown that the Wntcateninsignalling pathway plays a crucial role in cancer cellmetastasis and EMT2021 Therefore we examined whether DNER mediates the canonical Wntcatenin signalling pathway As shown in Fig 4a b compared withcontrol cells in DNER knockdown cells the protein levelsof Notch1 pGSK3 and catenin were increased andthose of GSK3 were unchanged Conversely DNERoverexpression dramatically shown the opposite effectNext we investigate whether there is a relationshipbetween Notch signal and catenin in the case of DNERoverexpressioncells weIn DNERoverexpressingknocked down Notch1 and found that catenin expression was decreased compared with DNER overexpressionalone Supplementary Fig 2B Notch1 functioned as animportant role in the Wntcatenin pathway and theactivation of Notch1 was positively related to the nucleartranslocation of catenin22 Theaccumulation ofcatenin in the nucleus plays an important role in themalignant progression of tumours We assessed the effectof DNER knockdown on nuclear catenin accumulationby western blotting and observed that upon the knockdown of DNER the levels of nuclear catenin and Snailwere reduced in BC cell lines Fig 4c and SupplementaryFig 2C The nuclear location of catenin detected byimmunofluorescence showed the same results as thoseOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER activates the Wntcatenin signalling pathway and is positively correlated with catenin a b Western blotting detected theexpression of Notch1 pGSK3 GSK3 and catenin after DNER knockdown or DNERoverexpressing in BC cells c Total proteins catenin andSnail nuclear proteins catenin and Snail in DNER knockdown cells were assayed with western blotting d The mRNA levels of Survivin cMyc andLEF1 were detected by qRTPCR e The staining of DNER and catenin in BC tissue by IHC magnification × f Correlation analyses of proteinexpression levels between DNER and catenin g Kaplan“Meier survival analysis of BC patients was performed with DNERHighcateninHigh andDNERLowcateninLow expression The values are the mean ± SD from three independent experiments p p vs thecorresponding groupdetermined by western blotting Supplementary Fig 2DTo further confirm the decrease in nuclear cateninaccumulation following DNER knockdown we examinedthe expression levels of catenin downstream targetgenes in BC cells by PCR Consistent with the westernblotting results the mRNA expression levels of SurvivincMyc and LEF1 were significantly downregulated uponDNER knockdown Fig 4d These data indicated thatDNER knockdown can inhibit nuclear translocation andtranscriptional activity of catenin thereby controllingthe Wntcatenin signalling pathwayTo verify the relationship between DNER and cateninwe measured the protein expression levels of DNER andcatenin in BC tissues IHC showed that catenin washighly expressed when DNER was overexpressed whilecatenin levels were low when DNER was knocked downFig 4e Interestingly correlation analyses showed thatcatenin expression was positively correlated with theexpression of DNER Fig 4f We also found a strongpositive correlation between DNER expression andnuclear catenin expression Supplementary Fig 2EFurthermore immunofluorescence analysis showed thatDNER overexpression promoted more nuclear accumulation of catenin in BC cells Supplementary Fig 2FFinally Kaplan“Meier analysis showed that the prognosisof BC patients with high levels of DNER and cateninwas worse than the prognosis of BC patients with lowlevels of both DNER and catenin Fig 4g In additionOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Table Clinicopathological associations of both DNERand catenin expression in breast cancerVariablesLowN HighN P valueAge at diagnosis years‰¤GradeWellModeratelyPoorlyTumour size cm‰¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveERNegativePositivePRNegativePositiveHER2NegativePositiveKi67 ‰¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically significant P ER oestrogen receptor PR progesterone receptor HER2 human epithelial growthfactor receptor2we continued to show the correlation between the highlevel expression of both DNER and catenin and BCpatient clinicopathologic features as shown in Table These data suggest a strong correlation between theexpression of DNER with that of catenin and high levelsof DNERcatenin with poor prognosis in BCOfficial journal of the Cell Death Differentiation AssociationThe Wntcatenin signalling pathway is involved in DNERinduced EMT and prometastatic phenotypesTo determine whether the Wntcatenin pathwayfunctions in DNERinduced EMT we assessed whetherCHIR a specific Wntcatenin pathway activator23 and XAV939 a Wntcatenin pathway inhibitor24 could reverse the effect of DNER overexpressionand DNER knockdown in BC cells Catenin levels in thetwo BC cell lines were significantly elevated after CHIR treatment and markedly suppressed after XAV939treatment Fig 5a b Compared with DNER knockdownalone levels of the EMTrelated proteins were dramatically exhibited the opposite effect after of the treatment ofDNER knockdown cells with CHIR Fig 5a Thetreatment of DNERoverexpressing cells with XAV939clearly show similar results Fig 5b These findingsindicated that CHIR partly rescued the inhibitoryeffect of DNER knockdown on EMT progression and thatXAV939 suppressed the activation of EMT induced byDNER overexpression To investigate the role of the Wntcatenin pathway in DNERmediated cell proliferationmigration and invasion we performed rescue experimentsby activating or inhibiting catenin in DNER knockdownor DNERoverexpressing cells respectively Consistentwith the effects of Wntcatenin pathway activation andinhibition on EMT in the presence of CHIR theproliferation migration and invasion of DNER knockdown cells were clearly elevated Fig 5c e f Similarlyinhibition ofin DNERoverexpressing cells distinctly decreased metastatic ability as shown by changes in cell growth migration andinvasion Fig 5d g h Altogether these data suggestedthat catenin is indispensable for DNERinduced BC cellEMT and prometastatic phenotypescatenin by XAV939DNER enhances the tumorigenic and metastatic ability ofBC cells in vivoTo verify our results in vitro we next examined the roleof DNER in vivo To that end MDAMB468 cells inwhich DNER was stably knocked down and MCF7 cellsstably overexpressing DNER were successfully establishedto use to establish xenograft models in mice Fig 6a b fg After a period of time the xenografts were removedphotographed and weighed DNER knockdown significantly inhibited tumour size and weight comparedwith those in NC group Fig 6c d Consistent with theeffect of DNER knockdown xenografts from DNERoverexpressing group were larger and heavier than thosefrom NC group More importantly XAV939 reversedchanges in the size and weight of xenografts Fig 6h iThe DNER catenin cMyc and Snail protein levels inxenograft tissue were measured to confirm the upregulation and downregulation by western blotting Fig 6e jSupplementary Fig 3A Moreover IHC results found 0cWang Cell Death and Disease Page of Fig The Wntcatenin signalling pathway is involved in DNERinduced EMT and metastasis a b The expression of EMTrelated proteinsand catenin were detected by western blotting in DNER knockdown or DNERoverexpressing cells with CHIR μM h or XAV939 μM h treatment respectively c d Cell growth was measured by CCK8 in BC cells treated as described above e g Wound healing assay was used toexamined migration ability in BC cells treated as described above f h Transwell assay showed the cell invasion abilities in BC cells treated asdescribed above Right Quantitative analysis of invasion ratio was shown The values are the mean ± SD from three independent experiments p p vs the corresponding groupthat DNER knockdown reduced nuclear location ofcatenin while DNER overexpression promoted thisnuclear translocation effect Supplementary Fig 3C Inaddition as shown in Supplementary Fig 3A C thewestern blotting and IHC results showed that DNERimpacted the tumour growth in vivo was related to thelevel of Ki67 which is consistent with the positive correlation between DNER expression and ki67 expression inBC patients of TCGA database Supplementary Fig 3BTo explore the role of DNER in BC metastasis to lungMDAMB231 cells with stably DNER knockdown wassuccessfully established Fig 6k As shown in Fig 6l theOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER enhances the tumorigenic ability of BC cells in vivo a f k The transfection efficiency of DNER knockdown or expression in MDAMB468 MCF7 or MDAMB231 cells respectively b g The knockdown or overexpression efficiency of DNER in MDAMB468 cells or MCF7 cellsrespectively c h The xenograft pictures of shDNER and NCDNER in MDAMB468 cells n d i Comparison of tumour weights from variousgroups e j The expression of DNER and catenin in xenograft tissue by western blotting h The xenograft pictures of NCDNER group OEDNERgroup and OEDNER treated with XAV939 group in MCF7 cells n l Schematic diagram of in vivo experimental procedure for lung metastasispotential in situ of BC m Bright imaging of the lungs metastasis left and quantification of the metastases tumour right generated by MDAMB231cells n p vs the corresponding groupOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of see figure on previous pageFig DNER reduces the chemosensitivity of BC cells to epirubicin in vitro a Cell proliferation was detected by CCK8 after treated withdifferent concentrations of epirubicin in two BC cell lines b c DNER was analyzed by western blotting in BC cells treated as described above Rightquantitative analysis of the optical density ratio of DNER compared with actin are shown d Expression of epirubicininduced DNER was detectedby PCR e Cell viability was assessed by CCK8 after DNER knockdown treated with epirubicin or not f Analysis of apoptosis with FACS in MDAMB cells treated as described in e Right Quantitative analysis of apoptosis ratio g The expression of PARP was detected by western blotting in BCcells treated as described above Right quantitative analysis of the optical density ratio of cPARP compared with actin are shown h Cell growthwas measured by CCK8 after DNER overexpression treated with epirubicin or not i Analysis of apoptosis with FACS in MDAMB468 cells treated asdescribed in h Right Quantitative analysis of apoptosis ratio j The expression of PARP was detected by western blotting in BC cells treated asdescribed above Right quantitative analysis of the optical density ratio of cPARP compared with actin are shown The values are the mean ± SDfrom three independent experiments p p p vs the corresponding groupcorresponding treated MDAMB231 cells were injectedinto the fourth mammary fat pad and tumours wereexcised when they reached about mm3 Lung metastasis was observed in each group after days Brightfieldpicture demonstrated that more lung metastasis wasfound in the NCDNER group compared with the shDNER group Fig 6m Similar t

Thyroid_Cancer

"Proteasomes are found in both the cell nucleus and cytoplasm and play a major role in the ubiquitindependent and independent nonlysosomal pathways of intracellular protein degradation Proteasomes are alsoinvolved in the turnover of various regulatory proteins antigen processing cell differentiation and apoptosis Todetermine the diagnostic value of serum proteasome in antineutrophil cytoplasmic antibody ANCAassociatedvasculitis AAV we investigated patients with AAV at various stages of the diseaseMethods Serum 20Sproteasome was measured by ELISA in patients with MPOANCAassociated microscopicpolyangiitis MPA and renal involvement Thirty of the patients provided serum samples before the initialtreatment and provided samples during remission provided samples at both time pointsResults The mean serum 20Sproteasome level was significantly higher in the activevasculitis patients ± ngmL n compared to the inactivevasculitis patients ± ngmL n p and controls ± ngmL p There were significant positive correlations between the serum 20Sproteasome level and the Birmingham Vasculitis Activity Score BVAS r p the ANCA titer r p the white blood cell WBC count r p the platelet count r p and the serum Creactive protein CRP level r p There were significant negative correlationsbetween the serum 20Sproteasome level and both the hemoglobin concentration r ˆ’ p and theserum albumin level r ˆ’ p In a multiple regression analysis there was a significant positivecorrelation between the serum 20Sproteasome level and only the BVAS results p In a receiveroperating curve analysis the area under the curve for the serum 20Sproteasome level was which is higherthan those of the WBC count and the serum CRP level Conclusion The serum level of 20Sproteasome may be a useful marker for disease activity in AAVKeywords ANCAassociated vasculitis 20Sproteasome Disease activity Microscopic polyangiitis Proteasome Correspondence khiratokyomedacjp1Department of Nephrology Tokyo Medical University Ibaraki Medical Center Chuo Ami Ibaraki JapanFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMaruyama BMC Rheumatology Page of BackgroundProteasomes are located in both the nucleus and cytoplasm of cells and they play a major role in theubiquitindependent and ubiquitinindependent nonlysosomal pathways of intracellular protein degradation[ ] Proteasomes are also involved in the turnover ofvarious regulatory proteins eg ratelimiting enzymes[] and proteins for cellcycle control [] or transcriptional regulation [] antigen processing [] cell differentiation [] and apoptosis [] The 26S proteasome is amulticatalytic enzyme with a highly ordered structurecomposed of at least different subunits arranged intwo subcomplexes a 20S core and a 19S regulator p [] The 20Sproteasome is composed of four ringsof nonidentical subunits two rings are composed ofseven alpha subunits and the other two rings are composed of seven beta subunits Three of the seven betasubunits have proteolytic sites the 1 2 and 5 subunits are associated with caspaselike trypsinlike andchymotrypsinlike activities respectively [] These 12 and 5 subunits cleave peptide bonds at postacidicˆ’basic and hydrophobic amino acid residues respectively [] However subunits 1 2 and 5 could be replaced with 1i 2i and 5i by interferongamma IFNÎ and this IFNÎinducible proteasome isotype is calledthe immunoproteasome []The serum proteasome levels of patients with malignanttumors are elevated because the proteasome is overexpressed in tumor cells In patients with multiple myelomaserum proteasome concentrations have been shown to beassociated with disease severity and activity [] theserum proteasome concentrations were significantly elevated in patients with multiple myeloma compared tocontrols in multiple myeloma versus monoclonal gammopathies of undetermined significance MGUS and in active versus smoldering multiple myeloma [] Similarlyelevated serum proteasome levels were also reported inautoimmune diseases characterized by Bcell abnormality[] In the present study to determine the diagnosticvalue of the serum proteasome concentration in antineutrophil cytoplasmic antibody ANCAassociated vasculitisAAV we investigated patients with myeloperoxidaseMPOAAV at various stages of the diseasePatientsPatients and controlsWe analyzed the cases of patients with MPOANCAassociated microscopic polyangiitis MPA and renal involvement The diagnosis of MPA was based on theEuropean Medicines Agency algorithm [] and patientswith other types of systemic vasculitis including eosinophlic granuromatosis with polyangiitis granulomatosis with polyangiitis and antiglomerular basementdisease were excludedOf the MPOAAV patients provided serumsamples before the initial treatment and providedsamples during remission provided samples both before the initial treatment and during remission The Birmingham Vasculitis Activity Score BVAS was used toevaluate patients™ disease activity and remission was defined as a BVAS of As controls healthy volunteersand patients with chronic kidney disease CKD wereinvestigated The causes of CKD were nephrosclerosisn chronic glomerulonephritis n diabeticnephrosclerosis n and autosomal dominant polycystic kidney disease n Sample collection and analysisThe serum samples measured by a commerciallyavailable enzymelinked immunosorbent assay ELISAkit Enzo Life Science Plymouth Meeting PA USin duplicate In brief 96well microtiter plates werecoated with a mouse anti20Sproteasome alpha6subunit monoclonal antibody and left overnight at °C followed by blocking with phosphatebuffered saline PBS containing bovine serum albumin for h atroom temperature RT A serum sample was thenadded to each well and the plates were incubated for h at RT A rabbit anti20Sproteasome polyclonalantibody was then added to each well and the plateswere incubated for h at RT followed by incubationwith a horseradishperoxidaselabeled goat antirabbitIgG antibody for h at RT The plates were finallyincubated with chromogen tetramethylbenzidine andhydrogen peroxide for min at RT and then addedwith N hydrochloride acid solutionBetween these steps the plates were washed five timeswith Trisbuffered saline The plates were immediatelyread on a microplate reader Sunrise Remote® TecanJapan Kanagawa Japan set at nm with nm as areference wavelength The inter and intraassay variationswere Statistical analysesAll statistical analyses were performed using PASW Statistics software ver IBM Japan Tokyo for Windows The data are expressed as means ± standarddeviations or as numbers with percentages of the totalThe chisquare test with Yates™ continuity correctionand Fisher™s exact test were used for differences in categorical variables and posthoc comparisons Bonferronicorrection were performed to detect differences amongthree groups The MannWhitney Utest was used fortwogroup comparisons and we conducted an analysisof variance ANOVA to assess differences among threeor more groups posthoc comparisons were made usingthe BonferroniDunn test Correlations were determinedusing Spearman™s univariate correlation test and a linear 0cMaruyama BMC Rheumatology Page of regression analysis The multiple linear regression analysis included the covariates shown to be significantly associated with the serum 20Sproteasome levelin thecorrelation analysis and the data are expressed as standardized regression coefficients We applied comparative receiveroperatingcharacteristic ROC curvesand the area under the curve AUC to assess the diseaseactivity accuracy of the the serum 20Sproteasome leveland inflammatory variables Pvalues were accepted assignificant at but in the comparisons of three ormore groups the critical pvalue was divided by thenumber of comparisons being madeResultsThe subjects™ characteristicsThe characteristics clinical symptoms and laboratorydata among the subjects of the three groups the activevasculitis patients the inactivevasculitis patients andthe controls are shown in Table At the testing therewas no patients treated with any immunosuppressant inboth active and inactive vasculitis but allinactivevasculitis patients had treated with corticosteroids dosesof prednisolone ± mgdaySerum 20Sproteasome levelsAs illustrated in Fig the mean level of serum proteasome in the activevasculitis patients ± ngmL was significantly higher than those in the inactivevasculitis patients ± ngmL p andthe controls ± ngmL p There weresignificant positive correlations between the serum 20Sproteasome levels and the BVAS results r p the MPOANCA titers r p theWBC counts r p the platelet countsTable Characteristics of subjectsAge yearsGender malefemaleBirmingham vasculitis activity scoreClinical symptomsFeverWeight lossArthralgiaEpiscleritis or uvitisSinusitisHearing lossAlveolar hemorrhageInterstitial lung diseaseArrhythmiaPericarditisHeart failureRapidly progressive glomerulonephritisPeripheral nerve damageLaboratory dataANCA titer UmLWhite blood cell mm3Hemoglobin conc gdLPlatelet count 104mm3Serum albumin gdLSerum creatinine mgdLSerum Creactive protein mgdLSerum 20Sproteasome mgdLDoses of prednisolone mg daily P vs Inactivevasculitis P vs ControlsMPOANCA associated vasculitisActivevasculitis n ± Inactivevasculitis n ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Controlsn ± ± ± ± ± ± ± ± 0cMaruyama BMC Rheumatology Page of Fig The serum levels of 20Sproteasome Closed circles means bars standard deviations circles the values for individual patientsr p and the serum CRP levels r p There were significant negative correlations between the serum 20Sproteasome levels and boththe hemoglobin concentrations r ˆ’ p and the serum albumin levels r ˆ’ p In the multiple regression analysis there was a significant positive correlation between the serum 20Sproteasome levels and only the BVAS results p Table In the activevasculitis patients there was no associationbetween the serum 20S proteasome levels and clinicalsymptoms except for pulmonary involvement Supplementary file S1 The mean serum 20S proteasome level inthe activevasculitis patients with interstitial lung diseasen ± ngmL was significantly higherthan those in the activevasculitis patients withoutpulmonary involvement n ± ngmLp and those in activevasculitis patients withalveolar hemorrhage n ± ngmL p There was no association between the serum 20Sproteasome levels and the percentages of crescent formation renal histological classification Berden™s classification [] or renal symptoms patients with chanceproteinuriahematuria and patients with rapidly progressive glomerulonephritisThe diagnostic potential for disease activityThe optimum cutoff levels for the disease activity ofvasculitis were identified from the ROC curves for theWBC count 7250mm3 serum CRP level mgdL and serum 20Sproteasome level ngmLFig The area under the curve AUC for the serumTable Correlation between the serum 20Sproteasome level and clinical parametersAgeBirmingham Vasculitis Activity ScoreANCA titerWhite blood cellHemoglobin concPlatelet countSerum albuminSerum creatinineSerum Creactive proteinUnivariate analysisrˆ’ˆ’Pvalue Multivariate analysis“ˆ’ˆ’ˆ’“ˆ’Pvalue““ 0cMaruyama BMC Rheumatology Page of polymyositis serum proteasome levels were correlatedwith serum creatinine kinase levels and serum proteasome levels were associated with disease activity [] Inthe present study elevated serum 20Sproteasome levelswere also demonstrated in patients with AAV Althoughthere was no relationship between the MPOANCA titersand the serum 20Sproteasome levels these elevationswere associated with disease activity ie the BVASTherefore the serum level of 20Sproteasome may be auseful marker for disease activity in AAVThe mechanisms that underlie the elevated serum proteasome observed in patients with AAV are not yetknown Several serum biomarkers are filtrated at theglomerulus and reabsorbed and catabolized by proximaltubular cells and the serum levels of such biomarkers inpatients with renal insufficiency are elevated due to lowurinary filtration Because we found no relationship between serum 20Sproteasome levels and serum creatinine in AAV patients we conclude that elevated serumproteasome is not associated with renal functionIn a previous investigationthe serum proteasomelevels in patients with multiple myeloma were elevateddue to overexpression in tumor cells but the mechanisms underlying these elevations in autoimmune diseases were not clarified [] On the other hand thestructure and function of serum proteasome in healthydonors and patients with autoimmune diseases SLE andRA were maintained in the same manner as the intracellular forms [] However rings of proteasomes inthe serum of patients with autoimmune diseases weredifferent from those in healthy donors and those ringscontained immunosubunits 2i and 5i [] Consideringthat proteasomes from nonimmunocompetent cells donot contain immunosubunits [] it was speculated thatserum proteasome in patients with autoimmune diseasesmay have its fraction structure added by an immunocompetent cell origin that is different from that in normal individuals Therefore the elevated serum proteasome levelsin AAV may also be associated with the activation of immunocompetent cells Further investigations are neededto clarify the mechanism by which the proteasome isreleased into the circulationBortezomib a proteasome inhibitor prevents the degradation of proteins marked by ubiquitination by inhibiting the 26S proteasome [] The main effects ofbortezomib are NFκB inhibition inhibition of cell proliferation by the stabilization of cyclindependent kinasesFig The comparative ROC curves for three measurements ofdisease activity Solid line serum levels of 20Sproteasome Dashdotted line WBC counts Dashed line serum CRP levels Dotted linereference line20Sproteasome level was which is higher thanthose of the WBC count and the serum CRP level On the ROC curve the serum 20Sproteasomehad sensitivity and specificity for the diseaseactivity Although the specificity of the serum 20Sproteasome level was less than that of the serum CRPlevelserum 20Sproteasome level was superior to that of the serumCRP level Table sensitivity ofthetheDiscussionPrevious studies have demonstrated that the serum20Sproteasome levels are elevated in individuals with autoimmune diseases In patients with various autoimmune diseases including systemic lupus erythematosusSLE polymyositis Sjögren™s syndrome antiphospholipidsyndrome rheumatoid arthritis RA systemic sclerosisautoimmune hepatitis and myasthenia gravis the serumproteasome levels were higher than in the controls[] The levels were especially and significantly high inthe patients with SLE polymyositis Sjögren™s syndromeRA and autoimmune hepatitis [] In patients withWhite blood cell countTable Comparative ROC curves for parameters of disease activity confidence interval“““Area under the curveSerum Creactive proteinSerum 20SproteasomePvalue Optimal cutoff levelsSensitivity Specificity 0cMaruyama BMC Rheumatology Page of the induction of apoptosis by the activation of cJunNH2terminal kinase the stabilization of proapoptotic proteinsand transcription factors and tumor suppressors and theinduction of cell death by activation of the terminalunfolded protein response [] Bortezomib has been approved for clinical use in patients with multiple myelomaand bortezomib treatment has implications for antibodymediated immune diseases as well []The efficacy of bortezomib was demonstrated in amouse model of MPOANCAassociated glomerulonephritis [] That is in antiMPOassociated glomerulonephritisinduced by immunizing MPOdeficientmice with murine MPO followed by irradiation and thetransplantation of wildtype bone marrow proteinuriaalbuminuria and hematuria were significantly reducedcompared to the controls by both standard steroidcyclophosphamide treatment and bortezomib treatment[] Moreover the percentage of glomeruli with crescent or necrosis formation was reduced by both treatments The clinical efficacy of bortezomib for AAV hasnot been determined because only one case of an AAVpatient treated with bortezomib was reported In thatcase complete remission could not be achieved by acombination treatment with corticosteroid cyclophosphamide and rituximab therefore bortezomib mgm2week for weeks was added [] After the additionof bortezomib the patient achieved complete remissionand the doses of corticosteroid could be withdrawn []Thusthe proteasome may be associated with thedevelopment of AAV and inhibition of the proteasomemay be effective for inducing the remission of AAVOur study has several limitations The study population was small and limited to MPOAAV patients withrenal involvement and thus further studies are neededto compare patients with PR3AAV or nonrenal vasculitis In addition this was a retrospective crosssectionalstudy a larger prospective longitudinal study includingvasculitis patients with relapse would provide more definitive results Since the present study was performed atone facility it is necessary to verify the accuracy of theELISA test Moreover allinactivevasculitis patientswere treated with corticosteroids at the testing so treatments themselves may affected to decreased levels in inactive vasculitis Therefore further studies are needed tocompare AAV patients without treatments at the testingor to investigate other diseases patients treated withwithout corticosteroids Finally although we did demonstrate that serum 20Sproteasome levels were elevated inour patients with AAV the cause of this elevation wasnot identified In patients with multiple myeloma elevation of serum 20Sproteasome may be associated withoverexpression in tumor cells or abnormal cellular turnover [] On the other hand elevation of serum 20Sproteasome was observed in septic patients and therelation between elevated serum 20Sproteasome levelsand increased lymphocyte apoptosis was demonstratedin critically ill patients [] In patinets with RA andSLE it was speculated that the expression of inflammatory cytokines may have influenced the elevation of theserum 20Sproteasome [] Althoug elevated serum20Sproteasome in active AAV may be reflected an acutephase response there was no significant correlation between the serum 20Sproteasome levels and serum CRPlevels in the multiple regression analysis To clarify themechanisms of the serum 20Sproteasome elevation invasculitis patients further in vitro and ex vivo investigations are neededConclusionThe serum levels of 20Sproteasome in our patients withactive MPOAAV were significantly elevated compared tothe levels in the patients with inactive MPOAAV and thecontrols Moreover the serum levels of 20Sproteasomewere related to the BVAS results The serum level of 20Sproteasome may therefore be a useful marker for diseaseactivity in AAVSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s41927020001374Additional file Supplementary file S1 The relationships betweenthe serum 20S proteasome levels and the patients™ clinical symptomsAbbreviationsAAV Antineutrophil cytoplasmic antibodyassociated vasculitisANCA Antineutrophil cytoplasmic antibody BVAS Birmingham VasculitisActivity Score MPA Microscopic polyangiitis MPO MyeloperoxidaseAcknowledgementsPart of this study was reported at the 18th International Vasculitis and ANCAWorkshop Tokyo and it was published in Rheumatology suppl as an abstractAuthors™ contributionsHM and KH designed the study executed the experiments and participatedin the data management statistical analyses and reporting logicalinterpretation and presentation of the results MY KO and RT participated inthe data collection MT and HS took part in the logical interpretation andpresentation of the results KH and MK coanized the course of the workAll authors read and approved the final manuscriptFundingThis study was supported in part by a research grant to KH from MSD KKTokyo Japan The funds for this study were used only to purchase ELISAkitsAvailability of data and materialsAll of the raw datasets used and analyzed in this study are available uponreasonable request from the corresponding authorEthics approval and consent to participateAll procedures performed in this study involving human participants were inaccordance with the ethical standards of the institutional committee andwith the Declaration of Helsinki and its later amendments orcomparable ethical standards The study protocol was approved by the 0cMaruyama BMC Rheumatology Page of Zoeger A Blau M Egerer K Feist E Dahlmann B Circulating proteasomesare functional and have a subtype pattern distinct from 20S proteasomes inmajor blood cells Clin Chem “Froment C UttenweilerJoseph S BousquetDubouch MP Matondo MBes JP Esmenjaud C Lacroix C Monsarrat B BurletSchiltz O Aquantitative proteomic approach using twodimensional gel electrophoresisand isotopecoded affinity tag labeling for studying human 20S proteasomeheterogeneity Proteomics “ Hideshima T Richardson P Chauhan D Palombella VJ Elliott PJ Adams JAnderson KC The proteasome inhibitor PS341 inhibits growth inducesapoptosis and overcomes drug resistance in human multiple myelomacells Cancer Res “ Boccadoro M Man G Cavenagh J Preclinical evaluation of theproteasome inhibitor bortezomib in cancer therapy Cancer Cell Int Fröhlich K Holle JU Aries PM Gross WL Moosig F Successful use ofbortezomib in a patient with systemic lupus erythematosus and multiplemyeloma Ann Rheum Dis “ Bontscho J Schreiber A Manz RA Schneider W Luft FC Kettritz RMyeloperoxidasespecific plasma cell depletion by bortezomib protectsfrom antineutrophil cytoplasmic autoantibodiesinducedglomerulonephritis J Am Soc Nephrol “ Novikov P Moiseev S Bulanov N Shchegoleva E Bortezomib in refractoryANCAassociated vasculitis a new option Ann Rheum Dis 2016751e9 Yousef AA Suliman GA Mabrouk MM The value of correlation of serum 20Sproteasome concentration and percentage of lymphocytic apoptosis incritically ill patients a prospective observational study Crit Care R215Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsEthics Committees of Tokyo Medical University Ibaraki Medical CenterWritten informed consent for inclusion in the study was obtained from allpatientsConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Nephrology Tokyo Medical University Ibaraki Medical Center Chuo Ami Ibaraki Japan 2Department of Intensive CareMedicine Tokyo Medical University Ibaraki Medical Center Ami IbarakiJapan 3Department of Nephrology Tokyo Medical University ShinjukuTokyo JapanReceived April Accepted May ReferencesArrigo AP Tanaka K Goldberg AL Welch WJ Identity of the 19S ˜prosome™p with the large multifunctional protease complex of mammaliancells the proteasome Nature “Hershko A Ciechanover A The ubiquitin system Annu Rev Biochem “ Murakami Y Matsufuji S Kameji T Hayashi S Igarashi K Tamura T Tanaka KIchihara A Ornithine decarboxylase is degraded by the 26S proteasomewithout ubiquitination Nature “Hershko A Roles of ubiquitinmediated proteolysis in cell cycle control CurrOpin Cell Biol “Kho CJ Huggins GS Endege WO Hsieh CM Lee ME Haber E Degradationof E2A proteins through a ubiquitinconjugating enzyme UbcE2A J BiolChem “Stoltze L Nussbaum AK Sijts A Emmerich NP Kloetzel PM Schild H Thefunction of the proteasome system in MHC class I antigen processingImmunol Today “Baz A Henry L Caravano R Scherrer K Bureau JP Changes in the subunitdistribution of prosomes MCPproteasomes during the differentiation ofhuman leukemic cells Int J Cancer “Pasquini LA Marta CB Adamo AM Pasquini JM Soto EF Relationshipbetween the ubiquitindependent pathway and apoptosis in different cellsof the central nervous system effect of thyroid hormones Neurochem Res“Jung T Grune T Structure of the proteasome Prog Mol Biol Transl Sci “ Groll M Ditzel L Lowe Löwe J Stock D Bochtler M Bartunik HD Huber RStructure of 20S proteasome from yeast at a resolution Nature “ Boes B Hengel H Ruppert T Multhaup G Koszinowski UH Kloetzel PMInterferon gamma stimulation modulates the proteolytic activity andcleavage site preference of 20S mouse proteasomes J Exp Med “Jakob C Egerer K Liebisch P Türkmen S Zavrski I Kuckelkorn U Heider UKaiser M Fleissner C Sterz J Kleeberg L Feist E Burmester GR Kloetzel PMSezer O Circulating proteasome levels are an independent prognosticfactor for survival in multiple myeloma Blood “Egerer K Kuckelkorn U Rudolph PE Rückert JC Dörner T Burmester GRKloetzel PM Feist E Circulating proteasomes are markers of cell damageand immunologic activity in autoimmune diseases J Rheumatol “ Watts R Lane S Hanslik T Hauser T Hellmich B Koldingsnes W Mahr ASegelmark M CohenTervaert JW Scott D Development and validation of aconsensus methodology for the classification of the ANCAassociatedvasculitides and polyarteritis nodosa for epidemiological studies AnnRheum Dis “ Berden AE Ferrario F Hagen EC Jayne DR Jennette JC Joh K Neumann INoël LH Pusey CD Waldherr R Bruijn JA Bajema IM Histopathologicclassification of ANCAassociated glomerulonephritis J Am Soc Nephrol“ 0c"

Thyroid_Cancer

Inconsistency of the results regarding the genetic variability within genes coding for receptor activator of nuclear factor κB RANK and its ligand RANKL in rheumatoid arthritis RA prompted us to study the RANK and RANKL polymorphisms as potential biomarkers associated with disease predisposition and response to antiTNF treatment in a group of Polish patients with RA This study enrolled RA patients and controls RANK rs8086340 C G rs1805034 C T and RANKL rs7325635 G A rs7988338 G A alleles were determined by realtime PCR with melting curve analysis and related with clinical parameters In addition RANKL serum levels were measured by ELISA The RANK rs8086340G allele was overrepresented among patients as compared to controls OD p Creactive protein CRP levels were significantly p associated with RANK rs8086340 polymorphism and were higher in the CChomozygotes at the baseline while lower in the GGcarriers at the 12th week of the treatment At the latter time point RANKL rs7325635GGpositive patients also showed significantly lower CRP concentrations Higher alkaline phosphatase levels before induction of antiTNF therapy were observed in RANK rs8086340 and RANK rs1805034 CC homozygotes p and p respectively The GG homozygosity of both RANKL single nucleotide polymorphisms was significantly associated with the number of swollen joints rs7988338 and rs7325635 before and at the 12th week of therapy respectively p in both cases These results imply that polymorphisms within the RANK and RANKL genes affect RA susceptibility and antiTNF treatment outcomeKeywords RANK a0· RANKL a0· Polymorphism a0· AntiTNF therapy a0· Rheumatoid arthritiskatarzynaboguniakubikhirszfeldpl Katarzyna BoguniaKubik Laboratory of a0Clinical Immunogenetics and a0Pharmacogenetics Hirszfeld Institute of a0Immunology and a0Experimental Therapy Polish Academy of a0Sciences Wroclaw Poland Department of a0Rheumatology and a0Connective Tissue Diseases Jan Biziel University Hospital No Bydgoszcz Poland Department of a0Rheumatology and a0Internal Medicine Wroclaw Medical University Wroclaw Poland Ludwik Rydygier Collegium Medicum in a0Bydgoszcz Nicolaus Copernicus University Torun PolandIntroductionRheumatoid arthritis RA is an autoimmune disorder that is present in approximately of the Caucasian population mostly women It is identified by inflammation of joint synovial membrane leading to the progression of cartilage and bone tissue damage eventually prompting disability RA is caused by a combination of environmental genetic and stochastic factors Smolen et a0al Introduction of the tumor necrosis factor TNF inhibitors to RA treatment had a positive effect on the quality of patients™ lives Biologic agents successfully suppress disease symptoms as well as stop further bone damage and reduce disability Geiler et a0al Our previous studies showed that some genetic features may influence disease susceptibility or antiTNF therapy outcome in RA patients BoguniaKubik et a0al Vol01234567891 0c Page of Archivum Immunologiae et Therapiae Experimentalis Gębura et a0al Iwaszko et a0al Świerkot et a0al 2015aWellcharacterized risk factors and early diagnosis are crucial for preventing bone loss and achieving therapeutic success However molecular mechanisms behind osteoporosis in RA have not been fully elucidated It is known that glucocorticoid therapy inflammatory cytokines reduced physical activity and low calcium intake are involved in RArelated osteoporosis Corrado et a0al Pathak et a0al demonstrated that sera from patients with active disease enhanced osteoblast proliferation and differentiation via receptor activator of nuclear factor NFκB ligand RANKL thus leading to bone loss Dysregulation of balance between bone resorption and formation was previously observed in RA Boyce and Xing Under chronic inflammation conditions and in presence of proinflammatory cytokines such as interleukin IL TNFα IL6 and IL17 RANKL is expressed by T cells fibroblastlike synoviocytes bone marrow stromal cells Braun and Zwerina Schett and B cells thereby promoting osteoclastdependent bone resorption Meednu et a0al Yeo et a0al RANKL plays a pivotal role in osteoclast activation development and survival Nemeth et a0al Nevertheless binding to its receptor RANK is required for the process to begin RANK is a transmembrane protein belonging to the TNF receptor superfamily TNFRSF Schett et a0al and is mainly expressed on the surface of the macrophagemonocyte lineage cells Besides that it is widely present on the surface of osteoclast progenitors and mature osteoclasts Liu and Zhang Typically those molecules do not have kinase activity and must engage factors able to activate signaling pathways Walsh and Choi During osteoclastogenesis the TNF receptorassociated factors are recruited and NFκB an essential transcription factor is activated Leibbrandt and Penninger The nature of the relationship between osteoclasts and TNFfamily proteins has been reported in numerous studies Kitaura et a0al Ono and Nakashima In our study we focused on the genetic variability of the RANK TNFRSF11A and RANKL TNFSF11 genes in RA patients subjected to antiTNF treatmentThe significance of chosen RANK and RANKL single nucleotide polymorphisms SNPs have been described before The role of RANK rs1805034 polymorphism has been analyzed in regard to RA Mohamed et a0al Yang et a0al cancer Yin et a0al Zhang et a0al hip osteoporotic fractures Zhang et a0al knee osteoarthritis Wang et a0al age at menarche Duan et a0al Pan et a0al and in Paget™s disease Chung et a0al The RANK rs8086340 was also previously documented in RA patients as well as with respect to age at natural menopause Lu et a0al It has been reported that RANKL rs7325635 may be a genetic marker in heart failure patients Schmitz et a0al and RANKL rs7988338 was significantly associated with femoral neck compression strength index Dong et a0al However according to our knowledge those polymorphisms has never been studied as potential markers associated with disease predisposition and response to treatment with TNF inhibitors in an independent cohort of Polish RA patientsMaterials and a0MethodsPatients and a0ControlsThreehundredeighteen Polish patients with diagnosed rheumatoid arthritis and qualified for treatment with antiTNF agents were enrolled in this study Patients were classified according to the American College of Rheumatology criteria as well as by the presence of active disease represented by the Disease Activity Score in joints [DAS28] ‰¥ prior to initiating biologic agent therapy The following inclusion criteria were applied age over a0years a complete medical history and physical examination of patients and resistance to treatment with at least two diseasemodifying antirheumatic drugs The exclusion criteria included clinically significant impairment of hepatic and renal function the coexistence of other systemic diseases of connective tissue besides RA infection with hepatotropic viruses infections resistant to therapy ongoing history of cancer or uncontrolled diabetes alcohol abuse pregnancy or breastfeeding insufficient clinical records and unwillingness or inability to cooperate Data collected from the patients comprise level of Creactive protein CRP presence of rheumatoid factor RF presence of anticyclic citrullinated peptide antiCCP antibodies disease activity score DAS28 and erythrocyte sedimentation rate ESR painful and swollen joint counts global health assessments by a patient and a physician and visual Table Characteristics of RA patientsNumber of RA patientsFemalesmales of femalesAge years [mean ± SD]Disease onset years [mean ± SD]Disease duration years [mean ± SD]DAS28 baseline [mean ± SD]CRP baseline [mean ± SD]RFpositive []AntiCCP positive []DAS28 disease activity score CRP Creactive protein RF rheumatoid factor antiCCP anticyclic citrullinated peptide antibodies ± ± ± ± ± 0cArchivum Immunologiae et Therapiae Experimentalis Page of analogue scale VAS of pain value The study was approved by the Wrocław Medical University Ethics Committee Identification Code KB6252016 and written informed consent was obtained from all participants Baseline characteristics of the patients are summarized in Table a0 The control group consisted of Polish unrelated healthy blood donors with no personal history of autoimmune diseasesA subgroup of patients was characterized by the following additional parameters levels of calcium alkaline phosphatase ALP vitamin D3 and thyroidstimulatinghormone TSH that together with the number of painful and swollen joints and VAS score might correlate with bone conditionAnti‘TNF Therapy OutcomeDisease activity in RA patients was estimated using the DAS28 score based on four components number of swollen and tender joints levels of CRP and ESR patients™ global assessment of general health expressed on the visual analogue scale The level of disease activity was interpreted as either low DAS28 ‰¤ moderate DAS28 ‰¤ or high DAS28 Clinical response was assessed according to the European League Against Rheumatism EULAR criteria at the 12th week after initiation of antiTNF treatment The patients were divided into three groups according to their response to treatment good moderate or nonresponders A good response was defined as improvement in the DAS28 score specifically ΔDAS28 and DAS28 at endpoint ‰¤ at the endpoint The moderate response was defined as either ΔDAS28 and DAS28 at endpoint or ΔDAS28 ‰¤ and DAS28 at endpoint ‰¤ A lack of response was defined as ΔDAS28 ‰¤ or ΔDAS28 ‰¤ and DAS28 at endpoint as previously described Iwaszko et a0al SNP Selection and a0GenotypingFor the present study the genetic variants of the TNFRSF11A and TNFSF11 genes were selected based on available literature analysis as well as search results from NCBI Database of Short Genetic Variations dbSNP Information regarding predicted functional consequences of SNPs was obtained using the SNPinfo Web Server Xu and Taylor The RANK gene is located on chromosome rs8086340 C G is placed within intron and rs1805034 C T missense substitution in exon leads to an amino acid change from alanine to valine The RANKL gene is located on chromosome and both rs7325635 G A and rs7988338 G A are situated in intron in possible transcription factor binding sites The minor allele frequencies MAF of all the selected SNPs were higher than Genomic DNA was isolated from peripheral blood of RA patients and controls using QIAamp DNA Blood Midi Kit Qiagen Hilden Germany according to recommendations of the manufacturer RANK rs8086340 rs1805034 and RANKL rs7325635 rs7988338 alleles were determined by realtime polymerase chain reaction PCR amplification and meltingcurve analysis using LightSNiP assay TIB MOLBIOL Berlin Germany on the LightCycler RealTime PCR system Roche Diagnostics Rotkreuz SwitzerlandRANKL Serum Level AnalysisSerum concentration of RANKL was measured by commercial ELISA kits DY626 RD Systems Minneapolis MN USA according to protocols provided by the manufacturer The analyses were performed for a subgroup of RA patients consisting of individuals before antiTNF treatment and controls The absorbance was measured in a Tecan Sunrise absorbance reader and Magellan software Tecan Trading AG Switzerland The optical density of each well run in duplicate was determined by microplate reader set to a0nm with wavelength correction set to a0nm Peptide concentration in the samples was measured by comparing the optical density of the sample with a computergenerated four parameters logistic curvefit standard curveStatistical AnalysisThe Hardy“Weinberg equilibrium was tested in patients and controls for each SNP Potential associations between examined SNPs and clinical parameters of RA patients were analyzed applying the Mann“Whitney U test for quantitative data and the Fisher™s exact test for parametric values The frequencies of respective genotype groups among RA patients in relation to the antiTNF outcome were investigated by comparing the EULAR scores using Fisher™s exact test Fisher™s exact test was also used to compare genotype variation distribution within patients and controls p values less than were considered statistically significant All statistical calculations were performed in the GraphPad7 Prism softwareResultsDistribution of a0RANK and a0RANKL Genotypes in a0Patients and a0ControlsGenotype distribution of all the studied SNPs in both groups patients and controls are presented in Table a0 MAF values were as follows RANK rs8086340 C patients vs controls RANK rs1805034 C vs 0c Page of Archivum Immunologiae et Therapiae Experimentalis Table Distribution of RANK rs8086340 C G rs1805034 C T and RANKL rs7325635 G A rs7988338 G A genotypes in RA patients and controlsGeneRANKrs8086340GenotypeCCCGGGCCCTTTGGGAAAGGGAAARA patients N 117a 489b Controls N RANKrs1805034RANKLrs7325635RANKLrs7988338N number of individuals with a given genotypea OR CI “ p b OR CI “ p RANKL rs7325635 A vs RANKL s7988338 A vs Genotype frequencies of the RANK rs8086340 polymorphism in RA patients were different from those in the control group Table a0 Both the presence of the G variant as well as CG heterozygosity were more frequent among patients than controls RANK heterozygous genotype was present in of patients and of controls OR CI “ p The RANK rs8086340 G allele was detected in of RA patients as compared to of controls CG GG vs CC OR CI “ p These results imply that the RANK rs8086340 SNP may affect disease susceptibilityThere were no differences in genotype distribution of RANK rs1805034 RANKL rs7325635 RANKL rs7988338 between patients and controlsRelationship between a0Patients™ Genotypes and a0Clinical ParametersAmong the patients included in this study the mean ± SD of the CRP concentration in RA patients™ blood was ± and the disease activity score DAS28 level was ± After a0weeks of antiTNF treatment both parameters significantly decreased to ± and ± respectively p in both casesExcept for CRP levels none of the clinical parameters such as RF antiCCP or DAS28 was found to be associated with any of the polymorphisms studied For details please see Table a0 Significant differences were observed between levels of CRP and RANK rs8086340 and RANKL rs7325635 SNPs In general both before and after treatment with TNF inhibitors CRP concentrations were lower in the RANK rs8086340 GG and RANKL rs7325635 GG homozygotes as compared to patients carrying other RANK rs8086340 or RANKL rs7325635 genotypes respectively These differences were especially noticeable after the 12th week of antiTNF administration RANK rs8086340 GG vs CG CC p RANKL rs7325635 GG vs GA AA p RANK rs8086340RANK rs1805034Table Distribution of the RANK and RANKL genotypes with respect to selected clinical parameters at baseline and at the 12th week of the therapyGeneCCPDAS28 at Baseline DAS28 at 12th week CRP at Baseline CRP at 12th week RFMean ± SDCC ± CG ± GG ± ± CCCT ± ± TTRANKL rs7325635 GG ± ± ± RANKL rs7988338 GG ± ± ± Mean ± SD311a ± ± ± ± ± ± ± ± ± ± ± ± Mean ± SD ± ± 715b ± ± ± ± 717c ± ± ± ± ± ± Mean ± SD ± ± ± ± ± ± ± ± ± ± ± ± Number Number GAAAGAAAa p b p c p 0cArchivum Immunologiae et Therapiae Experimentalis Page of Additionally RANK rs8086340 CC carriers had significantly higher CRP levels before treatment than carriers of other genotypes of this SNP CG GG vs CC p Response to a0Anti‘TNF Therapystudied polymorphisms were not found to affect the outcome of biological treatmentRelationship between a0Patients™ Genotypes and a0Bone Parameters or a0Thyroid DysfunctionResponse to antiTNF therapy after a0weeks was evaluated using EULAR criteria In general a good and moderate response was achieved by and of patients respectively while the remaining of RA patients investigated did not respond to treatment The We were able to analyze and compare the distribution of selected SNPs analyzed in the present study with regard to various bone parameters such as calcium and alkaline phosphatase levels vitamin D TSH levels Table a0 number of painful and swollen joints and VAS score Table a0Table Distribution of the RANK and RANKL genotypes with regard to biochemical parameters level presented as mean ± SDGeneRANK rs8086340GenotypeCCCGGGCCCTTTGGGAAAGGGAAACalcium [mgdl] ± ± ± ± ± ± ± ± ± ± ± ± Alkaline phosphatase [Ul] ± 659a ± ± ± 325b ± ± ± ± ± ± ± ± Vitamin D3 [ngml] ± ± ± ± ± ± ± ± ± ± ± ± TSH [ulUml] ± ± ± ± ± ± 115c ± ± ± ± ± ± RANK rs1805034RANKL rs7325635RANKL rs7988338a p b p c p Table Distribution of the RANK and RANKL genotypes with respect to the number of painful or swollen joints and visual analogue scale VAS for pain score presented as mean ± SD at baseline and at the 12th week of the therapyGeneVAS [mm] at baselineVAS [mm] at 12th weekNumber of painful joints at baselineNumber of painful joints at 12th week ± ± ± ± ± ± ± ± ± ± ± ± Number of swollen joints at baseline ± ± ± ± ± ± ± ± ± ± 559b ± ± Number of swollen joints at 12th week ± ± ± ± ± ± ± 641a ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± RANK rs8086340 CC ± CG ± GG ± RANK rs1805034 CC ± CT ± TT ± GG ± GA ± AA ± GG ± GA ± AA ± rs7325635RANKL RANKL rs7988338a p b p 0c Page of Archivum Immunologiae et Therapiae Experimentalis Analyses revealed that both polymorphisms in the RANK gene rs8086340 and rs1805034 are associated with alkaline phosphatase level Table a0 whereas RANKL variants correlated with the number of swollen joints before and after therapy Table a0 Please note that both numbers of painful ± and swollen joints ± as well as VAS scores ± significantly decreased following the biological treatment to ± and ± as well as ± respectively p in all casesThe RANK rs8086340 and rs1805034 CC homozygotes were characterized with higher alkaline phosphatase levels The statistically significant relationship was observed for rs1805034 CC vs CT TT p while a strong tendency was seen in the RANK rs8086340 SNP CC vs CG GG p The RANKL rs7988338 GG homozygotes were identified with a smaller number of swollen joints compared to patients carrying the A allele GG vs AG AA p Also a significant correlation between a number of swollen joints and RANKL rs7325635 was observed at the 12th week of the antiTNF therapy Interestingly RANKL rs7325635 GG homozygotes in this polymorphism had also more swollen joints after treatment GG vs AG AA p In addition RANK rs8086340 C allele was more commonly observed in a group of patients with higher TSH levels TT vs CT CC p although more TT homozygous patients had thyroid dysfunction TSH level below or above the norm “ ulUml vs p RANKL Serum ConcentrationSerum concentrations of RANKL were assessed in RA patients before antiTNF treatment patients after a0weeks of treatment and controls It appeared that only patients at baseline after treatment and controls presented with RANKL concentrations exceeding the minimum standard curve point a0pgml No statistically significant difference was observed between RANKL serum levels of patients and controls However it was noticed that average protein concentration equaled a0pgml and a0pgml for RA patients before induction of antiTNF agents and controls respectively Furthermore no correlation was found between RANKL genotypes and serum levels either in patients or in controlsInterestingly after a0weeks of antiTNF treatment RANKL serum levels decreased to an average serum concentration of a0pgml similar to that observed for the control group a0pgmlDiscussionIn recent years several studies investigated the association between the RANK TNFRSF11A and RANKL TNFSF11 gene polymorphisms and the risk for RA development in different populations However the results coming from these studies were conflicting For example Mohamed et a0al described RANK rs1805034 and RANKL rs9525641 nonsynonymous polymorphisms as potential genetic risk factors for osteoporosis in postmenopausal women with RA In other studies RANKL rs9525641 was found to be associated with younger age at onset of RA disease Tan et a0al Wu et a0al In German patients the major allele of RANKL rs2277438 and a minor variant of RANK rs35211496 both located within intronic regions were described to increase the risk for RA Assmann et a0al Moreover the RANKL rs2277438 G allele was found to be associated with radiographic progression of joint damage Furuya et a0al A very recent study of Yang et a0al documented that RANKL rs2277438 polymorphism increased RA risk and that the RANK rs1805034 SNP was not related to RA risk On the other hand Wang et a0al found the RANK rs1805034 SNP to be associated with susceptibility to knee osteoarthritisOur present study was conducted to evaluate the role of the RANK rs8086340 C G rs1805034 C T and RANKL rs7325635 G A rs7988338 G A SNPs as potential diagnostic biomarkers associated with the RA risk in the Polish population and prognostic biomarkers affecting the outcome of the biological treatment To the best of our knowledge the present study is the first one investigating relationships of these selected SNPs with clinical parameters in patients with RASimilarly to the results of Yang et a0al our study showed that the RANK rs1805034 SNP was not found to be related with RA risk However we observed a higher frequency of RANK rs8086340 heterozygotes and G allele carriers among patients than in controls showing that patients possessing the RANK rs8086340 G allele were more prone to RA development Such an association between RANK rs8086340 SNP and RA susceptibility was not previously describedRecently published results in French cohorts found the RANK rs8086340 SNP to be correlated with the presence of antiCCP RuyssenWitrand et a0al but we did not observe such a correlation in our patients of Polish origin either with antiCCP or RF Concerning some other clinical parameters we did notice a statistically significant decrease in the level of CRP and DAS28 during therapy but only changes in CRP serum concentrations were related to investigated polymorphisms We observed that GG homozygosity of both RANK rs8086340 and RANKL rs7325635 was 0cArchivum Immunologiae et Therapiae Experimentalis Page of associated with lower CRP levels especially after the 12th week of antiTNF treatment Our observations correspond with earlier studies considering the role of TNF blocker as an effective disease activity reducer Kurz et a0al SNPs investigated in this study may have a regulatory role in gene transcription Polymorphism rs8086340 of the RANK gene is located within an intronic region while rs1805034 results in an amino acid change from alanine to valine RANKL rs7325635 as well as rs7988338 are situated in an intronic area in a transcription factor binding site Therefore we also measured serum RANKL concentration in patients at baseline at the 12th week after induction of antiTNF agents and in the control groupPrevious studies reported significantly increased RANKL levels in RA patients compared to controls and in antiCCPpositive individuals Boman et a0al Higher RANKL concentration was also detected before the onset of RA Johansson et a0al Interestingly in our study we observed some differences in serum concentrations between patients before initiation of biological treatment and controls with a higher average protein concentration in RA patients a0pgml vs a0pgml in controls However this difference did not reach statistical significanceNo significant relationship was detected between RANKL concentration and the both SNPs within the RANKL encoding gene Although an average RANKL concentration was over a0pgml higher at the baseline in patients carrying the RANK rs8086340 G allele found in the present study to be associated with an increased risk for RA development Patients carrying the G allele presented with an average of a0pgml RANKL in serum vs a0pgml for patients lacking this genetic variantAs for RANKL SNPs investigated in our study RuyssenWitrand et a0al described RANKL rs7325635 as significantly associated with antiCCP antibody presence and erosions whereas RANKL rs7988338 was significantly associated with femoral neck compression strength index a phenotypic parameter integrating bone density bone size and body size having significant potential to improve hip fracture risk assessment Dong et a0al In our group of RA patients the GG homozygosity of both RANKL SNPs rs7988338 and rs7325635 was significantly associated with the number of swollen joints while the RANK rs8086340 and rs1805034 CC homozygotes were significantly more frequent among patients with higher ALP levels These relationships have not been previously described Surprisingly a significant difference was also noted with respect to glucocorticosteroid dose The RANKL rs7988338 G allele carriers received a significantly lower dose of this drug than the AA homozygous patients individual data not shown Despite antiinflammatory effects glucocorticosteroids also affect the delayed progression of erosive lesions in the joints Świerkot and Madej This finding is in line with our observation on the lower number of swollen joints before treatment in the GG homozygous patientsWe also observed a decrease in RANKL serum level during the therapy At the 12th week of the treatment the average protein concentration equaled a0pgml which corresponds to RANKL level of the control group This observation may reflect effectiveness of the antiTNF treatment which is in line with the results of GonzálezAlvaro et a0al They also showed a decrease in RANKL serum concentration during antiTNF therapy and further suggested that serum level of RANKL may be helpful in predicting outcome in patients receiving biologic agents GonzálezAlvaro et a0al Also Świerkot et a0al 2015b demonstrated decreasing RANKL concentration in the subgroup of RA patients with goodmoderate MTX therapy response after months of treatmentThe results of our study indicate a possible role of the RANK TNFRSF11A and RANKL TNFSF11 gene polymorphisms as diagnostic andor prognostic factors in Polish patients with RA They imply that RANK rs8086340 SNP may affect disease susceptibility Furthermore RANK rs8086340 and RANKL rs7325635 polymorphisms may have a prognostic role in modulating the clinical outcome of antiTNF therapyNevertheless further genetic studies on larger groups of patients of various origins subjected to biological treatment are necessary to confirm these findingsAcknowledgements This work was supported by a grant from the National Science Centre Poland 201621BNZ501901 The authors thank the Regional Centre of Transfusion Medicine and Blood Bank in Wroclaw for providing control samples We are also grateful to Piotr Łacina for his assistanceCompliance with ethical standards Conflict of interest The authors declare that they have no competing interestOpen Access This article is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this article are included in the article™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the article™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons orglicen sesby40 0c Page of ReferencesAssmann G Koenig J Pfreundschuh M et a0al Genetic variations in genes encoding RANK RANKL and OPG in rheumatoid arthritis a casecontrol study J Rheumatol “BoguniaKubik K Świerkot J Malak A et a0al IL17A IL17F and IL23R gene polymorphisms in Polish patients with rheumatoid arthritis Arch Immunol Ther Exp “Boman A Kokkonen H Ärlestig L et a0al Receptor activator of nuclear factor kappaB ligand RANKL but not sclerostin or gene polymorphisms is related to joint destruction in early rheumatoid arthritis Clin Rheumatol “Boyce BF Xing L Functions of RANKLRANKOPG in bone modeling and remodeling Arch Biochem Biophys “Braun T Zwerina J Positive regulators of osteoclastogenesis and bone resorption in rheumatoid arthritis Arthritis Res Ther Chung PY Beyens G Riches PL Genetic variation in the TNFRSF11A gene encoding RANK is associated with susceptibility to Paget™s disease of bone J Bone Miner Res “Corrado A Maruotti N Cantatore FP Osteoblast role in rheumatic diseases Int J Mol Sci Dong S Liu X Chen Y et a0al Association analyses of RANKLRANKOPG gene polymorphisms with femoral neck compression strength index variation in Caucasians Calcif Tissue Int “Duan P Wang ZM Liu J et a0al Gene polymorphisms in RANKLRANKOPG pathway are associated with ages at menarche and natural menopause in Chinese women BMC Womens Health Furuya T Hakoda M Ichikawa N et a0al Associations between HLADRB1 RANK RANKL OPG and IL17 genotypes and disease severity phenotypes in Japanese patients with early rheumatoid arthritis Clin Rheumatol “Gębura K Świerkot J Wysoczańska B et a0al Polymorphisms within genes involved in regulation of the NFκB pathway in patients with rheumatoid arthritis Int J Mol Sci Geiler J Buch M McDermott MF AntiTNF treatment in rheumatoid Arthritis Curr Pharm Des “GonzálezAlvaro I Ortiz AM Tomero EG et a0al Baseline serum RANKL levels may serve to predict remission in rheumatoid arthritis patients treated with TNF antagonists Ann Rheum Dis “Iwaszko M Świerkot J Kolossa K et a0al Influence of NKG2D genetic variants on response to antiTNF agents in patients with rheumatoid arthritis Genes Johansson L Ärlestig L Kokkonen H et a0al An increased concentration of receptor activator of nuclear factor kappaB ligand predates the onset of rheumatoid arthritis Rheumatology “Kitaura H Kimura K Ishida M et a0al Immunological reaction in TNFαmediated osteoclast formation and bone resorption in a0vitro and in a0vivo Clin Dev Immunol Kurz K Herold M Russe E et a0al Effects of antitumor necrosis factor therapy on osteoprotegerin neopterin and sRANKL concentrations in patients with rheumatoid arthritis Dis Mark https doiorg101155201527696 Leibbrandt A Penninger JM RANKRANKL regulators of immune responses and bone physiology Ann N Y Acad Sci “Liu W Zhang X Receptor activator of nuclear factorκB ligand RANKLRANKosteoprotegerin system in bone and other tissues Review Mol Med Rep “Lu Y Liu P Recker RR et a0al TNFRSF11A and TNFSF11 are associated with age at menarche and natural menopause in white women Menopause “Archivum Immunologiae et Therapiae Experimentalis Meednu N Zhang H Owen T et a0al Production of RANKL by memory B cells a link between B cells and bone erosion in rheumatoid arthritis Arthritis Rheumatol “Mohamed RH Mohamed RH ElShahawy EE Relationship between RANK and RANKL gene polymorphisms with osteoporosis in rheumatoid arthritis patients Genet Test Mol Biomark “Nemeth K Schoppet M AlFakhri N et a0al The role of osteoclastassociated receptor in osteoimmunology J Immunol “Ono T Nakashima T Recent advances in osteoclast biology Histochem Cell Biol “Pan R Liu YZ Deng HW et a0al Association analyses suggest the effects of RANK and RANKL on age at menarche in Chinese women Climacteric “Pathak JL Bravenboer N Verschueren P et a0al Inflammatory factors in the circulation of patients with active rheumatoid arthritis stimulate osteoclastogenesis via endogenous cytokine production by osteoblasts Osteoporos Int “RuyssenWitrand A Degboé Y Cantagrel A et a0al Association between RANK RANKL and OPG polymorphisms with ACPA and erosions in rheumatoid arthritis results from a metaanalysis involving three French cohorts RMD Open 2e000226Schett G Effects of inflammatory and antiinflammatory cytokines on the bone Eur J Clin Invest “Schett G Ha

Thyroid_Cancer

"Early detection of capecitabineresistance could largely increase overall survival of colorectal cancerCRC patients Previous studies suggested examination of immune cells in peripheral blood would help to predictefficacy of chemotherapyMethods We examined the immunological characteristics of peripheral blood in CRC patients with capecitabinetreatment We analyzed the relationships between the abnormal immune cell population in capecitabineresistancepatients and major clinical features Furthermore RNA sequencing analyses of cell surface marker expression andthe correlations with other major immune cell populations were performed using this population to explore thepossible function of these cellsResults The expression level of CD16 on neutrophils was downregulated in capecitabineresistant CRC patientsPatients with CD16lowˆ’neutrophils after capecitabine therapy had adverse clinical features What™s important thechange of CD16 expression level on neutrophils appeared much earlier than CT scan RNA sequencing revealedthat CD16lowˆ’neutrophils in capecitabineresistant patients had lower expression level of neutrophilrelated genescompared to CD16neutrophils in capecitabinesensitive patients suggesting this CD16lowˆ’population might beimmature neutrophils Furthermore the expression level of CD16 on neutrophils in patients with capecitabinetreatment was positively correlated with the number of antitumor immune cell subsets such as CD8T cell CD4Tcell NK cell and monocyteConclusions Our findings indicated that CD16 expression on neutrophils in peripheral blood was a goodprognostic marker for predicting efficacy of capecitabine in CRC patientsKeywords CD16 Neutrophils Capecitabineresistance Colorectal cancer Correspondence drzhongming1966163com gaoweiqiangsjtueducnyanzhsjtueducnYu Lu Yizhou Huang and Lei Huang share first authorship2Department of Gastrointestinal Surgery Renji Hospital School of MedicineShanghai Jiaotong University Shanghai China1State Key Laboratory of Oncogenes and Related Genes RenjiMed X StemCell Research Center Renji Hospital School of Medicine Shanghai JiaotongUniversity Shanghai ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLu BMC Immunology Page of BackgroundColorectal cancer CRC is one of the leading cause ofdeath worldwide More than million patients are diagnosed with CRC every year [“] What™s more this lifethreaten disease kills nearly million people annually []In north America and Europe the morbidity and mortalityremain at high level [] despite developments of cancerscreening and endoscopy [ ] In China CRC becomesthe 5th most diagnosed cancer and 5th most deadly cancer[“] Nearly million new cases are diagnosed andabout million people die from the disease every year []Postoperative adjuvant chemotherapy is firstline treatment for CRC patients [ ] Capecitabine a carbamatederivative of fluoropyrimidine is the backbone of CRCchemotherapy [ ] Asthe oral prodrug of fluorouracil 5FU it is widely used for postoperative adjuvant chemotherapy due to its long stable durationlower toxicity and convenient dosing compared to infusional 5FU [ ] However this chemotherapeutic drughas only modest efficacy the response rates of 5FU foradvanced CRC is only for single treatment and for combined chemotherapy [ ] The chemoresistance is recognized as a principal obstacle for cancer therapy [“] leading to tumor recurrence or metastasisespecially liver and lung metastasis and cause over ofCRC mortality [] Intense researches on the mechanisms underlying the resistance revealed that changes oftumor cells themselves cause resistance although thesefindings are mainly restricted to tumor specimen examinewhich is not that suitable for posttreatment surveillanceWhat™s more CT computed tomography scan and colonoscopy are insensitive to micro metastasis despite theirgoodrecurrenceCapecitabineresistant patients could only be diagnosedwith cancer recurrence by CT scan or colonoscopy about“ years after capecitabine therapy [] when tumorsare big enough to be discovered Thus good prognosticmarkers are indispensable for predicting capecitabineresistance in the early stage after capecitabine therapydetection ofaccuracytheforCancer cells and their microenvironment could interactwith each other Immune cells could dynamically reflectcancer status and display multifaceted functions in cancerdevelopment [“] Myeloid cells including monocytesmacrophages granulocytes neutrophils eosinophils basophils and mast cells play critical roles in cancer progression [“] Myeloidderived suppressor cells MDSCs aheterogeneous population of myeloid cells remain at different stages of differentiation are immature counterparts ofmyeloid cells in cancer MDSCs acquire immunosuppressive features and mainly inhibit lymphocytes including Tcells and NK cells [“] Recent studies report that chemotherapeutic agents like 5FU could interact with myeloid cells and influence antitumor efficacy [“]Vincent J reported that 5FU selectively inducedMDSC apoptotic cell death and increase IFNÎ productionby tumorspecific CD8T cells [] Other researchersshowed that activation of NLRP3 inflammasome and increased amount of HSP70 exosomes on MDSC by 5FUlead to MDSC activation [ ] Yuan Y found thattumorassociated macrophages secret IL6 to induce 5FUchemoresistance []ImportantlyIn this study we discovered that the expression ofCD16 on CD11bmyeloid cells was dramatically decreased in capecitabineresistant CRC patients after capecitabine adjuvanttherapy The expression level ofCD16 was closely related to poor prognosis after capecitabine therapythe downregulation ofCD16 on CD11bmyeloid cells appeared as early as month after capecitabine therapy in patients who werediagnosed with capecitabineresistance by CT scansabout “ years after the treatment The cutoff value ofCD16 expression would be helpful for the prediction of capecitabine chemoresistance Further analysisdemonstrated that these CD11bCD16lowˆ’myeloid cellswere mainly immature neutrophils and expression levelof CD16 on neutrophils had a positive relationship withfrequencies of antitumor immune cell populations suchas CD8T cells and NK cellsResultsCD16 expression levels on CD11bmyeloid cells inperipheral blood of capecitabineresistant CRC patientsare different from capecitabinesensitive CRC patientsafter capecitabine therapyTo explore if myeloid cells in peripheral blood could predict the treatment efficacy of capecitabine we chose CRC patients with capecitabine adjuvant treatment whoseimmune cells populations in peripheral blood were examined by flow cytometry before and about “ months afterthe treatment Patients were divided into capecitabinesensitive and capecitabineresistant groups based on thediagnosis of recurrence by CT scan in about “ years aftercapecitabine treatment Table Additional file Fig S1ENo significant change was observed in major myeloid cellsubsets such as monocytes CD11bCD14CD15ˆ’ neutrophils CD11bCD15CD14ˆ’ or CD11bCD66bCD14ˆ’and MDSCsbetweencapecitabinesensitive patients and capecitabineresistantpatients Additional file S1A B C and D But we foundthat the frequency of CD11bCD16myeloid cells was decreased in capecitabineresistant patients after capecitabinetreatment compared to that before the treatment Fig 1aWhat™s important a dramatic lower expression level ofCD16incapecitabineresistant patients compared to that of drugsensitive patients Patient and patient are representative patientsgroup andcapecitabineresistant group respectively Fig 1b TheCD11bHLADR\\lowCD33from capecitabinesensitiveon CD11bmyeloidcells wasobserved 0cLu BMC Immunology Page of Table Baseline characteristics of CRC patients in Fig GroupNumber of PatientsAgeSexTNM StageLocationCEA ngmlCA199 ngml Diagnosis of Recurrence AfterCapecitabinesensitiveCapecitabineresistantMMMMMFFFMFMFMMMFMMFFFMFMFMMMMFMMMFFMIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIRectumRectumColonColonRectumRectumRectumColonRectumColonColonColonRectumColonRectumRectumRectumRectumColonColonRectumRectumRectumRectumColonRectumRectumColonColonRectumRectumRectumRectumRectumRectumColonCapecitabine TreatmentNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoYesYesYesYesYesYesYesYesYesYesdiagnosis of capecitabine resistance was determined by CTscan Additional file Fig S1E However when we analyzed these CD11bCD16myeloid cells in healthy donorsHDs and CRC patients before capecitabine therapy wefound no difference between these two cohorts Additionalfile Fig S1F and G This indicated that change of CD16expression on CD11bCD16myeloid cells was particular inCRC patients who were resistant to capecitabine therapyDecreased CD16 expression is correlated with poorpathological features in CRC patients after capecitabinetherapyTo determine whether the expression level of CD16 onCD11b myeloid cells is related to treatment efficacy of capecitabine we collected peripheral venous blood of CRCpatients “ months after capecitabine treatment and divided these patients into two groups CD16 group and 0cLu BMC Immunology Page of Fig CD16 expression of peripheral blood myeloid cells were differential in CRC patients after capecitabine therapy Peripheral venous bloodfrom CRC patients received singleagent oral capecitabine adjuvant therapy was collected before the therapy and “ months after the therapyand analyzed for myeloid cellrelated markers Attention Blood were collected “ months after capecitabine treatment unless particularlynoted a Frequencies of CD11bCD16myeloid cells were compared before and after capecitabine therapy in capecitabinesensitive andcapecitabineresistant patients n in sensitive group and n in resistant group respectively b Representative images of CD16 expressionon CD11bmyeloid cells before and after capecitabine therapy in two CRC patients from capecitabinesensitive group or capecitabine resistantgroup respectively Diagnosis of drugresistance was proved by CT scan during the followup in Fig S1e Mean ± SEM P005 by t tests aCD16lowˆ’ group Firstly Kmean clustering algorithm wasused to determineto divideCD11bCD16myeloid cells into CD11bCD16highcells andthe boundaryvalueCD11bCD16lowcells based on mean fluorescent intensityMFI of CD16 on CD11bCD16myeloid cells in peripheralblood after capecitabine therapy Additional file Fig S2A 0cLu BMC Immunology Page of ROCanalysisThe boundary value of CD16 MFIfor division ofCD11bCD16high cells and CD11bCD16low cells was × Next we analyzed frequency of CD11bCD16high cellsin peripheral blood after capecitabine therapy Additional file Fig S2B and determined the cutoff value for CD16 expression on CD11bmyeloid cells by receiver operating characteristicand Youden Index valuesAdditional file Fig S2C and S2D The cutoff value was Patients of CD16 group or CD16low group were determined if their frequencies of CD11bCD16highcells werehigher or lower than the cutoff value Additional file FigS2B S2C and S2D Then we assessed correlations betweenthe expression level of CD16 and CRC clinicopathologicalcharacteristics by χ2 test The data revealed that patients inCD16lowˆ’ group had more cancer recurrence P and high level of carcinoembryonic antigen CEA P as well as carbohydrate antigen CA199 P compared to patients in CD16 group Table There were CRC patients developing recurrenttumor in CD16lowˆ’ group whereas only cases were observed in CD16 group Among CRC patientswith high CEA level patients belonged toCD16lowˆ’ group while only patients wereCD16 And patients with high CA199level were found in CD16lowˆ’ group compared with cases in that of CD16 However no significant difference was observed between these twogroups on age gendertumor sizeand Tumor Node Metastasis TNM stage Table tumor locationTo further confirm these results we divided CRCpatients after capecitabine treatment into two groupsbased on the level of CEA or CA199 and compared theexpression level of CD16 on CD11bCD16myeloid cellsbetween CEAhigh CEA ng and CEAlow CEA ‰¤ Table Relationship between CD16 expression on CD11bmyeloid cells after capecitabine therapy and clinicopathologiccharacteristicsCharacteristicsCD16lowˆ’ after therapy n nAll patients n nCD16 after therapy n nAge years‰¥GenderMaleFemaleTumor locationRectumColonTumor Size‰¥ cm cmCEA level after therapy‰¤ ngml ngmlCA199 level after therapy‰¤ ngml ngmlTNM stage AJCCStage IIStage IIILocation of recurrenceLocoregionalliver lungliverlungperitoneumPvalue 0cLu BMC Immunology Page of ng groups or between CA199high CA199 ngand CA199low CA199 ‰¤ ng groups The boundaryvalue of CEA and CA199 were decided by clinical guidelines The results showed that the expression level ofCD16 was dramatically decreased in either CEAhigh orCA199high groups compared to CEAlow or CA199low groups Fig 2a and b suggesting that the decreasedexpression level of CD16 on CD11bmyeloid cells aftercapecitabine treatment was related to the poor pathological features In conclusion low level of CD16 expression was related to poor pathological features such astumor recurrence CEA and CA199in CRC patientswith capecitabine therapyCD16 serves as a prognostic marker for CRC patientsreceived capecitabine adjuvant chemotherapyTo further explore the prognostic significance of CD16expression on CD11bmyeloid cells in predicting thetreatment efficacy of capecitabine chemotherapy wecompared the differences of overall survival OS anddisease free survival DFS between CD16 group andCD16lowˆ’ group The survival curves revealed that therewere significant association between the expression levelof CD16 and OS P 00006Fig 3a or DFS P 00023Fig 3b suggesting that low expression level ofCD16 was associated with shorter survival Next weused univariate analysis to further elucidate the significance of CD16 expression in predicting prognosis ofCRC patients receiving capecitabine The result demonP HR strated that CD16 expression level was prognostic factor for OS Table What™simportant Cox multivariate analysis also demonstratedthat expression level of CD16 P HR wasindependent predictors of OS Table Thesestillresults demonstrated that the expression level of CD16on CD11bmyeloid cells may serve as a good prognosticmarker for overall survival in CRC patients with capecitabine adjuvant chemotherapy[] Next we wondered ifDownregulation of CD16 expression on CD11bmyeloidcells appears earlier than diagnosis of capecitabine byimaging testsAs we know adjuvant chemotherapy remains the firstline therapy for CRC patients Capecitabine the oralprodrug of 5fluorouracil is one of the primary drugsfor the treatment A number of CRC patients becomeinsensitive to the therapy and suffer from cancer recurrence In clinic capecitabineresistance is mainlydiagnosed by cancer recurrence discovered throughcolonoscopy or CT scan in about “ years after capecitabine treatmentthechange of CD16 expression level on CD11bmyeloidcells appeared earlier than CTshowed recurrence Weselected CRC patients with capecitabine treatmentwhose blood samples were examined before and aftercapecitabine treatment Table The results showedin patients in capecitabineresistant groupthefrequency of CD11bCD16myeloid cells was decreased “ months after treatment compared to thatbeforecapecitabineresistance was diagnosed by CT scan about yearsafter the treatmentfile Fig S1E What™s important in a resistant patient decreased expression level of CD16 was found as earlyas month after capecitabine treatment Fig 4a Thefrequency of CD11bCD16high cell population waslargely lower than the cutoff value Neverthelesstumor monthsTable and Additional1a whiletreatmentFigafterthecapecitabinetherapyFig CD16 expression of CD11bCD16myeloid cells related to pathological features of CRC patients with capecitabine therapy CRC patientsreceiving capecitabine therapy were divided into different groups according to their CEA or CA199 level n in CEAhigh CEA ng groupand n in CEAlow CEA ‰¤ ng group n in CA199high CA199 ng group and n in CA199low CA199 ‰¤ ng group CD16MFI of CD11bCD16myeloid cells in CRC patients acquired from flow cytometry analysis was compared between different groups Mean ± SEMP001 P0001 by t tests a b 0cLu BMC Immunology Page of Fig CD16 high expression on CD11bmyeloid cells was good prognostic marker for CRC patients™ survival KaplanMeier analysis of overallsurvival OS and disease free survival DFS was performed in CD16 group and CD16lowˆ’ group p values were calculated by logrank test n in CD16 group and n in CD16lowˆ’ grouprecurrence was found in the liver from CT scan Fig 4bThese data suggested that downregulation of CD16on CD11bmyeloid cells served as a more sensitiveexamine than CT in CRC patientstreated withcapecitabineCD11bCD16lowˆ’myeloid cells are mainly immatureneutrophils after capecitabine therapyTo further characterize the population of CD11bCD16lowˆ’myeloid cells we isolated CD11bCD16myeloid cells fromcapecitabinesensitive patients and CD11bCD16ˆ’myeloidcells from capecitabineresistant patients after capecitabinetherapy Fig 5a The data from flow cytometry revealed thatthese two populations were mainly neutrophils provedby their CD15 and CD66b expression Additional file Fig S3A To further verify these CD11bCD16ˆ’myeloid cells and CD11bCD16myeloid cells were bothneutrophils we sorted these cells from capecitabineresistant patients and capecitabinesensitive patientsrespectively Characteristics ofthese patients werelisted in Additionalfile Table S1 We comparedour data of RNA sequencing with published data ofneutrophils from Jiang K [] using gene set enrichment analysis GSEA The results revealed thatin gene sets of neutrophil signature the expressionpattern of these cells was similar to that of the neutrophils provided by other group Additionalfile Fig S3B Additionalfile Table S2 Neverthelessthe decline of CD15 and CD66b expression combinewith the elevation of hematopoietic progenitorrelatedmarkers especially CD33 and CD117 suggested thatthese CD11bCD16ˆ’myeloid cellsin capecitabineresistant patients became more immature after thetherapy compared with CD11bCD16myeloid cells fromcapecitabinesensitive patients Fig 5b The data of RNA sesomequencing also revealed declined expression ofTable Univariate and multivariate analyses for survival in CRC patients after capecitabine therapyPrognosticparameterUnivariate analysisHRCD16 expressionGenderAgeTumor locationTumor sizeCEACA199TNMRecurrenceHR Hazard ratio CI Confident interval95CI“““““““““p valueMultivariate analysisHR“95CI““““““““““““““p value““““““ 0cLu BMC Immunology Page of Fig Analysis of CD16 expression was more sensitive than CT scan after capecitabine therapy a Peripheral venous blood from CRC patientsreceiving singleagent oral capecitabine adjuvant therapy was collected at different time before capecitabine therapy month and years afterthe therapy Frequencies of CD11bCD16highmyeloid cells were analyzed by flow cytometry b CT scan was performed during followup afteradjuvant chemotherapy in same patients as that of a respectively Sensitive patient normal operation site with no recurrence Resistant patientresectable metachronous liver metastases red arrowsand ATP wereneutrophilrelated genes in CD11bCD16ˆ’myeloid cells fromcapecitabineresistant patients after capecitabine therapywhich implied immature status of these neutrophils Fig 5cIn addition active metabolism of nitrogen species purinenucleosidetheseCD11bCD16ˆ’myeloid cells which are tightly related toimmunosuppressive role of MDSC [ ] Fig 5d To verify the immunosuppressive role of these CD11bCD16ˆ’myeloid cells we sorted peripheral blood CD11bCD16ˆ’myeloidcellsandCD11bCD16myeloid cellsfrom capecitabinesensitiveCRC patients or HDs and autologous T cells as well Aftercoculture T cells with these myeloid cells in the presence offrom capecitabineresistant CRC patientsinalsofoundleukocyte activators proliferation of T cell was significantlydeclined in resistant CRC patients group compared withsingle T cell group HD group and sensitive CRC patientsgroup Fig 5e ThetheseCD11bCD16ˆ’myeloid cells in capecitabineresistant patientsmight exert immature cell status and play immunosuppressive role like MDSCsuggested thatresultsThe low expression level of CD16 on neutrophils isrelated to protumor status in CRC patients aftercapecitabine therapyAs we know immature myeloid cells are usually MDSCswhich could exert powerfulimmunosuppressive role 0cLu BMC Immunology Page of Fig CD11bCD16myeloid cells became immature neutrophils after therapy in capecitabineresistant patients a Peripheral venous blood fromcapecitabineresistant and capecitabinesensitive CRC patients was collected after the treatment in “ months CD11bCD16myeloid cells insensitive patients and that of CD11bCD16ˆ’ in resistant patients were sorted for further analysis in b c and d b Expression of myeloidassociated and hematopoietic progenitorassociated markers on CD11bCD16myeloid cells in sensitive patients and on CD11bCD16ˆ’myeloidcells in resistant patients was analyzed by flow cytometry c Peripheral blood CD11bCD16myeloid cells in sensitive patients andCD11bCD16ˆ’myeloid cells in resistant patients were sorted and analyzed by RNA sequencing Expression of neutrophilrelated and monocyterelated genes derived from the results of RNA sequencing was shown in the heatmap d GO enrichment terms of differentially expressed MDSCrelated immunosuppressive biological processes derived from RNA sequencing e Autologous T cells were cultured alone cocultured withperipheral blood CD11bCD16myeloid cells from HDs and sensitive CRC patients or CD11bCD16ˆ’myeloid cells from resistant CRC patientsfor h respectively Proliferation of T cells were analyzed by flow cytometry after incubation n for each group CD16N HD CD11bCD16myeloid cells from HDs CD16N CRC S CD11bCD16myeloid cells from sensitive CRC patients CD16ˆ’N CRC R CD11bCD16ˆ’myeloid cells from resistant CRC patients Mean ± SEM P005 P001 by t tests epatientscapecitabinesensitiveespecially in inhibiting T cells and NK cells [ ]As our results showed that CD11bCD16myeloid cellsfromandCD11bCD16ˆ’myeloid cells from capecitabineresistantpatients were mainly neutrophils we tried to find out therelationship between the expression level of CD16 on neutrophils and other major immune cell subsets We collected peripheral venous blood from colorectal cancerpatients “ months after capecitabine therapy and analyzed frequencies of immune cells by flow cytometry Therelationships between expression level of CD16 on neutrophils and frequencies ofimmune cell subsets wereanalyzed by Pearson™s correlation test The results showedthat CD16 expression was positively related to CD8T cellCD4T cell monocyte and NK cell frequencies Fig 6a bc and d but not that of cDC and pDC in patients aftercapecitabine therapy Fig 6e and f suggesting thatCD16lowˆ’neutrophils might have immunosuppressive activity as MDSCsDiscussionOver the past few decades numerous researchers haveattempted to improve the efficacy of capecitabine adjuvant therapy to ameliorate prognosis of CRC patients 0cLu BMC Immunology Page of Fig CD16 low expression on neutrophils predicted protumor immune status in CRC patients with capecitabine therapy Peripheral venousblood from CRC patients received singleagent oral capecitabine adjuvant therapy was collected “ months after the therapy and analyzed fordifferent immune cell subsets by flow cytometry CD16 MFI of peripheral blood neutrophils was calculated by flow cytometry analysis and thecorrelations between CD16 MFI of neutrophils and frequencies of CD8 T cells a CD4 T cells b monocytes c NK cells d cDCs e and pDCsf among total peripheral blood leukocytes were analyzed by Pearson™s correlation testHoweverit remains one of the principal obstacle forcancer therapy at present In this study we demonstrated that the expression level of CD16 was downregulated in capecitabineresistant patients and lower expression level of CD16 on neutrophils in peripheralblood was correlated with poor prognosis in CRC patients with capecitabine adjuvant therapy Importantlydownregulation of CD16 was observed as early as month after capecitabine treatment which was moresensitive than CT scan indicating its great value in clinical application We determined the cutoff value ofCD16 expression on neutrophils for the prediction of capecitabine chemoresistance which would behelpful for clinical application and further researchesAnalyzationincapecitabineresistant patients revealed their immaturestatus and the expression of CD16 on neutrophils waspositively correlated with frequencies of antitumor immune cell populationsCD16lowˆ’neutrophilstheseofrecurrence which is vitalTo this day coloscopy and CT scan are still themain examines to supervise CRC progression and discoverfor capecitabineresistance diagnosis Unfortunately these two methodscould only provide evidence untiltumors are bigenough to be discovered patients won™t have enoughtime to adjustthe treatment CEA and CA199 arewidely used to CRC surveillance as well especiallyCEA [] However CEA and CA199 cannot predictcancer progression so precisely and the false positivelead to anxiety and excessiveor negative results willtherapy What™s more some clinicaltrial also suggested that combining CEA and CT got no advantagecompared with single examine [] In this study ourresults showed that CD16 expression could serve as agood prognostic marker for poor CRC progressionafter capecitabine therapy Analyzation of CD16 expression hasthe downregulation of CD16 expression on neutrophils couldbe observed atcapecitabineresistance after the treatment Fig Previous studieshave demonstrated that CRC patients had primary resistance to 5FU single treatment[ ]thus the marker is essential for the drugselection inthese patients Second this marker is quite accuratefor predicting capecitabineresistance after the therapy In our study we collected totally CRC patients with capecitabinetheexpression level of CD16 on neutrophils Among patients who werecapecitabineresistance patients were observed to have downadvantages Firstto examinediagnosedtherapyasgreattheearlystage of 0cLu BMC Immunology Page of regulation of CD16 in “ months after capecitabinetreatment Table Third the examination of CD16expression only takes about ml peripheral bloodand it is noninvasive and has nearly no effect on patients™ healthCapecitabine the oral form of 5FU which is widelyused in CRC therapy has only modest efficacy due tothe chemoresistance Great efforts have been taken tofind out the mechanism Previous studies mainly concentrated on tumor cells themselves such as expressionof specific genes or generation of particular tumor cells[ ] In this research we worked on the correlationbetween changes on immune system and capecitabinechemoresistance and illustrated the conversion fromneutrophilsto immunosuppressive PMNMDSClikeneutrophils in these capecitabine insensitive patients byRNA sequencing and flow cytometry Our conclusioncould also be supported by other studies that 5FUcould promote MDSC protumor function The study byBruchard M found that 5FU could activate NLRP3inflammasome in MDSC and promote tumor growth[] Gobbo J also discovered that 5FU facilitatedproduction of tumorderived HSP70 exosomes whichfavored MDSC activation [] Thus prevention ofMDSC function after capecitabine or 5FU therapyholds great promise for improving drug efficacyreceptorResearchers have revealed that CD16myeloid cellswere tightly related to CRC development[ ]Giulio S found that CD16myeloid cell infiltration in CRC tumor tissue represented favorable prognosis [] and by using in vitro studies these studiesalso demonstrated that colon cancer infiltrate neutrophils enhance the responsiveness of CD8 T cells byTcelltriggering [] Our work differedfrom theirs in some ways Firstly our study focusedon CRC patients who received capecitabine adjuvanttreatment after surgery while Giulio Spagnoli groupfocused on all CRC patients and some healthy donorsSecondly biopsies from different positions were analyzed Peripheral blood was used in our study whileGiulio Spagnoli group mainly focused on tumor biopsies Exceptthese differences some of our resultswere also consistent with studies from Giulio Spagnoligroup Firstly both our data and Giulio Spagnoligroup™s data found that phenotype of peripheral bloodCD11bCD16myeloid cells had no difference betweenhealthy donors and CRC patients without capecitabinetherapy Fig S1F and G Secondly our work indicated that CD16 highpositive expression after capecitabine therapy predicted sensitivity to the therapyand good prognosis These results were consistentwith the work from Giulio Spagnoli groupthatCD16myeloid cells related to good prognosis of CRCpatientsMDSCs are a heterogeneous population of myeloidcells stay at different stages of differentiation PMNMDSCs are a great part of MDSCs that could be considered as counterparts of immature granulocytes chieflyimmature neutrophils []In this study we founddownregulation of CD16 expression on myeloid cells incapecitabineinsensitive CRC patients after capecitabinetreatment These CD16lowˆ’myeloid cells after the therapy were mainly immature neutrophils CD16 is a lowaffinity FcÎ receptor which could activate antibodydependent process like phagocytosis in neutrophils andother phagocytes [] It is expressed on neutrophilsduring the maturation Researchers also revealed thatCD16 is typically associated with PMN activation andphagocytosis and its expression will change in differentmaturation status [ ] MDSCs could exert protumor roles mainly through inhibition of effective Tcells and NK cells [ ] Our study demonstrated thatlow expression of CD16 on neutrophils after the therapywas related to decreased frequencies of antitumor immune cells like CD8T cells and NK cells suggestingthatthey may have immunosuppressive activity asMDSCs The mechanism underlying the changes induced by capecitabine would be investigated further andit could be a good target to compete against capecitabinechemoresistanceConclusionsIn conclusion CD16 seems to be a promising target forCRC progression surveillance after capecitabine therapyStudies of CD16 expression on neutrophils may light thepath for not only predicting prognosis but also solvingcapecitabine resistance in CRC patientsMethodsPatients and peripheral bloodPeripheral venous blood of CRC patients in Departmentof Gastrointestinal Surgery Renji Hospital ShanghaiChina from January to December was gottenbefore capecitabine adjuvant treatment and at differenttime after the treatment as indicated in figure legendPeripheral venous blood of healthy donors was gotten inRenji Hospital The pathological information of patients was retrieved from the Pathology Department ofRenji Hospital These peripheral blood was used for flowcytometric analysis All the patients were provided withwritten informed consent before enrolment and thestudy was approved by the Research Ethics Committeeof Shanghai Jiao Tong University School of MedicineRenji Hospital Approval No Renji [] N013 Noneof patients had received radiotherapy or chemotherapybefore surgery All patients were followedup until deathor until the final followup May 0cLu e

Thyroid_Cancer

Gastric cancer GC persists as a worldwide public health crisis According to the American Cancer Society the 5yearsurvival rate of GC remains at worldwide and withinthe United States1 These survival statistics have increasedoverall since the 1980s when the 5year survival rate for stageII disease was below and near for stage IIIB andhigher1 With the development of chemotherapies such as platinums and taxanes survival beyond stage II increased steadilyto Although chemotherapies improved overall survivalthis is not as dramatic as that in other solid malignancies suchas prostate or breast Furthermore even with the identificationof molecular targets such as BRCA mutations and HER2amplifications clinical success with available therapies hasbeen minimal23 A recent clinical trial with olaparib a polyADP ribose polymerase inhibitor showed little efficacy compared to standard of care4 Although a subset of gastric diseasehas HER2 amplification monoclonal antibodies against HER2have demonstrated very limited success in GC unlike theresponse seen in HER2 positive breast cancer5 It is clear that Department of Oncology Wayne State University School of MedicineDetroit MI USACorresponding AuthorAsfar S Azmi PhD Department of Oncology Wayne State University Schoolof Medicine Karmanos Cancer Institute John R HWCRC DetroitMI USAEmail azmiakarmanosCreative Commons CC BY This is distributed under the terms of the Creative Commons Attribution License creativecommonslicensesby40which permits any use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cTechnology in Cancer Research Treatmentmore work is needed to elucidate the underlying moleculardrivers and resistance mechanisms in GCGastric cancer is classified mainly using either the Laurenclassification or the World Health anization WHO criteria The Lauren classification compares tumors based ongrowth invasion pattern with subtypes intestinal well differentiated diffuse poorly differentiated and intermediatemixed67 The majority of patients outside US with GC areyounger 60years old and have the poorly differentiated diffuse subtype which is located within the distal portion of thestomach characterized by poor cellular differentiation and highintratumor heterogeneity This subtype has poorer outcomesdue to its widespread infiltration and invasive nature of thedisease78 Conversely within the United States the pathologyof GC is similar to that of malignancies found within the gastroesophageal junction8 Older patients are primarily impactedand the disease is commonly well differentiated intestinalThe welldifferentiated subtype is found in the cardia or lowerregion of the stomach with welldefined glandular structuresand growth pattern The WHO designation for GC was createdin and expands vastly on the Lauren classification67There are subtypes tubular adenocarcinoma papillary adenocarcinoma mucinous adenocarcinoma poorly cohesiveSignet ring cell carcinoma and mixed carcinoma67 Similarities exist between the Lauren and WHO classificationsSignetring cell carcinoma comparable to poorly differentiatedGC is steadily increasing in incidence within the United Statesand around the world9 This increase is attributed to eradication efforts of Helicobacter pylori a pathogen known toinduce intestinal type GC increases in genetic predisposition to genes such as Ecadherin CDH1 hypermethylationand less screening and detection due to the œlow riskpopulation within the United States compared to other regionssuch as Japan10Here we aim to analyze the molecular signatures as well asdifferences between Lauren classified GCs We also aim tounderstand the molecular differences between male and femalepatients with GC We chose to look solely at Lauren classifiedcancers within this due to its established use within themedical community as well as its availability and relevancewithin publicly available data sets Our overarching goal is toidentify and dissect some of the heterogeneous aspects of GCthat are commonly overlooked within the literatureMethodsOncomine Database SearchOncomine Compendia Bioscience was used for analysis andvisualization Three separate data sets were used to explore theup and downregulation of Lauren subtypes of GC Chen Gastric Mol Biol Cell mRNA DErrico Gastric EuropeanJournal Dataset2 mRNA and Cho Gastric ClinicalCancer Research mRNA For the nonsubtyped GC analysis we have used separate data sets Cui Gastric NucleicAcids Research mRNA Wang Gastric MedicalOncology mRNA and Cho Gastric Clinical CancerResearch mRNA To find highly ranked genes weselected our subtype of interest or GC compared to normaland assessed upregulated or downregulated genes We averaged the fold changes for genes in the individual analyses andhave used the computed P values provided by the OncominesoftwareKyoto Encyclopedia of Genes and Genomes PathwayAnalysisTo identify pathways involved in the genes found to be upregulated or downregulated from our Oncomine analysis weutilized the Kyoto Encyclopedia of Genes and GenomesMiRWalk Database AnalysisMiRWalk Database University of Heidelberg was used foranalysis of gene“microRNAs miRNA interactions11Drug“Gene Interaction AnalysisDGIdb database was used to identify druggable targets withinour genes found to be differentially expressed12Protein DatabaseThe Human Protein Atlas available from httpwwwproteinatlas was used to identify survival curves in stomach cancerwith the following proteins CWD43 Stage IIV Survivalcurves wwwproteinatlasENSG00000109182CWH43pathologystomach¾cancer METLL7A Stage IIVwwwproteinatlasENSG00000185432METTL7Apathologystomach¾cancer SLC2A12 Stage IIV wwwproteinatlasENSG00000146411SLC2A12pathologystomach¾cancer MAL Stage IIV wwwproteinatlasENSG00000172005MALpathologystomach¾cancer DMRT1 Stage IIV wwwproteinatlasENSG00000137090DMRT1pathologystomach¾cancerAll are available from v19proteinatlasProtein“Protein Interaction NetworksSTRING Database was used to identify protein“proteininteractions for the following genes CWH43 METLL7ASLC2A12 MAL BTD CAPN9 ADAM17 EPB41 TOM1L1and DMRT113GEO Database AnalysisThe data discussed within this publication have been previously deposited in NCBI™s Gene Expression Omnibus andare accessible through GEO Series accession numberGSE118916 wwwncbinlmnihgovgeoqueryacccgiacc¼GSE118916 0cSexton et alTable Top Upregulated Genes Found in Gastric Cancer Cohort viaOncomine DatabaseaGene nameFold change diffusevs normal averagePublicationsP valuefoundINHBACOL1A2CLDN1CDH11COL3A1COL5A2COL1A1TIMP1SULF1SPON2549E7949E12664E6117E10241E6289E6299E6383E6465E6644E10aP values were calculated using Oncomine softwareStatisticsOncomine software and Human Protein Atlas providedStatisticsEthical ApprovalThe data are not obtained from patients and does not requireinstitutional review board approvalResultsGenetic Analysis of Upregulated GC GenesWithin the literature various genetic aberrations have beenproposed that can serve as prognostic or therapeutic markersincluding SOX17 hypermethylation BCL2 transforminggrowth factor beta TGFb vascular endothelial growth factorVEGFR and HER21418 Many of these proposed markersare studied extensively and do not serve as ideal targets dueto their limited clinical utility as either drug targets or predictors of therapeutic response Some examples of this include lesssuccessful attempts to target HER2 with monoclonal antibodiesand the use of TGFb inhibitors which although promisinghave proven to be highly toxic1920 Additionally these targetshave demonstrated limited clinical utility due to the crosstalkbetween TGFb and other signaling pathways such as RAS aknown nontargetable protein2122 While VEGF inhibitors areused as a therapeutic modality in GC they do not improveoverall survival23 An indepth investigation of the molecularmechanisms are urgently and investigations need to be distinctfrom the commonly studied and clinically intractable targetsAlthough this is the case discrepancies exist within the literature as some groups look at the molecular composition of GC asa whole while others focus on differences within the Laurenclassification systemUsing the Oncomine database we have found significantupregulation in several understudied genes in all GCs including COL3A1 COL5A2 SPON2 and CDH11 Table Wealso have confirmed the upregulated status of many of thegenes found within the literature that are somewhat well knownsuch as INHBA a gene associated with poor overall outcomes24 but are still understudied Claudin CLDN1 hasbeen found to be highly expressed in GC and is a poor predictive disease marker by mediating tumor necrosis factorainduced cell migration enhancement of proliferation andmetastasis while SULF1 has been found to be significantlyhypomethylated causing significant downregulated proteinexpression2528 This SULF1 downregulation may be indicativeof a posttranslational modification feedback loop or degradation event via protein“protein interactions but is still unclearNot surprisingly a significant underrepresentation was notedwhen comparing publications related to these genes over publications to the commonly studied genes such as MAPKPI3K and TP53 over total publicationsGenetic Analysis of Upregulated GC Genes Using LaurenType Classified GCsWe stratified the data sets based on the respective Lauren distinguished subtype and have highlighted the vast heterogeneticmolecular landscape within the poorly differentiated diffusewell differentiated intestinal and mixed GC subtypesTable Poorly differentiated GC shares many similaritieswith GC overall including perturbations in various collagentranscribing genes stimulation of PI3KAKT signaling andperturbations in cellular structural components This is a dominant subtype throughout the world for reasons we have previously mentioned Due to the overabundance of collagentranscribing genes we wanted to explore whether a potentialgenetic link exists Literature search identified a study correlating EhlersDanlos syndrome EDS a disease caused bycollagen gene perturbations to the development of GC29EhlersDanlos syndrome also presents with gastrointestinalinvolvement such as increased rates of heartburn which is arisk factor for developing esophageal cancer3031 Based on thelocation of these gastric tumors within the stomach that is inthe proximal stomach near the esophagus and the connectionbetween gastric and esophageal cancers it is quite possiblethere may be a much stronger correlation between EDS anddiffuse GC than previously thoughtWe have found GC overall does not share many molecularsimilarities with the welldifferentiated subtype of GC withinthe scope of our analysis We have found only a similarityCLDN1 expression Claudin is a gene involved in coding forthe protein involved in epithelial barrier functions and is part ofthe claudin family Within GC CLDN1 has found to be differentially expressed in GC and has been found to be upregulatedin a small patient population being linked to poor survival outcomes indicative of an oncogenic function32 Other groupshave found claudin1 has tumor suppressive activities and canreverse the epithelialtomesenchymal transition in GC cellsand was found to be downregulated in intestinal type GC in aof patients cohort3334 It is clear that work needs to be donein order to elucidate the role CLDN1 plays within intestinaltype gastric tumors as it has differing functions based on the 0cTechnology in Cancer Research TreatmentTable Top Significantly Upregulated Genes Based on Molecular Subtype of Gastric Cancer Well Differentiated Poorly DifferentiatedMixed Subtype Based on Oncomine DatabaseaFold change diffusevs normal averageP valueKEGG pathway analysisGastric cancersubtypeGene nameTHY1TIMP1BGNCOL1A2SULF1COL6A3OLFML2BRAB31THBS2COL1A1TTYH3THY1CADUBE2CCLDN1PRC1DAZAP1ATP11ADCAF13MTHFD1LCOL6A3FBN1RCC2AHCYTGIF1FN1MYO9BVCANLUMMCM4PI3KAKT focal adhesion ECM receptor proteoglycansPI3KAKT focal adhesion ECM receptor““Immune component161E12124E11 HIF signaling238E11223E10139E9 Metabolism585E9404E8361E9 Membrane trafficking118E8165E7232E23 Transporter346E21202E8Phagosome PI3KAKT focal adhesion ECM“receptor interactionPI3KAKT focal adhesion ECM receptor proteoglycansImmune componentPhenylpropanoid biosynthesis metabolic pathways biosynthesis ofsecondary metabolitesmRNA surveillance262E20 Ubiquitinmediated proteolysis650E15 Cell adhesion tight junction134E14 Tubulin binding protein680E8768E19 Metabolism translocase971E8893E9109E7191E7161E9213E6733E9943E9Ribosome biogenesisOne carbon metabolismPI3KAKT signaling focal adhesion ECM“receptor interactionTGFb signaling“Cysteine and methionine metabolismTGFb signalingPI3KAKT signaling focal adhesion ECM“receptor interactionregulation of actin cytoskeleton proteoglycans and pathways in cancer224E6 Membrane trafficking260E6380E6833E6Cell adhesion molecules CAMsProteoglycans in cancerDNA replication cell cycleDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalMixedMixedMixedMixedMixedMixedMixedMixedMixedMixedAbbreviations ECM extracellular matrix KEGG Kyoto Encyclopedia of Genes and Genomes TGFb transforming growth factor betaaP values were calculated via Oncomine software and KEGG pathway analysis was used to analyze gene functionliterature Many of the processes underlying intestinal GCinvolve alterations in metabolism and cellular crosstalkTable It is not surprising that the intestinal and diffuse GCsare distinctly different but we did find similarity with THY1expression both having similar fold changes Although thisgene has not been investigated in GC it is overexpressed inthe pancreatic cancer microenvironment35 Further investigation may be needed as this gene may have importance in GCdevelopmentWe finally investigated the mixed subtype of GC a subtypethat is commonly overlooked within the literature Table Interestingly mixed GC has some similarities to the diffusesubtype including PI3KAKT signaling a collagen transcribinggene and upregulation of cellular anizational componentsInterestingly we have found the genes perturbed within thissubtype are involved in driving a number of genetic diseasessuch as Marfan syndrome FBN1 and hypermethioninemiaAHCY Research has shown Marfan syndrome due to aberrant TGFb signaling can induce GC development in a murinemodel36 Hypermethioninemia which can go undetected foryears was found to induce aggressive cancers by protectingtumors from 5flurouracil 5FUinduced death a chemotherapy commonly used to treat GC3738 It is likely the diffusesubtype is not the only subtype with a strong genetic link butthe mixed subtype may have a stronger genetic component thanpreviously thought We hypothesize some of the genetic diversity within GC is masked when analyzed as a whole whichfurther supports the notion of this disease being highlyheterogeneousGenetic Analysis of Downregulated GC GenesThere are about twice as many published studies looking atupregulated GC genes compared to downregulated vs The most common downregulated GC genes are influenced in part by aberrant DNA methylation3940 Other thanthis much less is studied pertaining to highly significant downregulated genes in GC Using the Oncomine database we have 0cSexton et alTable Top Significantly Downregulated Genes According to Oncomine Database in Gastric CanceraGene nameLIFRCWH43RDH12MFSD4METTL7AATP4BSLC2A12GHRLMALADH7Fold change diffusevs normal averageP valueKEGG pathway analysis 251E6Cytokine“cytokine receptor interaction signaling for pluripotency in stem cells279E9136E8220E5227E5165E10365E10617E8119E9947E8JAKSTAT signaling“Retinol metabolism metabolic pathways““Oxidative phosphorylation metabolic pathways gastric acid secretionTransportercAMP signaling neuroactive ligand“receptor interaction growth hormonesynthesis secretion and action“Glycolysisgluconeogenesis fatty acid degradation tyrosine metabolism retinolmetabolism chemical carcinogenesisAbbreviation KEGG Kyoto Encyclopedia of Genes and GenomesaP values were calculated via Oncomine software and KEGG pathway analysis was used to analyze gene functionfound the most significant downregulated genes were LIFRRDH12 MSFD4 ATP4B GHRL and ADH7 All of these arepoorly represented within the literature Table We haveinvestigated the survival outcomes of select genes from table that have not been investigated in gastric cancer to the best ofour knowledge These genes include METTL7A MALSLC2A12 and CWH43 Figure 1A We found a trend towardimproved survival with upregulated CWH43 and downregulated METLL7AWe have included protein interaction networks for the genes we have obtained using the STRING databaseFigure 1BE SLC2A12 interacts with AKT1 a commonly studied gene of interest within GC known to contribute to chemoresistance41 Although many of the interacting proteins are not aswell studied as AKT1 various genes such as MTUS1 PGAP3ALDOA and PMP22 have been shown within the literature toonly influence GC but pancreatic cancer as well4246 It is clearthat further investigation into these understudied specific geneticinteraction networks are needed We then wanted to look intowhether any of these genetic aberrations or their interactor proteins were targetable To do this we utilized the DGIdb METTL7A is a methyltransferase that is located primarily in lipiddroplets and is silenced via DNA methylation in thyroid cancer47 There is a variety of drug interactions within the networkof METTL7A including CDA gemcitabine cytaribine deoxycytidine LTA4 H Kelatophan Ubenimex and a variety of preliminary drug compounds B2 M pembrolizumab QPCTpramipexole ALDOA a variety of preliminary compoundsand HP Estradiol pyridoxine Pembrolizumab has been FDAapproved for the treatment of advanced staged GC with positivePDL1 expression B2M acquired mutations were found to conferresistance to pembrolizumab in other malignancies48 but little isknown in GC Downregulation of these genes may partiallyexplain why there is some efficacy issues with pembrolizumabor other chemotherapies MAL encodes a membrane proteinwithin the endoplasmic reticulum ER of Tcells and is involvedin myelin biogenesis49 Drug interactions within the networkinclude ACTA1 kabiramide c latrunculin ab aplyronine a anda variety of preclinical compounds LIMK1 dabrafenibPMP22 progesterone and MAG GSK249320 CWH43 isinvolved in cell wall biogenesis and involved in lipid remodeling50 Drugs that interact with the protein network include UPP2fluorouracil brivudine Understanding the genetic landscape ofGC gene interaction networks and how those genes respond totherapies may explain partially why this disease is highly resistant to conventional chemotherapies However more work isneeded to understand the possible underlying resistance mechanisms within subsets of GC that would bring forward the idealpopulations that benefit from conventional and commonly usedtherapiesIncreasing interest has been placed around small RNAsincluding miRNAs involvement within GC development5152We wanted to investigate the interaction networks betweenthese uncharacterized genes of interest bold and miRNAsUsing the miRWalk database we found miRNA to interactwith our genes of interest Figure 1FI Many of the miRNAsare uncharacterized in GC but we did find that miRNA612miR612 a METTL7A interacting miRNA induces PAX8 atumorsuppressor and represses FOXM1 to inhibit angiogenesis and metastasis of GC53 Our lab™s work in part involves studying the role of nuclear export and miRNA expression and uncovering ways in which tumor suppressive miRNAs canbe upregulated within the nucleus by manipulating nuclearexport Nuclear export via XPO1 has a limited role in exportingmiRNA from the nucleus to the cytosol rather than its nuclearexport family member XPO5 which exports the majority ofcellular miRNAs54 XPO1 overexpression was found to be atherapeutic target in GC and we have found blocking the protein with the small FDA approved molecule selinexor XPOVIO influences the expression of a subset of tumorassociatedmiRNAs55 Furthermore we have found via small RNAsequencing that after XPO1 inhibition with selinexor as well 0cTechnology in Cancer Research TreatmentFigure Gastric cancer is a highly heterogeneous disease A Survival curves taken from the human protein atlas for CWH43 METTL7ASLC2A12 and MAL BD Protein interaction networks for CWH43 METTL7A SLC2A12 and MAL taken from the STRING Database EHmiRNA interaction networks found from top interactions with CWH43 METTL7A SLC2A12 and MAL in the miRDb as the second generation inhibitor KPT8602 miR7977CWH43 interacting miRNA is significantly upregulated foldchange P ¼ 392E23 and fold change P ¼ 546E in the early stage diffuse gastric cell line SNU1 suggestiveof the tumor suppressive role of this miRNA The connectionbetween nuclear export and cancerspecific miRNAs in GC has 0cSexton et alTable Top Significantly Downregulated Genes Based on Molecular Subtype of Gastric Cancer Well Differentiated Poorly DifferentiatedMixed Subtype Based on Oncomine DatabaseaGenenameMT1GMT1FGCNT2SLC9A1PPFIBP2DBTMT1MPXMP2MT1HGSTA2MALPGA3SIDT2ADRB2BRP44 LSSTGCNT2CKMT2RAB27ASTK32BFGAPXMP2NRXN1GSTA2PKIBPOU2AF1SLC22A23AQP4MLXCXCL14Fold change diffuse vsnormal averageP value KEGG pathway analysisGastric cancersubtype 143E4 Mineral absorption213E10 Mineral absorption597E7 Glycosphingolipid biosynthesis metabolism762E7 Transporter150E9654E4 Valine leucine isoleucine degradation propionate metabolism metabolicpathwayPeroxisome903E7172E9113E6 Mineral absorption231E9 Glutathione metabolism drug metabolism platinum drug resistancepathways in cancer chemical carcinogenesis881e11 Ribosome biogenesis454e12 Protein digestion and absorption199E10 103E12 cAMP signaling neuroactive ligand“receptor interaction188E12 Mitochondrial biogenesis422E8cAMP signaling neuroactive ligand“receptor interaction gastric acidsecretion growth hormone synthesis secretion and action206E12 Glycosphingolipid biosynthesis metabolic pathways537E8 Arginine and proline metabolism metabolic pathways258E12 Membrane trafficking156E9 Metabolism318E10 Membrane trafficking175E8190E7 Cell adhesion molecules CAMs155E6 Glutathione metabolism drug metabolism platinum drug resistancePeroxisomepathways in cancer chemical carcinogenesis184E6890E7111E5 anic acid transporters384E6 Bile secretion vasopressinregulated water absorption139E5139E5 Cytokine“cytokine receptor interaction viral protein interaction chemokineInsulin resistance nonalcoholic fatty liver disease NAFLDDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseDiffuseIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalIntestinalMixedMixedMixedMixedMixedMixedMixedMixedMixedMixedAbbreviation KEGG Kyoto Encyclopedia of Genes and GenomesaP Values were calculated via Oncomine software and KEGG pathway analysis was used to analyze gene functionsignaling pathwaynot been investigated in depth We are working toward not onlycharacterizing this novel interaction but also using this information to uncover novel genes pertinent to GC growth anddevelopmentGenetic Analysis of Downregulated GC Genes UsingLauren Type Classified GCsWe stratified the data sets based on the respective Lauren distinguished subtype as we did previously and have highlightedthe vast heterogenetic molecular landscape within the diffuseintestinal and mixed Table GC subtypes All subtypes areexpectedly distinct from one another within our molecular analysis The diffuse and intestinal type GCs seem to have moreprominent downregulation of metabolism related genes suchas GSTA2 and DBT GSTA2 is involved with chemoresistancedue to the action of glutathione metabolism an antioxidant andthis observation suggests that this subtype may be more sensitiveto platinum drugs56 This overall downregulation of metabolicpathways may also point to an increase in the Warburg effectThis alternative metabolic pathway has been suggested to contribute phenotypically to high rates of invasion and aggressiveGCs57 We also observed downregulation of ADRB2 in theintestinal type GC Table Zhang et al described ADRB2signaling as essential in GC and is likely related to stressinduced tumor induction58 They suggest treating with antagonists of ARDB2 likely will provide survival benefit This may beimportant to note and be beneficial for nonintestinal like GCsbecause there is a clear trend of significant downregulation ofthis gene fold differenceWe next assessed the molecular aberrations in the downregulated genes of mixed subtype GC Table Interestingly 0cTechnology in Cancer Research Treatmentwe found various genes that are significantly downregulatedwith no pathway analysis and no real evidence of a mechanismat the protein level Table PKIB function has not beenexplored within the literature in regard to GC but has beenshown to promote proliferation through PI3KAKT pathwayin breast cancer59 POU2AF1 is another gene that has not beencharacterized within the GC literature but has been found to bea highrisk gene in gastrointestinal stromal tumors a type ofsoft tissue sarcoma and rheumatoid arthritis6061 Again themixed subtype is molecularly different from the intestinal anddiffuse gastric subtypes based on this genetic pathway analysiswith notably less involvement of metabolism related genesAlthough this is expected due to its difference in subtypingthe mixed gastric subtype has a much smaller representationwithin the literature than the intestinal and diffuse types and itis clear that further investigation is needed A better understanding of the diverse nature of downregulated genes in allaspects of GC is needed as a first step to identify new therapeutic options that will benefit patients with GCGastric Cancer Exhibits High Molecular DifferencesBetween GendersWithin the United States men and women older than are athigher risk for developing GC while the male population ishigher in risk for welldifferentiated GC development than thefemale population mainly due to the protective effect of estrogen against developing H pylori induced gastric carcinogenesis62 Females have higher incidence of poorly differentiatedGCs compared to their male counterparts for reasons largelyunknown Various environmental factors play a role in diseasedevelopment as a whole including obesity smoking drinkingand a poor diet6366 A retrospective study by Kim et al hasshown that women not only have a higher incidence of diffusetype GC but have a worse overall prognosis as well as geneticdifferences compared to men including ERb expression67 suggesting a hormonal component may also be a contributing factor to this subset of disease Due to the evident genderdisparities in GC we investigated the underlying moleculardifferences between male and female patients by preformingGEO2R analysis on the GSE118916 data set Our results showstriking differences in differentially expressed genes betweenmales and femalesOverall both male and female patients with GC showed anabundance of upregulated genes Figure 2A After stratifyingbased on gender the female patients with GC have a higherabundance of upregulated genes oncogenic like genes genes greater than 5fold upregulation compared to downregulated genes Figure 2B while male patients with GC have agreater abundance of downregulated genes tumor suppressorlike genes Figure 2B This trend can also be seen from just thetop differentially expressed genes in the provided tables Current treatment options for GC are somewhat limited in achieving a longterm survival benefit and we wanted to use ourcohorts to identify whether there are differences in actionabletargets between gendersFemale Patients With GC Are Vastly UnderrepresentedWithin Clinical StudiesWe found no direct druggable targets according to the DGIdbdatabase with the top differentially expressed genes Therefore we looked further into the individual protein“proteininteraction networks using STRING database Figure 2CFBroadening the scope of our search allowed us to find manypotential druggable targets Table We narrowed the scopeof our search to inhibitorsantagonist type compounds due tothe substantial genes found to be upregulated Many of thedruggable targets such as estimated glomerular filtration rateEGFR tyrosine kinase inhibitors TKIs are currently beingexplored in a variety of malignancies including GC Erlotinibwas investigated in a phase II clinical trial in combination withoxaliplatinleucovorin5FU in metastatic GC68 Lapatinib aTKI responsible for inhibiting HER2neu and EGFR wastested in a phase III clinical trial TyTAN Trial in Asianpatients with GC69 There was no statistically significant difference in overall survival for Paclitaxel plus Lapatinib overPaclitaxel alone70 We looked further into the patient demographics of the TyTAN trial and noticed a large underrepresentation of female patients within all arms of the study total female patients Another example of this is a trial withBortezomib which interacts with the ADAM17 pathway andhas been tried unsuccessfully in Phase II clinical trials in combination with paclitaxel and carboplatin in metastatic patientswith GC71 As with the Lapatinib trial this one had an overrepresentation of male patients compared to femalepatients A common occurrence within many of theGC clinical trials is combination of new therapies with paclitaxel or some type of Taxol We have found the female cohortto have an abundance of druggable targets interact with paclitaxel including EPB41L4B and CAPN9 Table but largelythis demographic is underrepresented within clinical trial studies It is clear that based on the molecular profile of femalepatients with GC this issue demands further investigationMale Patients With GC May Benefit From HormoneInhibiting TherapiesAs we have previously mentioned the male cohort has anopposite molecular profile compared to the female cohort withWhen screening for actionable drug targets we limited thescope of our analysis to agonists due to the substantial geneticdownregulation already occurring naturally and notion thatmale patients with GC have an abundance of tumor suppressorlike genes In doing so we have found direct druggable targetssuch as SSTR1 and GPT Table GPT is a gene that encodesthe alanine aminotransaminase protein and catalyzes thereversible transamination between alanine and 2oxoglutaratewithin the tricarboxylic acid TCA cycle to generate pyruvatea TCA intermediate and glutamate72 Glucagon and tacrolimus interact with GPT but the stimulation of this gene wouldlikely enhance glucose metabolism through the TCA cyclelikely being nonbeneficial as a treatment option Furthermore 0cSexton et alFigure Male and female patients with gastric cancer have different molecular signatures A Density plots of differentially expressedgenes in the GSE118916 data set for all gastric cancer cases within the cohort B Male and female cohort density plots of the differentiallyexpressed genes in the GSE118916 data set CG STRING Database interaction networks for protein networks from genes found to bedifferentially expressed in female gastric cancer cases within the cohort BTD CAPNS9 EPB41L4B ADAM17 TOMIL1Tacrolimus can influence the development of lymphomas73Although targeting GPT would not be beneficial targetingSSTR1 may have more benefit Hypermethylation of SSTR1was found to contribute to the pathogenesis of GC by actingin a tumor suppressive manner This hypermethylation wasfound to be caused by EpsteinBarr virus infection74 a positive 0cTechnology in Cancer Research TreatmentTable Genes Found to Be Significantly Differentially Expressed Within the Female Cohort From the GEO Database GSE118916aFold change diffuse vsGene namenormal averageP value DrugFBX13DMRTA1BTDPFDN2GRAMD1CCAPN9PBLDEPB41L4BADAM17 109E9 201E8 Testosterone Tretinoin LY294002201E8 Biotin Hydrocortisone Aspartic Acid Celiponase alfa319E9 533E8 620E8 Emricasan Paclitaxel Rizatriptan Celecoxib Idronoxil956E8 961E8 Paclitaxel Vindesine Colchicine Docetaxel Cabzitaxel Erbulin mesylate IxabepiloneLexibulin Tamoxifen Ornithine144E7 Cetuximab Nimotuzumab Tesevatinib Infliximab Etanercept Adalimumab GolimumabHydrocortisone Everolimus Methotrexate Mercaptopurine Bortezomib PrednisoloneDexamethasone Ribociclib Nitrogacestat Dacomitinib Lapatinib Erlotinib PoziotinibIbrutinib PelitinibTOM1L1155E7 Erlotinib Afatinib Gefitinib Cetuximab Lapatinib Panitumumab Rociletinib IcotinibLacomi

Thyroid_Cancer

poorest prognoses of all malignancies with little improvement in clinical outcome over the past years Pancreatic ductal adenocarcinoma is responsible for the vast majority of pancreatic cancer cases and is characterised by the presence of a dense stroma that impacts therapeutic efficacy and drives protumorigenic programs More specifically the inflammatory nature of the tumour microenvironment is thoughtto underlie the loss of antitumour immunity and development of resistance to current treatments Inflammatory pathways are largely mediated by the expression of and signallingthrough cytokines chemokines and other cellular messengers In recent years there hasbeen much attention focused on dual targeting of cancer cells and the tumour microenvironment Here we review our current understanding of the role of IL6 and the broader IL6cytokine family in pancreatic cancer including their contribution to pancreatic inflammationand various roles in pancreatic cancer pathogenesis We also summarise potential opportunities for therapeutic targeting of these pathways as an avenue towards combating poorpatient outcomesIntroductionPancreatic cancer is a devastating malignancy with a 5year relative survival rate of only “ dependenton the geographical location surveyed [“] with these statistics exhibiting only modest improvementover the last four decades [“] The median survival postdiagnosis ranges from just “ months forlocally advanced disease and “ months for metastatic disease [] It was estimated by the World Healthanisation that pancreatic cancer is currently the 7th leading cause of cancerrelated death being responsible for over deaths worldwide in [] With incidence increasing pancreatic cancerhas been predicted to be the third leading cause of cancerrelated death in the European Union by [] and the second leading cause of cancerrelated death in the United States of America and Germanyby []Several factors contribute to the poor survival of pancreatic cancer patients A current lack of reliablediagnostic markers that would enable early screening [] coupled with largely nonspecific symptoms ofdisease results in over of patients presenting with metastatic disease at diagnosis [] This subgroupof patients have limited therapeutic options and are thus typically administered palliative chemotherapyaimed at prolonging survival and reducing symptoms during endoflife care [“] Moreover whilstapproximately “ of patients present with localised disease that is eligible for potentially curativesurgery [] disease recurs in over of patients postresection [] Ultimately these factorsculminate in more than of patients diagnosed with pancreatic cancer succumbing to disease []These harrowing statistics highlight that despite research efforts there remains a lack of understandingof the pathogenesis of disease which in turn limits the development of new therapeuticsReceived March Revised July Accepted August Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Pancreatic ductal adenocarcinomaPancreatic ductal adenocarcinoma is the predominant pancreaticmalignancyPancreatic cancer can arise from either the endocrine or the exocrine region of the pancreas Tumours arising fromthe exocrine compartment are termed pancreatic ductal adenocarcinoma PDAC and account for over of allpancreatic cancers []PDAC develops via a stepwise progression from normal tissue through to invasive lesions which is associated withdistinct morphological characteristics [“] It has been proposed that this process begins with a phenomenontermed acinartoductal metaplasia ADM which is a normal homeostatic mechanism whereby acinar cells transdifferentiate into a ductal phenotype in response to particular stimuli [] However if compounded by an oncogenic˜hit™ cells are pushed towards a pathogenic phenotype that develops into pancreatic intraepithelial neoplasia PanIN[“] Disease progresses through preinvasive stages termed PanIN1A PanIN1B PanIN2 and PanIN3 priorto invasive PDAC [] This progression is associated with increasing nuclear atypia whereby the nuclei are no longerpositioned basally and loss of normal architecture as cells become more papillary in nature with PanIN3 lesionsdemonstrating increased mitoses [] As disease progresses to PDAC cells become invasive and breach the basement membrane growing through the extracellular matrix and metastasising to distant ans Figure Less common precursor lesions include intraductal papillary mucinous neoplasms IPMNs and mucinous cysticneoplasms MCNs that also develop through multistep processes [] Whilst they share some common featureseach lesion is morphologically and genetically distinct In contrast with PanINs that form within small ducts IPMNsdevelop within the primary or secondary branches of the main pancreatic duct whilst MCNs lack ductal involvement[]An ‚ammatory tumour microenvironment contributes to PDACpathogenesisAn archetypal feature of PDAC is the development of extensive stromal networks within the tumour microenvironment TME that can account for up to of the total tumour volume [“] This unique characteristic drives theinflammatory nature of PDAC that contributes to its aggressive phenotype [] Desmoplasia is driven by pancreaticstellate cells PSCs and cancerassociated fibroblasts CAFs that upon activation produce a range of extracellularmatrix ECM components such as collagen laminin and fibronectin which in turn form a physical barrier preventing the penetration of therapeutics [“] Though PSCs and CAFs have been shown to support cancer metastasis and drug resistance they interact with cancer cells in a bidirectional manner with each promoting the survivalgrowth and proliferation of the other [“] Quiescent fibroblasts are able to differentiate into two unique subtypes termed myofibroblastic CAFs myCAFs and inflammatory CAFs iCAFs [] These two subtypes are distinct whereby myCAFs express high levels of αsmooth muscle actin αSMA and are located adjacent to cancercells while iCAFs express low levels of αSMA and instead secrete high levels of inflammatory mediators including IL6 and are distributed distant from cancer cells within desmoplastic tumour regions [] Broadly myCAFsappear to have roles in epithelialtomesenchymal transition EMT and ECM remodelling whilst iCAFs appear tobe involved in inflammation and ECM deposition [] A third less abundant subtype termed antigenpresentingCAFs apCAFs has more recently been defined [] These cells express low levels of both αSMA and IL6 andinstead express high levels of major histocompatibility complex class II MHCII and related genes [] As such allthree subtypes are transcriptionally and functionally distinctThe wider TME contains a plethora of other cell types including endothelial cells tumourassociated macrophagesTAMs and neutrophils TANs mast cells regulatory Tcells myeloidderived suppressor cells MDSCs dendriticcells natural killer NK cells and nerve cells [] Interactions between various cells within the TME can driveeither proor antitumorigenic functions of others for example cancer cells can induce PSCs to secrete inflammatorycytokines that drive immune cells towards an immunosuppressive phenotype and also form a positive feedback loopby increasing the tumorigenic potential of cancer cells [] The ECM itself has also been suggested to modifyPSC behaviour in particular that ECM stiffness promotes the myCAF phenotype indicated by increased αSMAexpression [] This results in substantial complexity that ultimately determines tumour phenotype []The components of the microenvironment modify tumour behaviour through the production of cytokines growthfactors and other signalling molecules that predominantly drive a proinflammatory and immunosuppressive program that enhances PDAC tumour growth and progression [“] Figure The ability of the TME toinhibit therapeutic efficacy through both molecular mechanisms and physical fibrotic barriers contributes to the The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Figure Our current understanding of the contribution of IL6 family cytokines to PanIN and PDAC developmentPreinvasive PanIN lesions develop from normal ductal epithelia through PanIN stages 1A 1B and to stage invasive andormetastatic PDAC This process is associated with acinartoductal metaplasia ADM early in disease combined with an accumulation of oncogenic mutations most common mutations are indicated A number of cells within the tumour microenvironment havebeen shown to secrete IL6 family cytokines which in turn results in the activation of a protumorigenic signalling cascade A betterunderstanding of the relationship between each of these cells within the tumour microenvironment may reveal new therapeuticopportunities to manage cancer progressionintrinsic resistance of disease [] Thus dual targeting of cancer cells and the TME may be required to induce afavourable therapeutic response although this poses a signficant scientific and clinical challenge [“]Molecular basis of pathogenesisPDAC development is associated with accumulation of mutationsThe progression of tumorigenesis through PanIN and PDAC stages is associated with the stepwise accumulation ofspecific genetic mutations that drive malignant transformation The most frequent genetic alteration is an activatingKRAS point mutation codon that occurs early on in tumour development [] and is detected in over ofPDAC tumours [“] Mutations in KRAS have been shown to drive development of precursor PanIN lesions andwhen combined with an appropriate tumour suppressor mutation these lesions progress to invasive or metastaticPDAC [] Figure Patient tumours harbouring wildtype WT KRAS often carry activating mutations indownstream effector molecules such as BRAF or PIK3CA [] The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Inactivation of a range of tumour suppressor proteins is also common including mutations in TP53 CDKN2Aand SMAD4 in approximately and of tumours respectively [] Whilst each mutation has uniquemechanistic outcomes all three proteins are either directly or indirectly involved in the regulation of the G1S cellcycle checkpoint Analysis of patient tumours indicates that two or more of these mutations often occur together withCDKN2A mutation being combined with either TP53 SMAD4 or both usually in the background of KRAS mutation [] This suggests that by disrupting this checkpoint cancer cells are able to overcome inhibitory mechanismsallowing continued progression to invasive diseaseUnbiased sequencing efforts have also enabled identification of low prevalence PDAC mutations observed in lessthan of cases [] These include mutations in genes involved in chromatin modification KDM6A DNAdamage repair ATM and other tumourrelated processes such as growth TGFBR1 or TGFBR2 []Furthermore it is important to note that technical advances are continuously uncovering epigenetic mechanisms thatfurther modulate the PDAC transcriptional landscape and ultimately influence disease heterogeneity and tumourprogression []Molecular subtypesThe PDAC epithelial compartment is typically divided into two subtypes including a classical subtype exhibiting anepitheliallike expression profile and a squamous or mesenchymallike subtype [] An additional third exocrinesubtype is outlined in some analyses and is characterised by a gene expression profile related to digestive enzymeproduction [] The classical or epithelial subtype has also been further divided into a pancreatic progenitor andan immunogenic subtype whereby the immunogenic subtype is distinguished by significant immune infiltration andassociated gene programmes [] Though there is no consensus on which classification system will allow the mostvaluable stratification of patients the mesenchymal subtype is invariably associated with a poor prognosis []The stromal compartment has also been classified into either normal or activated subtypes reflecting the proandantitumorigenic capabilities of the TME with the activated subtype associated with reduced survival [] This isparticularly valuable as the extensive heterogeneity of PDAC complicates clinically relevant stratification of patientsThus the identification of molecularly unique subtypes may enable development of tailored therapeutic regimensthat will provide improved clinical outcomesCurrent treatment optionsRegardless of disease stage at time of diagnosis patients have relatively limited treatment options For the majorityof patients disease will be locally advanced or metastatic disqualifying them from undergoing potentially curativesurgery [] In these cases patients are typically offered chemotherapy with palliative intent [“]Surgery provides the only potentially curative treatmentSurgical resection remains the only potentially curative treatment option due to minimal efficacy of standardofcarechemotherapy and radiotherapy Due to its aggressive nature the majority of patients present to clinic with locallyadvanced or metastatic disease with only “ of patients presenting with localised tumours that are eligiblefor surgical resection [] Even for those able to undergo surgical intervention over of patients relapsepostresection [] with median survival improving to months and 5year relative survival rate increasingmodestly to “ [] The use of neoadjuvant therapy is generally reserved for borderline resectable disease inan effort to enable patients to become eligible for surgery [] However a range of recent trials have shown improved clinical outcomes including overall survival for neoadjuvant treatment of patients with resectable tumours[] Following surgical resection patients are typically treated with adjuvant gemcitabinebased chemotherapy []although a recent study showed improved diseasefree survival and overall survival with a modified FOLFIRINOXtherapy combination of oxaliplatin irinotecan leucovorin and fluorouracil []Radiotherapy provides variable clinical outcomeWhilst the use of radiotherapy and chemoradiotherapy combination chemo and radiotherapy in the neoadjuvantand adjuvant settings have been investigated there remains a lack of consensus regarding therapeutic benefit []This is due to issues such as insufficient radiation dose and low participant numbers as well as low uptake of moderntechniques [] In the neoadjuvant setting preliminary studies reported reduced lymph node positivity and rates oflocal recurrence for chemoradiotherapy compared to surgery with adjuvant chemotherapy [] However the useof radiotherapy in the palliative setting was reported to modestly reduce overall survival [] More recent studiesusing ablative doses of radiation have shown a survival benefit highlighting that technological advancements mayprovide an avenue for improved clinical outcomes following radiotherapy [] These contrasting results highlight the The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211need to determine which subset of patients may benefit from the inclusion of radiotherapy approaches in standardtreatment regimensChemotherapy remains the cornerstone of treatmentDespite modest improvements in overall survival palliative chemotherapy remains the standard treatment optionfor patients with locally advanced or metastatic disease Gemcitabine monotherapy has been the mainstay treatmentfor pancreatic cancer since when it was demonstrated to improve median survival by just over month compared with fluorouracil [] Within the last decade there have been some further improvements in clinical outcomewith combination chemotherapies gemcitabinenabpaclitaxel and FOLFIRINOX providing median overall survivalbenefits of and months respectively compared with gemcitabine alone [] Although FOLFIRINOX treatment resulted in a lower percentage of patients experiencing reduced quality of life it also had increased toxicity andadverse events thus preventing its administration to patients with multiple comorbidities []Therapeutic resistance remains a significant barrier to patient survivalDespite advances in chemotherapeutic options treatment efficacy and patient prognosis remain poor due to the inherent therapeutic resistance of pancreatic cancer It has been proposed that this drug resistance may be driven by theTME including changes to cytokine signalling and metabolic pathways [“] This intrinsic resistance is demonstrated by patients experiencing similar overall survival for chemotherapy treatment “ months compared withbest supportive care “ months which encompasses the use of palliative surgery psychological support painmanagement and other methods of symptom control [] Whilst a range of targeted treatments such as EGFR orcheckpoint inhibitors have been trialled with or without chemotherapy they have shown limited success [“]Emerging roles for the IL6 family of cytokines in PDACCytokines are soluble molecular messengers that enable efficient communication between a range of cell types andhave been recognised to be major contributors to the growth and metastasis of cancers [“] In pancreatic cancer cytokines mediate signalling between cancer cells and the cells of the TME including PSCs CAFs endothelialcells and a range of immune cells including macrophages mast cells neutrophils and regulatory Tcells [“]It is the specific signalling pathways active within this community of cells that dictates the balance of pro andantitumorigenic functions []The IL6 family of cytokinesThe interleukin IL6 family of cytokines includes IL6 IL11 leukaemia inhibitory factor LIF oncostatin MOSM ciliary neurotrophic factor CNTF cardiotrophin1 CT1 cardiotrophinlike cytokine CLC neuropoietin NP IL27 and IL31 [“] These cytokines are grouped as they share structural similarity forming a fourαhelical bundle termed helices AD with an upupdowndown topology []IL6 and IL11 utilise a hexameric signalling complex consisting of two molecules each of the cytokine αreceptoreither IL6R or IL11R respectively and βreceptor glycoprotein gp130 [“] IL6 and IL11 are ableto signal via two distinct mechanisms termed classic and transsignalling Classic signalling involves the formation of a complex including membranebound IL6R or IL11R with gp130 and the respective cytokine Converselytranssignalling utilises soluble IL6R or IL11R molecules which are able to form a signalling complex with gp130and the respective cytokine [“] In this way classic signalling relies on the responding cell™s intrinsic expressionof IL6R or IL11R whilst transsignalling is able to activate any cell expressing gp130 []LIF OSM IL27 and IL31 signal through trimeric complexes with a single cytokine molecule engagingthe respective receptor LIFR OSMR IL27R WSX1 or IL31R and either gp130 or OSMR for IL31[“] CNTF CT1 CLC and NP form tetrameric signalling complexes composed of one cytokinemolecule one LIFR one CNTFR and one gp130 receptor [] In each case the active signalling complex consists of two chains that are signalling competent with a combination of either gp130 LIFR OSMR IL27R or IL31R[] The requirement for multiple receptor subunits means that although gp130 is ubiquitously expressed the expression of other receptor subunits dictates the ability for any given cell to respond to cytokine as signalling initiationrequires the presence of cytokine and a compatible receptor complex [] Figure 2ASignalling complex assembly leads to transphosphorylation and activation of receptorassociated Janus tyrosinekinases JAKs largely JAK1 and to a lesser extent JAK2 and TYK2 [] In the case of gp130mediated signalling this results in phosphorylation of the cytoplasmic domain of gp130 at tyrosine Y and [] Phosphotyrosine pY and of gp130 provide docking sites for signal transducer and The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Figure IL6 family cytokine signalling pathwayA Schematic representation of the stepwise binding process for the IL6 family members with IL6 as an example The site interaction involves cytokine binding to the respective receptor with the site interaction generally between the cytokine and thecommon gp130 receptor chain finally site interactions involve formation of the final active signalling complex in this case formation of the IL6IL6Rgp130 hexameric complex B General outline of the IL6 family cytokine signalling pathway Formation ofan active hexameric complex leads to activation of JAKs with subsequent activation of the STAT3 MAPK and PI3K pathways leftThis results in upregulation of the negative regulator SOCS3 as well as a range of inflammatory and protumorigenic moleculesThe pathway is inhibited by SOCS3 PIAS3 and PTPs via dephosphorylation ubiquitinmediated proteasomal degradation andSUMOylation right The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211activator of transcription STAT molecules leading to their subsequent phosphorylation by JAK1 and formation ofactive STAT dimers [“] Phosphorylated STAT pSTAT dimers then translocate to the nucleus and modulatetarget gene expression including upregulation of a range of genes involved in inflammatory and protumorigenicpathways [“] Figure 2B Broadly these STAT3regulated genes can be categorised into pathways associatedwith inhibition of apoptosis increased cell proliferation modulation of immunity and inflammation increased angiogenesis and increased invasive and metastatic potential [“]Although JAKSTAT signalling is the predominant pathway activated downstream of IL6 family cytokines themitogenactivated protein kinase MAPK and phosphoinositide 3kinase PI3K pathways can also be activated[] The MAPK pathway has been suggested to be activated by a Src homology domain 2containing phosphatase SHP2mediated mechanism whereby SHP2 is recruited to pY759 on gp130 allowing JAKmediated phosphorylation of SHP2 [] This promotes association with the adaptor protein growth factor receptor bound protein Grb2 leading to activation of the Gprotein Ras via son of sevenless SOS with a subsequent phosphorylationcascade including Raf MEK and ERK12 activity [] Following this a MAPKdependent phosphorylationevent leads to the recruitment of Grb2associated binding protein Gab1 to the plasma membrane where Gab1 issuggested to act as a scaffold or adaptor protein to allow binding of PI3K and SHP2 leading to activation of the PI3Kand MAPK pathways respectively [] Figure 2BThe suppressor of cytokine signalling SOCS3 is largely responsible for regulation of signalling and is directlyupregulated by STAT3 [] SOCS3 contains an SH2 domain allowing it to bind to pY residues within the gp130receptor [] with preferential binding to Y759 [] Once bound SOCS3 recruits an E3 ubiquitin ligasecomplex containing Cullin5 Rbx2 and adaptors Elongin B and C via its SOCS box domain [] This complexubiquitinates the gp130 receptor inducing its internalisation and targeting it for proteasomal degradation [“]and is also able to ubiquitinate JAK2 in vitro [] SOCS3 also mediates direct inhibition of the kinase activityof JAK12 via its kinase inhibitory region [“] Thus SOCS3 is able to downregulate IL6 family cytokinesignalling pathways through two distinct mechanismsThe phosphotyrosine phosphatases PTPs and protein inhibitors of activated STATs PIASs also limit the strengthand duration of cytokine signalling [] A range of PTPs including SHP2 are responsible for dephosphorylatingtyrosine phosphorylated substrates including JAKs STATs and other SHP2 molecules [] PIAS3 preferentially binds pSTAT3 and inhibits activity either by preventing STAT3 interaction with DNA by recruiting transcriptional repressors to STAT3 target genes or by SUMOylating STAT3 to prevent its activity [] Figure 2BInterleukin in PDACElevation of serum IL6 is a negative prognostic marker in human PDACSerum IL6 levels were increased in PDAC patients compared with healthy patients [“] or those withchronic pancreatitis [] and were also increased in patients with metastatic PDAC compared to thosewith locally advanced disease [“] Moreover elevated serum IL6 positively correlated with increased diseaseburden weight losscachexia and metastasis [““] however there are conflicting observations inthe literature regarding IL6 and cachexia [] Although increased serum IL6 levels correlate with increased disease stage and in metastatic patients correlates with poor overall survival [] As such it has been suggestedthat IL6 may be a superior marker for diagnostic and prognostic purposes compared with the standard Creactiveprotein CRP carcinoembryonic antigen CEA and carbohydrate antigen CA199 markers []IL6 is expressed within the TMElL6IL6 was overexpressed in human PDAC tumours in comparison with adjacent normal tissue [] Whilstthis tumourspecific elevation has been correlated with reduced survival in some studies [] othersshowed no significant correlation with survival [] similar to the data available in The Cancer Genome AtlasTCGA dataset for both IL6 and IL6R Figure 3AB The TCGA comprise aggregate sequencing data which doeshave limitations regarding interpretation of contributions of individual cell populations to disease outcome howeverit remains a widely used resource for exploratory investigations However overexpression of IL6 has been observedat the mRNA and protein level in the pancreata of PDAC mice [] with Il6 expression increasing with agewhich is indicative of disease stage in these models []Despite the presence of IL6 in tumours primary human and commercial pancreatic cancer cell lines have been reported to exhibit variable expression levels of IL6 and secreted cytokine albeit consistently higher than normal pancreatic ductal epithelial cells [] In an anoid model minimal IL6 was expressed by pancreaticcancer cells PCCs or PSCs in monoculture however in coculture PCCs expressed only Il6ra whilst iCAFs expressedhigh levels of IL6 with this activating STAT3 within PCCs [] iCAFs also demonstrate an upregulation of The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211Figure IL6 family cytokine expression in PDAC patientsOverall survival for patients with high top quartile and low bottom quartile level expression of A IL6 B IL6R C IL11 D IL11RE LIF F OSM G CNTF H CTF1 CT1 I CLCF1 CLC and J IL27 n per group Data and graphs obtained fromOncoLnc [] using data from The Cancer Genome Atlas TCGA Statistical significance determined by MantelCox Logranktest The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BYNCND 0cClinical Science “101042CS20191211the JAKSTAT pathway with expression of IL6 being dramatically increased in vitro when incubated with PCC conditioned media indicating that soluble factors trigger IL6 production [] More recently PCCderived IL1αhas been shown to induce autocrine LIF secretion and thereby promote the iCAF phenotype including activation ofthe JAKSTAT signalling pathway and IL6 production []In addition TAMs have been identified as producers of IL6 in pancreatic cancer by correlative immunohistochemistry and expression analysis of isolated cell populations [] Production of IL6 by TAMs was shownto influence tumour development via bonemarrow chimeras as mice reconstituted with IL6 knockout KO Il6myeloid cells developed lowgrade PanINs whilst those reconstituted with IL6 WT cells developed PanIN3 lesions[]IL6 is a driver of PDAC pathogenesisBoth in vitro and in vivo studies suggest that the presence of IL6 in the TME can drive activation of STAT3 []with IL6 inhibition reducing STAT3 phosphorylation [] This IL6STAT3 program has been proposed tobe a driver of PDAC pathogenesis by enhancing tumour initiation and progression angiogenesis regulation of cytokine expression and immune cell behaviour resistance to apoptosis and promotion of metastasis [“] In aninducible KRASdriven mouse model genetic deletion of Il6 resulted in a reduction of ADM and PanIN formationwhen KRAS mutation was initiated embryonically compared with controls suggesting a role for IL6 in tumour initiation [] This was also observed in a constitutive KRAS mutant model where genetic deletion of IL6 preventedtumour initiation in vivo with a reduction in the number of PanIN and lesions [] Interestingly oncogenicKRAS and hypoxic conditions both features of PDAC tumours [] were shown to induce IL6 production[] perhaps representing a feedforward pathway enhancing tumorigenesis [] However IL6 is notabsolutely required for PanIN formation as induction of KRAS mutation at weeks of age in conjunction with anexperimental pancreatitis model drove formation of PanIN lesions that were not significantly different between IL6WT and KO mice []Il6 mice exhibited reduced tumour progression with decreased proliferative capacity of both cancer and stromal cells enabling regression of precursor lesions [] Furthermore this inhibition of tumour progression by IL6deletion was due at least in part to the reversal of ADM with ductal cells reverting to an acinarlike phenotype[] Increased apoptosis of cancer and stromal cells was also shown to contribute to this reduced tumour progression as demonstrated by appropriate immunohistochemical analyses with upregulation of proapoptotic anddownregulation of antiapoptotic BCL2 family members [] This is mirrored by in vitro data whereby IL6 stimulation increased the expression of antiapoptotic BCL2BCL2 and BCL2L1BCLXL [] with blockade of IL6signalling or STAT3 activation inducing apoptosis [] Collectively these data suggest that whilst IL6 contributes it is not required for PDAC initiation and progressionThe process of angiogenesis supports tumour growth and progression by enabling adequate blood supply whichis enhanced by IL6 signalling Upon IL6 stimulation PDAC cell lines upregulate key angiogenic factors such asvascular endothelial growth factor VEGFVEGF and neurophilin1 NRP1NRP1 [] with significant correlation observed between the expression of IL6R and VEGF on human PDAC sections [] IL6inducedupregulation of VEGF correlated with a growth advantage in PCCs with both features inhibited by treatment witha JAK2 inhibitor []Another facet of the protumorigenic effects of IL6 is the regulation of cytokine expression that enables modulationof the immune system [] In particular it has been shown that IL6 is able to upregulate a type cytokine profile invitro that may inhibit antitumour immunity in disease [] IL6 suppressed the differentiation of human CD14cells into dendritic cells DCs in vitro whilst combination treatment with IL6 and granulocyte colonystimulatingfactor GCSF inhibited the ability of DCs to respond to alloantigen a process that is required for DC maturationand antigen presentation where these effects were reversed by blockade of IL6 andor GCSF [] IL6 has alsobeen implicated in driving increased apoptosis of type I conventional DCs cDC1s leading to cDC1 dysfunctionea

Thyroid_Cancer

"Evidence on the association between exposure to perfluoroalkyl and polyfluoroalkyl substancesPFASs and blood glucose concentrations in pregnant women is inconsistent This study aimed to examine theassociation between PFAS exposure and the concentrations of fasting plasma glucose FPG and onehour plasmaglucose hPG after a 50g oral glucose tolerance test in pregnant womenMethods The study was based on the ShanghaiMinhang Birth Cohort in which pregnant women were recruitedAmong them women provided blood samples at “ gestational weeks for PFAS measurement FPG data collectedfrom women at “ GW and hPG data collected from women at “ GW were obtained through medicalrecords from the routine prenatal care system High FPG or hPG was defined as ‰¥90th percentile of FPG or hPG Theanalysis of eight PFASs was conducted in this study perfluorohexane sulfonate PFHxS perfluorooctane sulfonate PFOSperfluorooctanoic acid PFOA perfluorononanoic acid PFNA perfluorodecanoic acid PFDA perfluoroundecanoic acidPFUdA perfluorododecanoic acid PFDoA and perfluorotridecanoic acid PFTrDA The odds ratios ORs and associated confidence intervals CIs were estimated to determine the associations of each PFAS compound with high FPG and hPG from a logistic regression modelResults After adjustment for potential confounders most PFASs were positively associated with high hPG concentrationsThe OR for high hPG concentrations was CI “ with a one log unit increase of PFOS similar associationswere observed for PFNA OR CI “ PFDA OR CI “ PFUdA OR CI “and PFDoA OR CI “ When the PFAS concentrations were categorized into three groups by tertiles thehighest tertiles of PFOS PFOA PFNA PFDA PFDoA and PFTrDA had a statistically significant increase in the risk of high hPG concentrations compared with the lowest tertiles No statistically significant association was observed between PFASexposure and high FPGConclusion PFAS exposure was associated with an increased risk of high hPG among pregnant women but no suchassociation was observed for FPGKeywords Perfluoroalkyl and polyfluoroalkyl substances Plasma glucose Cohort study Pregnancy Correspondence miaomaohua163com Yanfeng Ren and Longmei Jin contributed equally to this work4NHC Key Lab of Reproduction Regulation Shanghai Institute of PlannedParenthood Research Fudan University Shanghai ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cRen Environmental Health Page of IntroductionPerfluoroalkyl and polyfluoroalkyl substances PFASs agroup of manmade chemicals with water stain andgreaseresistant properties are used in a wide range ofconsumer products including fast food packaging stainresistant carpets windshield washing fluid firefightingfoam insecticides and paints [] Humans are widely exposed to PFASs through the ingestion of contaminateddrinking water and food as well as the inhalation ofcontaminated indoor air and dust [] Some PFASs havebeen shown to bioaccumulate in anisms [] Themean halflives of PFASs in adult humans vary from to years [ ] The most commonly studied PFASsincluding perfluorohexane sulfonate PFHxS perfluorooctane sulfonate PFOS perfluorooctanoate PFOAand perfluorononanoate PFNA are detected in the majority of human serum samples []Animal studies have shown that PFAS exposure is associated with a wide range of adverse health effects including the disruption of endocrine hormones such astestosterone estrogen and thyroid hormones [ ] alterations in serum lipid levels [] impaired glucose metabolism and insulin hypersensitivity [] and immune systemdisturbance [ ] Human studies have also suggested theadverse effects of PFASs on the immune system [] carcinogenesis [] pregnancyinduced hypertension arterialatherosclerosis [ ] and glucose metabolism [“]Although epidemiological studies have suggested thatPFASs are associated with impaired glucose toleranceand homeostasisinsulin resistance betacell dysfunction and a higher risk of diabetes [“] the associations observed in the general population cannot begeneralized to metabolically vulnerable pregnant womenowing to their specialinsulinresistant state duringpregnancy The current evidence on the effects of PFASson glucose metabolism in pregnant women is limitedand inconclusive In the Odense Child Cohort studyPFHxS and PFNA concentrations were associated withimpaired glycemic status in pregnant women and maytherefore enhance the risk of developing gestational diabetes mellitus GDM [] In another prospective studyof women higher prepregnancy PFOA concentrations were associated with an increased risk of GDMbut the associations for six other PFASs were not statistically significant [] In contrast Valvi found noassociations between PFOA PFOS PFHxS PFNA orperfluorodecanoic acid PFDA concentrations and therisk of GDM in pregnant women []In the present study we sought to evaluate the associations between PFAS exposure and fasting plasma glucose FPG and 1h plasma glucose concentrations hPG measured after a 50g oral glucose tolerance testOGTT in pregnant women by using data from theShanghaiMinhang Birth Cohort Study SMBCSMethodsStudy participantsAll study participants were recruited from the SMBCSbetween April and December Pregnantwomen attending their first routine antenatal care at theMaternal and Child Health Hospital of Minhang districtin Shanghai were consecutively recruited if they wereat “ gestational weeks GW of pregnancy theywere registered residents of Shanghai they had nohistory of chronic disease of the liver kidney or otherans they planned to give birth in the study hospital and they were willing to participate in specifiedinterviews during pregnancy and after delivery Among pregnant women who were invited pregnantwomen were recruited corresponding to a response rateof Exposure assessment and quality controlBlood samples for PFASs measure were collected at recruitment and plasma samples were separated andstored at ˆ’ °C before they were transported to theCenter for Disease Control and Prevention in HubeiProvince for the assay of PFASHighperformanceliquid chromatographycoupledwith tandem mass spectrometry Agilent TechnologiesInc USA was used for the quantitative measurement ofPFASs The information on sample collection separation reservation transportation quantification limit ofdetection LOD and quality control has been detailedpreviously [] Among the PFASs measured in ourstudy eight PFASs with detection rates above including PFHxS PFOS PFOA PFNA PFDA perfluorododecanoic acid PFDoA perfluoroundecanoic acidPFUdA and perfluorotridecanoic acid PFTrDA wereincluded in the final analysesAn internal standard approach was used to aidquantification MilliQ water was used to performprocedural blank analysis for each batch of samplesThe concentrations of each detected congener shouldbe more than three times of that in the proceduralblank and were corrected by subtracting the procedural blank concentration in the present study LODwas defined as the concentration with a signaltonoise ratio equal to or greater than All the recoveries ranged from “ A five point calibration curve was drawn and each precursor rangedfrom “ ngmL Calibration curves presenteda linear pattern over the concentration range of theprecurslucose and covariate measurementThe information on plasma glucose concentrations inpregnant women was collected from the medical recordsof the prenatal care system and included results for FPG 0cRen Environmental Health Page of and hPG In the study hospital within the studyperiod pregnant women were asked to provide bloodafter overnight fasting for FPG testing at their earliestconveniences generally within week after their firstantenatal care It was suggested that pregnant womenunderwent a h 50g OGTT at GW in order toscreen for gestational diabetes if they were consideredto have a high risk of GDMn ie FPG ‰¥ mmolL mgdL [] or overweight and obeseieBMI ‰¥ kgm2 [] otherwise it was suggested thatthe examination of hPG was performed between and GW The 50g OGTT was performed after anovernight fasting also The distribution of gestationalweeks in which the FPG and hPG examination wasperformed is shown in Supplemental Table S1 Information on whether the women had been diagnosed withGDM was extracted through medical records at birthA structured questionnaire was used by trained interviewers to collect information on the covariates Thewomen were asked about age per capita household income education level passive smoking height prepregnancy weight parity history of abortion and stillbirth pregnancy complications etc Prepregnancy BMIkgm2 was calculated as body weight divided by bodyheight squaredStatistical analysisAmong the pregnant women recruited womendelivered singleton live births and women providedblood samples at enrollment for PFAS measurement FPGconcentrations measured at “ GWs were obtainedfor women and hPG concentrations measured at“ GWs were obtained for pregnant womenPregnant women who had data on PFASs and FPG concentrations were included in this study Fig We firstdescribed and compared the demographic characteristicsofthe included and excluded pregnant women Themeans and standard deviations SD were used to describethe distributions of FPG and hPG according to thedemographic characteristics ofthe included pregnantwomen A logistic regression model was used to examinethe association between PFAS exposure and plasma glucose with the 90th percentiles of FPG mmolL ie mgdL and hPG mmolL ie mgdL usedlogarithm lntransformedas the cutoff value NaturalPFAS concentrations were firstincluded in logisticFig Study population of the present study from SMBCS FPG fasting plasma glucose hPG hplasma glucose after a 50g oral glucosetolerance test SMBCS ShanghaiMinhang Birth Cohort Study 0cRen Environmental Health Page of regression models and those with concentrations belowthe LOD were assigned a value of LODˆš PFAS concentrations were also categorized into three groups by tertilesT1 lowest tertile T2 middle tertile and T3 highest tertile and included in the logistic regression models withthe lowest tertile as the reference group Odds ratiosORs and associated confidence intervals CIs wereestimated for the association between each PFAS and highFPG1 hPG ie ‰¥90th of FPG concentration or ‰¥ 90th of hPG concentration Based on tertiles the concentrations were transformed to ordinal data and assigned to allpersons to calculate ptrend values In addition multiplelinear regressions were used to analyze the associationbetweenglucoseconcentrationscontinuousplasmaPFASsandPotential confounders were identified a prior according to the previous literature Age of pregnant womeneducation economic income prepregnancy BMI passive smoking parity history of abortion and stillbirthand pregnancy complicationsincluding bleeding thyroid disease and pregnancyinduced hypertension [ ] were identified and a directed acyclic graphsSupplemental Figure S1 was used to evaluate the appropriation of covariates We did not adjust for alcoholconsumptionn in the final models because of thelow prevalence The statistical assumptions of logistic regressions were evaluated and met including linear relationship of independent variables with logitp outliersand colinearity of independent variablesSeveral sensitivity analyses were performed to test therobustness of the primary results1 Considering the potential effect of prepregnancy BMI on GDM [] andthe variation in PFAS concentrations across BMI we repeated the analysis in women with a prepregnancy BMIof kgm2 to eliminate the confounding effect ofBMI To test the generalizability of the results we repeated the analyses in pregnant women without GDM To examine whether the associations of PFASs withFPG1 hPG were timedependent we performed subgroup analyses for different spans of GW at glucosemeasurement for FPG at “ GWs and “ GWsfor hPG at “ GWs and “ GWs StatisticalAnalysis System SAS software version SAS Institute Inc Cary NC USA was used for statistical analysis P values of were considered statisticallysignificantResultsTable presents the characteristics of the included pregnant women are compared with those of the excludedwomen in the study The majority of women included inthe present analyses were nulliparous “years of age with a BMI between “ kgm2 with a household income per capita of Table Characteristics of the included and excluded pregnantwomenCharacteristicsPvalue of Student™sttest or Chisquare testIncludedN N Mean ± SDExcludedN N Mean ± SDMaternal age at enrollment yearsMean ± SD ± “‰¥ Prepregnancy BMI kgm2Mean ± SD ± “‰¥ Maternal education ± ± Below highschool High School College orabove Per capita household income CNY “ Passive smokingYesNo Pregnancy complicationYesNo History of abortion and stillbirthYesNoParity‰¥ CNYmonth well educated collegeleveleducation or above without pregnancy complication without history of abortion and stillbirth Approximately of women were exposed topassive smoking during pregnancy The distributions ofthese demographic characteristics were not significantlydifferent between the included and excluded womenexcept parityTable presents PFHxS PFOS PFOA PFNA andPFDA were detected in all maternal plasma sampleswhile PFUdA PFDoA and PFTrDA were detected in 0cRen Environmental Health Page of Table PFASs concentrations ngmL of the includedpregnant women N PFASLODLOD NGMGSDPercentiles5th25th 50th 75th 95thPFHxS PFOSPFOAPFNAPFDAPFUdA PFDoA LOD PFTrDA Note LOD limit of detection GM geometric mean GSD geometricstandard deviationLOD about samples PFOA and PFOS had the highestconcentrations PFOA GM ngmL PFOS GM followed by PFHxS GM ngmL ngmLPFDA ngmL PFNA ngmL and PFUdA ngmL while PFDoA and PFTrDA had the lowestconcentrationsTable presents the concentrations of FPG and hPGaccording to the demographic characteristics of the subjects The mean SD FPG and hPG concentrationswere mmolL ie mgdL and mmolL ie mgdL respectively Theconcentrations of FPG and hPG were comparableacross pregnant women with different BMI household income passive smoking status pregnancy complicationand history of abortion and stillbirth The concentrationof hPG was higher in pregnant women who were olderor had higher education levels but not in those with FPGThe concentration of FPG was lower in nulliparous pregnant women but not in those with hPGTable presents that higher concentrations of PFOSPFOA PFNA PFDA PFDoA and PFTrDA were associatedwith an increased risk of high FPG however the associationswere not statistically significant AORPFOS CI“ AORPFOA CI “ AORPFNA CI “ AORPFDA CI “AORPFDoA 95CI “ AORPFTrDA CI “ Higher concentrations of PFASs were associated with an increased risk of high hPG except for PFHxSand the associations with PFOS PFNA PFDA PFUdA andPFDoA were statistically significant after adjustment for potential confounders AORPFOS CI “AORPFNA CI “ AORPFDA CI “ AORPFUdA CI “ AORPFDoA CI “ In addition multiple linearregressions were also used to analyze the association betweenPFASs and plasma glucose Similar results were found inmultiple linear regression as in logistic regression modelalthough the association of PFDoA with hPG is not statistically significant Supplemental Table S2We further examined the associations between the categorized PFAS concentrations and FPG1 hPG Weak associations between the highest tertiles of PFASs and anincreased risks of high FPG were observed but the associations were not statistically significant Fig Comparedwith pregnant women with the lowest tertiles of PFASsthe risk of high hPG was increased in women with thehighest tertiles of PFASs with statistically significant associations observed for PFOS PFNA PFDA PFUdA andPFDoA AORPFOS CI “ AORPFNA CI “ AORPFDA CI “ AORPFUdA CI “ AORPFDoA CI “ Fig Linear trends were observed between the tertiles of PFOS PFNA PFDAPFUdA and PFDoA and high hPG P for trend and respectivelyWe repeated the analysis after excluding women withGDM The pattern of associations between PFASs andhigh FPG and hPG did not change substantially except that the association between PFDoA and high hPG was no longer statistically significant SupplementalTable S3 In addition the analysis among pregnantwomen with a BMI of kgm2 produced similar results Supplemental Table S4thatIn the subgroup analysis for different GW spans theassociations between PFASs and high FPG remainednonsignificant disregard of the timing of FPG measurements exceptthe increased concentrations ofPFNA were associated with an increased risk of highFPG at “ GWs AORPFNA CI “ The pattern of association between PFASs andhigh hPG did not substantially change disregard ofmeasurement time of hPG with the exception thatthe association with high hPG became nonsignificantfor PFOS PFUdA PFDoA at “ GWs and PFOSPFNA PFDA and PFUdA at “ GWs largely owingto the reduced sample size Supplemental Table S5DiscussionIn this prospective cohort study PFAS exposures inpregnant women were found to be associated with high hPG but not FPG and the association persisted forpregnant women without GDM or with BMI kgm2Many studies have demonstrated that PFASs wereassociated with impaired glucose homeostasis and anincreased risk of diabetes in the general population [“] However in pregnant women the associations between PFASs and glucose homeostasis have not beenwell investigated Wang et al™s study showed that severalPFAS compounds were associated with increased postpartum FPGincluding perfluoro1metylheptylsulfonat mPFOS perfluoro34metylheptylsulfonat m 0cRen Environmental Health Page of Table The distribution of FPG and hPG mmolL according to participant™s demographic characteristicsCharacteristicsFPGN Mean ± SD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± PvalueTotalMaternal age at enrollment years “‰¥ Prepregnancy BMI kgm2 “‰¥ Maternal educationBelow high schoolHigh SchoolCollege or abovePer capita household income CNY “ Passive smokingYesNoPregnancy complicationYesNoHistory of abortion and stillbirthYesNoParity‰¥ hPGN Mean ± SD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Pvalue FPG fasting plasma glucose hPG hplasma glucose after a 50g oral glucose tolerance test p compared with the first groupmPFOS perfluoro5metylheptylsulfonat mPFOSand PFHxS [] The Longitudinal Investigation of Fertility and the Environment LIFE study reported thateach SD increment in PFOA concentrations was associated with a 187fold increase in GDM risk [] In theOdense Child Cohort study in metabolically vulnerablepregnant women ie BMI ‰¥ kgm2 family history ofdiabetes mellitus previous GDM multiple pregnancy ordelivery of a macrosomic child PFHxS and PFNA concentrations were associated with impaired glycemic status however no associations were found in women withlow GDM risk [] It™s a pity that the absence of information on history of family diabetes and subjects withprevious GDM limited our ability of examining the[]association in subjects with high risk Higher concentrations of PFASs in our study may partially contributeto the differences with other studies In our studyconcentrations of most PFASs were much higher thanthose in the Odense Child Cohortthe LIFEStudy [] and Wang et al™ study [] except thatPFOS is higher in the LIFE Study compared to thecurrent study The differences in concentrations aswell as outcome indices of impaired glucose homeostasistiming of measurement and population included make the comparison between these studiesdifficult nevertheless the potential for PFAS exposureto disturb glucose homeostasis has been supported inmost studies 0cRen Environmental Health Page of Table Association between PFAS concentrations lntransformed and high FPG and hPG in pregnant womenInPFASngmlPFHxS hPGN COR CIFPGN COR CI AOR CIPFOSPFOAPFNAPFDAPFUdAPFDoA AOR CI PFTrDACOR crude odds ratio AOR adjusted odds ratio CI confidence interval FPG fasting plasma glucose hPG hplasma glucose after a 50g oral glucosetolerance testModels were adjusted for maternal age at enrollment years prepregnancy BMI kgm2 per capita household income education level passive smokingpregnancy complication history of abortion and stillbirth and parity Although the underlying mechanism linking PFASs toglucose homeostasis is not yet clear it has been suggestedthat inhibition of phosphorylation of protein kinase BAkt and the activation of peroxisome proliferator activated receptors PPARs may play a role [ ] Studies using animal models and HepG2 cells have indicatedthat PFAS compounds reduce the expression of the phosphatase and tensin homolog protein and affect the Aktsignaling pathway [ ] The inhibition of Akt a keymediator of cellular insulin sensitivity may stimulate gluconeogenesis and hepatic insulin resistance [] BothPFOA and PFOS have been certified to affect glucose metabolism by AKT signaling pathway However the otherPFASs were not investigated in these studies [ ] Inaddition studies have demonstrated that PFASs can bindto and activate the PPAR α and γ receptors [] PPAR anuclear transcription receptor is known to play essentialroles in the regulation of gene expression glucose homeostasisfatty acid metabolism and inflammation []Therefore PFASactivated PPAR could disturb glucosehomeostasis by influencing insulin resistance [] and insulin secretion [] PFOA have the highest potential ofPPARα activation than the other PFASs with a shortercarbon chain length including PFHxS PFNA PFDA andPFDoA [] Moreover PFAS exposure may interferewith secretion and function of glucocorticoids andthyroid hormones via hypothalamic“pituitary“adrenalaxis and hypothalamic“pituitary“thyroid axis whichmay further disturb glucose metabolism [ ]The physiologicaleffects of PFASs on glycemichomeostasis may depend on the potency and concentration of individual PFASs [ ]Fig Association between PFAS concentrations divided by tertiles and high FPG Notes All the ptrend values for PFASs with FPG were insignificant 0cRen Environmental Health Page of Fig Association between PFAS concentrations divided by tertiles and high hPG Notes p for trend The strengths of the present study were the prospective nature of the study design the large sample size andthe measurement of a wide range of PFAS compoundsHowever the potential limitations of the study shouldbe considered First a considerable proportion of subjects was lost to followup which may have led to selection bias However the characteristics of the includedsubjects were similar to those excluded in terms of ageeducation prepregnancy BMI and household incomeand thus a substantial selection bias was not expectedSecond the followup period from the measurement ofPFAS exposure to the endpoints FPG and hPG wasshort but the singlepoint measurement of PFAS concentration may reflect PFAS exposure long before thedate of blood collection owing to their long halflifeThirdthe relationships between PFASs and bloodglucose measures may have been confounded by unmeasured confounders such as family diabetes historyand dietary habits this should be examined in futurestudiesinformation on maternal activesmoking was not collected since the proportion of activesmoking was quite low in Chinese women [] For example only of pregnant women have been exposedto active smoking during pregnancy in a Shanghai BirthCohort [] Thus the current result is not expected tobe severely biased by the unadjustment of active smoking Fourth not all the subjects had information on hPG after the 50g OGTT which may have led to missedcases of GDM and affect the association between PFASsand outcome indicesin the sensitivity analysis ofpregnant women with GDM However the absence ofGDM cases if any would have attenuated the observedassociation Fifth data for FPG “ GWs or hPG“ GWs were collected over a long time span andIn additionthus the associations between PFASs and FPG and hPG may have been confounded by the gestational weekHowever we performed subgroup analyses using different GWs spans at glucose measurement and found thatthe results did not change significantlyConclusionExposure to certain PFASs ie PFOS PFNA PFDAPFUdA and PFDoA was associated with an increasedrisk of high hPG among pregnant women Furtherstudies are needed to clarify the effect of PFASs ongestational glycemic homeostasis and the underlyingmechanismSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12940020006408Additional file Table S1 The distribution of gestational week atglucose measurement for the included pregnant women Table S2Association between PFAS concentrations lntransformed and high FPGand hPG using multiple linear regression Table S3 Associationbetween PFAS concentrations lntransformed and high FPG and hPGin pregnant women without GDM Table S4 Association between PFASconcentrations lntransformed and high FPG and hPG in pregnantwomen with BMI kgm2 Table S5 Subgroup analysis of theassociation between PFAS concentrations lntransformed and high FPGand hPG in pregnant women by gestational age Figure S1 Assumeddirected acyclic graph for PFASs and plasma glucoseAbbreviationsPFASs Perfluoroalkyl and polyfluoroalkyl substances FPG Fasting plasmaglucose hPG Onehour plasma glucose GWs Gestational weeksPFHxS Perfluorohexane sulfonate PFOS Perfluorooctane sulfonatePFOA Perfluorooctanoic acid PFNA Perfluorononanoic acidPFDA Perfluorodecanoic acid PFUdA Perfluoroundecanoic acidPFDoA Perfluorododecanoic acid PFTrDA Perfluorotridecanoic acidORs Odds ratios CIs Confidence intervals GDM Gestational diabetesmellitus OGTT Oral glucose tolerance test SMBCS ShanghaiMinhang Birth 0cRen Environmental Health Page of Cohort Study LOD Limit of detection SD Standard deviations HOMAIR Homeostasis model of assessment for insulin resistance Akt Proteinkinase B PPARs Peroxisome proliferator activated receptorsAcknowledgementsThe authors thank fieldworkers involved in the survey for their efforts in datacollection and quality control and all the pregnant women investigatedAuthors™ contributionsWY HL and MM conceived and designed the study YR LJ and MMperformed data analysis and drafted the WY MM YR FY HL XS ZZand JD revised the manuscript and critically discussed the results All authorswere involved in interpreting the data and approved the final FundingThis work was supported by grants from the National key research anddevelopment program [grant numbers 2016YFC1000505 2018YFC1002801]Shanghai Municipal Commission of Health and Family Planning [grantnumber ] Innovationoriented Science and Technology Grantfrom NHC Key Laboratory of Reproduction Regulation [grant numbersCX2017“] and Shandong Medical and Health Science and Technology Development Project [grant numbers 2018WS060]Availability of data and materialsThe datasets used during the current study are available from thecorresponding author on reasonable requestEthics approval and consent to participateThe study was approved by the ethical review committee of ShanghaiInstitute of Planned Parenthood Research SIPPR Written informed consentwas obtained before the data collection and analysis and the survey wasconducted in accordance with the Declaration of Helsinki PrinciplesConsent for publicationNot applicableCompeting interestsThe authors declare they have no actual or potential competing financialinterestsAuthor details1Department of Health Statistics School of Public Health Weifang MedicalUniversity Weifang Shandong China 2Minhang District Maternal and ChildHealth Hospital Shanghai China 3Department of Global Public HealthKarolinska Institute Stockholm Sweden 4NHC Key Lab of ReproductionRegulation Shanghai Institute of Planned Parenthood Research FudanUniversity Shanghai ChinaReceived May Accepted August ReferencesLau C Anitole K Hodes C Lai D PfahlesHutchens A Seed J Perfluoroalkylacids a review of monitoring and toxicological findings Toxicol Sci “Tittlemier SA Pepper K Seymour C Moisey J Bronson R Cao XL DabekaRW Dietary exposure of Canadians to perfluorinated carboxylates andperfluorooctane sulfonate via consumption of meat fish fast foods andfood items prepared in their packaging J Agric Food Chem “Conder JM Hoke RA De Wolf W Russell MH Buck RC Are PFCAsbioaccumulative A critical review and comparison with regulatory criteria andpersistent lipophilic compounds Environ Sci Technol “Olsen GW Burris JM Ehresman DJ Froehlich JW Seacat AM Butenhoff JLZobel LR Halflife of serum elimination of perfluorooctanesulfonateperfluorohexanesulfonate and perfluorooctanoate in retired fluorochemicalproduction workers Environ Health Perspect “Bartell SM Calafat AM Lyu C Kato K Ryan PB Steenland K Rate of declinein serum PFOA concentrations after granular activated carbon filtration attwo public water systems in Ohio and West Virginia Environ HealthPerspect “Tian Y Zhou Y Miao M Wang Z Yuan W Liu X Wang X Wang Z Wen SLiang H Determinants of plasma concentrations of perfluoroalkyl andpolyfluoroalkyl substances in pregnant women from a birth cohort inShanghai China Environ Int “Biegel LB Liu RC Hurtt ME Cook JC Effects of ammoniumperfluorooctanoate on Leydig cell function in vitro in vivo and ex vivostudies Toxicol Appl Pharmacol “Fuentes S Colomina MT Rodriguez J Vicens P Domingo JL Interactions indevelopmental toxicology concurrent exposure to perfluorooctanesulfonate PFOS and stress in pregnant mice Toxicol Lett “Seacat AM Thomford PJ Hansen KJ Olsen GW Case MT Butenhoff JLSubchronic toxicity studies on perfluorooctanesulfonate potassium salt incynomolgus monkeys Toxicol Sci “ Yan S Zhang H Zheng F Sheng N Guo X Dai J Perfluorooctanoic acidexposure for days affects glucose homeostasis and induces insulinhypersensitivity in mice Sci Rep Goudarzi H Araki A Itoh S Sasaki S Miyashita C Mitsui T Nakazawa HNonomura K Kishi R The Association of Prenatal Exposure to Perfluorinatedchemicals with glucocorticoid and androgenic hormones in cord bloodsamples the Hokkaido study Environ Health Perspect “ Barry V Winquist A Steenland K Perfluorooctanoic acid PFOA exposuresand incident cancers among adults living near a chemical plant EnvironHealth Perspect ““Lind PM Salihovic S van Bavel B Lind L Circulating levels of perfluoroalkylsubstances PFASs and carotid artery atherosclerosis Environ Res “ Darrow LA Stein CR Steenland K Serum perfluorooctanoic acid andperfluorooctane sulfonate concentrations in relation to birth outcomes in themidOhio Valley Environ Health Perspect “ MatillaSantander N Valvi D LopezEspinosa MJ ManzanoSalgado CBBallester F Ibarluzea J SantaMarina L Schettgen T Guxens M Sunyer J Exposure to Perfluoroalkyl substances and metabolic outcomes inpregnant women evidence from the Spanish INMA birth cohorts EnvironHealth Perspect Zeng XW Lodge CJ Dharmage SC Bloom MS Yu Y Yang M Chu C Li QQHu LW Liu KK Isomers of per and polyfluoroalkyl substances and uricacid in adults Isomers of C8 Health Project in China Environ Int 133Pt A105160Lin CY Chen PC Lin YC Lin LY Association among serum perfluoroalkylchemicals glucose homeostasis and metabolic syndrome in adolescentsand adults Diabetes Car

Thyroid_Cancer

cancer is still one of the most prevalent and highmortality diseases summing more than million deaths in This has motivated researchers to study the application of machine learningbased solutionsfor cancer detection to accelerate its diagnosis and help its prevention Among several approaches one is toautomatically classify tumor samples through their gene expression analysisMethodsstomach breast and lung To do so we have adopted a previously described methodology with which we comparethe performance of different autoencoders AEs used as a deep neural network weight initialization technique Ourexperiments consist in assessing two different approaches when training the classification model ” fixing theweights after pretraining the AEs or allowing finetuning of the entire network ” and two different strategies forembedding the AEs into the classification network namely by only importing the encoding layers or by inserting thecomplete AE We then study how varying the number of layers in the first strategy the AEs latent vector dimensionand the imputation technique in the data preprocessing step impacts the network™s overall classification performanceFinally with the goal of assessing how well does this pipeline generalize we apply the same methodology to twoadditional datasets that include features extracted from images of malaria thin blood smears and breast masses cellnuclei We also discard the possibility of overfitting by using heldout test sets in the images datasetsResults The methodology attained good overall results for both RNASeq and image extracted data Weoutperformed the established baseline for all the considered datasets achieving an average F1 score of Continued on next pageCorrespondence mafaldafferreirafeuppt1Faculty of Engineering University of Porto Rua Dr Roberto Frias sn Porto Portugal2INESC TEC Institute for Systems and Computer Engineering Technology andScience Porto Portugal The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes weremade The images or other third party material in this are included in the ™s Creative Commons licence unlessindicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 The CreativeCommons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of Continued from previous pageand and an MCC of and for the RNASeq when detecting thyroid cancer the Malaria and theWisconsin Breast Cancer data respectivelyConclusions We observed that the approach of finetuning the weights of the top layers imported from the AEreached higher results for all the presented experiences and all the considered datasets We outperformed all theprevious reported results when comparing to the established baselinesKeywords Cancer Classification Deep learning Autoencoders Gene expression analysisBackgroundCancer is a label for a group of diseases that is characterized by abnormal and continuous cell growth with thepotential to spread through its surrounding tissues andother body parts [] During cancer was the secondleading cause of death globally accountable for milliondeaths where around were in developing countries[] Throughout the years and given the evolution of techniques technology and treatments in medicine cancersurvival rates have been improving [] However there arestill some types that have survival rates of under suchas pancreatic esophagus and liver cancers Its prevalencemakes it more crucial to correctly and accurately classify such diseases For tackling this need many researchgroups have been trying to help on accelerating cancerdiagnosis by experimenting and studying the applicationof machine learning algorithms to this problem []When automatically classifying tumor samples oneapproach is to analyze the samples derived molecularinformation which is its gene expression signatures Geneexpression is the phenotypic manifestation of a gene enes by the processes of genetic transcription and translation [] By studying it this gene map can help to betterunderstand cancer™s molecular basis which can have adirect influence on this disease™s life cycle prognosis diagnosis and treatment There are two main cancer genomicsprojects ” The Cancer Genome Atlas TCGA [] andThe International Cancer Genome Consortium ICGC[] ” that aim to translate gene expression systematizing thousands of samples across different types of cancersWith this elevated number of features each representing a particular gene one may find genomewide geneexpression assays datasets in these projects However thistype of data presents some challenges because of alow number of samples an unbalanced class distribution with few examples of healthy samples and a high potential of underlying noise and errors due toeventual technical and biological covariates [] This difficulty in gathering data accurately is underlying for everydataset creation The equipment used to collect the datahas intrinsic errors associated mechanical of acquisitionand others hence the dataset will reflect these errorsSeveral authors have chosen the previously mentionedapproach of analyzing the gene expression of tumor samples Many of the developed methodologies in this scopeuse straightforward supervised training especially whenusing deep neural networks DNNs relying on theirdepth to produce the best results Gao [] proposedDeepCC a supervised deep cancer subtype classificationframework based on deep learning of functional spectra quantifying activities of biological pathways robust tomissing data The authors conducted two studies eachwith a different cancer detection colorectal and breastcancer data The authors claimed that the describedmethod achieved overall higher sensitivity specificity andaccuracy compared with other classical machine learningmethods widely used for this kind of task namely randomforests support vector machine SVM gradient boostingmachine and multinomial logistic regression algorithmswith an accuracy higher than Sun [] proposed Genome Deep Learning GDLa methodology aiming to study the relationship betweengenomic variations and traits based on DNNs Thisstudy analyzed over six thousand samples of Whole ExonSequencing WES mutations files from different cancer types from TCGA and nearly two thousand healthyWES samples from the one thousand genomes projectsThe main goal of GDL was to distinguish cancerous fromhealthy samples The authors built models to identify each type of cancer separately a totalspecific modelable to detect healthy and cancerous samples and a mixedmodel to distinguish between all types of cancerbasedon GDL All the experiments were evaluated througha three performance metrics ” accuracy sensitivityand specificity ” and b Receiver Operating Characteristic curves with the respective Area Under the CurveROCAUC This methodology achieved a mean accuracy of on the specific models on mixturemodels and on total specific models for canceridentificationIn [] Kim compared the performances of a neural network a linear SVM a radial basisfunctionkernel SVM a knearest neighbors and arandom forest when identifying types of cancers and 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of healthy tissues The classifiers were trained with RNAseq and scRNAseq data from TCGA where they selectedup to the most significant genes expressed for eachof the cancer variations To determine the optimal number of genes for each classifier™s binary classification taskthe methods mentioned above were trained with different sizes of gene expression datasets from to genes When learning with genes the neural network the linear SVM and the radial basis functionkernelSVM models achieved their best performance with a witha Matthews Correlation Coefficient MCC of and respectively The knearest neighbors and random forest models achieved an MCC of and accordingly when using genes Furthermore theauthors identified classes with an accuracy of over and achieved a mean MCC of and a mean accuracy of with the neural network classifierHowever many DNNs besides the known challenges regarding their training setting [] have a highertendency to overfit which one can detect when applying the same architecture to unseen data or to a heldouttest Thus our motivation focuses on exploring unsupervised pretraining methods based on a lowerdimensionallatent representation with the usage of an autoencoderAE This approach is grounded in the hypothesis thata there is unessential information in high dimensionality datasets and b the acquisition and processing errorspotentially present in the dataset are discarded contributing to a lower probability of overfitting [] Furthermorepretraining AEs and using the learned weights as priorsof the supervised classification task not just improves themodel initialization but also often leads to better generalization and performance [] This may be one of thereasons why AEs are found to be the most predominantstrategy when analyzing RNASeq data []To support our motivation and choices we presentsome works that include unsupervised training in theirmethodologies In [] the authors designed a solution by combining a Multilayer Perceptron and StackedDenoising Autoencoder MLPSAE aiming to predicthow good genetic variants can be a factor in gene expression changes This model is composed of layers inputtwo hidden layers from the AEs and output and trained itto minimize the chosen loss function the Mean SquaredError MSE The authors started by training the AEs witha stochastic gradient descent algorithm to later use themon the multilayer perceptron training phase as weight initialization crossvalidation was used to select the bestmodel The performance of the chosen model was compared with the Lasso and Random Forest methods andevaluated on predicting gene expression values for a different dataset The authors concluded that their approach outperformed both the Lasso and Random Forest algorithms with an MSE of versus and respectively and was able to capture the change ingene expression quantificationThe authors in [] described a study of four different methods of unsupervised feature learning ” PrincipalComponent Analysis PCA Kernel Principal Component Analysis KPCA Denoising AE DAE and StackedDenoising AE ” combined with distinct sampling methods when tackling a classification task The authorsfocused on assessing how influential the input nodes areon the reconstructed data of the AE™s output when feeding these combinations to a shallow artificial networktrained to distinguish papillary thyroid carcinoma fromhealthy samples The authors highlighted two differentresults in their 5fold cross validation experiment thecombination of a SMOTE [] with Tomek links and aKPCA was the one with the best overall performancewith a mean F1 score of while the usage of a DAEachieved a mean F1 score of In [] presented a stacked sparse autoencoder SSAEsemisupervised deep learning pipeline applied to cancer detection using RNASeq data By employing layerwise pretraining and a sparsity penalty this approachhelps to capture more significant information from theknown high dimensionality of RNASeq datasets usingthe filtered information to the sequent classification taskThe SSAE model was tested on three different TCGARNASeq datasets ” corresponding to lung stomach andbreast cancers ” with healthy and cancerous samplesand compared it to four others classification methodsan SVM a Random Forest a neural network supervisedlearning only and a vanilla AE The authors performed5fold cross validation and evaluated the model™s performance through four metrics accuracy precision recalland F1 score The results show that the semisuperviseddeep learning approach achieved superior performanceover the other considered methods with an average F1score of across the three used datasetsThe authors in [] developed a methodology for detecting papillary thyroid carcinoma They analyzed how theusage of AEs as a weight initialization method affectedthe performance of a DNN Six types of AEs were considered Basic AE Denoising AE Sparse AE DenoisingSparse AE Deep AE and Deep Sparse Denoising AEBefore being integrated into the classifier architecture allAEs were trained to minimize the reconstruction errorSubsequently they were used to initialize the weights ofthe first layers of classification neural network meaningthat the AE layers become the top layers of the wholeclassification architecture using two different strategieswhen importing the weights just the encoding layersand all the pretrained AE Moreover in the training phase the authors studied two different approacheswhen building the classifier a fixing the weights ofthe AE and b allowing subsequent finetuning of all 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of the network™s weights The authors used stratified 5foldcrossvalidation and evaluated the model through distinct metrics Loss Accuracy Precision Recall and F1score The authors reported that the overall best resultwas achieved through a combination of Denoising AEfollowed by its complete import into the classification network and by allowing subsequent finetuning throughsupervised training yielding an F1 score of in which the main goalIn [] the authors present a transfer learning methodologyis to explore whetherleveraging the information extracted from a large RNASeq data repository with multiple cancer types leadsto extract important latent features that can help complex and specific prediction tasks such as identifyingbreast cancer neoplasia The authors used the TCGAPanCancer dataset which is composed of approximately RNASeq gene expression examples of distincttumor types This data was split into two sets breast cancer and nonbreast cancer data The nonbreast data isfirstly used to train the three selected architectures forthis study a sparse AE a deep sparse AE and a deepsparse denoising AE models Then the breast data isused to finetune the resulting AEs After pretrainingthese models the authors aim to predict the breast tumorintrinsicsubtypes which is given by the PAM50 subtype information included in the clinical data included inthe PanCancer data The extracted features from the AEbased architectures are then fed as input to three differentmachine learning classifiers namely Logistic RegressionSupport Vector Machine and a shallow Neural NetworkTo assess the deep AEs performance as feature extractionmethods the authors compared them to other classical feature extraction methods combining them with theclassification algorithms previously mentioned ANOVAMutual Information ChiSquared and PCA A 10foldcross validation was performed and all the combinationswere compared through the accuracy metric The resultsshowed the deep sparse denoising AE performs best whenusing the AE extracted features where the combinationwith a shallow neural network leads to the best overall of ±In [] Ferreira used the same methodologydescribed in [] to discriminate different types of cancerinstead of distinguishing cancerous samples from healthyones In this case they aimed to identify thyroid skin andstomach cancer correctly Given that a Denoising AE wasthe AE that lead to the best results in previous studiesthe authors chose to single it out instead of the original The rest of the experiments remained the same strategies for importing the pretrained AE into the top layersof the classifier two approaches when training the classifier to detect different types of cancer same evaluation ofthe obtained results Although in a different domain thebest outcome was reached with a combination of the samestrategy and the same approach in the previous work []with an F1 score of when identifying thyroid cancerMethodsWe extend the previously described work in [] byassembling three different types of experiments dividedinto two main parts where we use three different AEsand five types of cancer samples In the first one we analyze the performance of a deep neural network DNNusing the same pipeline to identify different types of cancer In the second part we choose one of the used AEsto assess how the variance of its latent vector dimension impacts the essential information capture and therefore possibly influencing the classifier™s performance and different data imputation strategies can influence theoverall performance in the classification task Moreoverwe study if the network architecture is correlated withits overall performance and how the model reacts whentraining with a different data type dataset We built thispipeline in Python using the Numpy [] and Pandas []packages for the data preprocessing step the Keras deeplearning library [] running on top of TensorFlow andthe ScikitLearn [] package to train and evaluate themodels and the Matplotlib [] library for visualizationAdditionally we used an NVIDIA GeForce RTX TiGPU on a Ubuntu operating systemThis section is anized as follows œThe data subsection describes the used data and its inherent preprocessing œAutoencoders subsection overviews the AEs considered to this study œMethodology subsection outlinesthe pipeline for each of the referred experiments œEvaluation subsection details how we evaluate the results toprovide statistical evidence Finally œBaseline subsectionpresents the established baseline results for all the useddatasetsThe dataIn our experiments we use two different types of datawhich are described in the subsections that followRNASeq dataWe used five different RNASeq datasets from The Cancer Genomes Atlas TCGA [] each representing a typeTable Five instances of the thyroid RNASeq dataset we have usedTPTEP1 AKR1C6PUBE2Q2P2 HMGB1P1 LOC155060 ZZZ3 NANANANANANANANANANA NA NA NA NA NAThe first line the header contains the genes names and the column valuesrepresent its expression samplewise except for the first column which is thesample ID NA stands for missing value for a particular gene and sample 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of of cancer thyroid skin stomach breast and lung Onecan find a sample of the described data in Table The datasets were downloaded from the cBioPortal []which gathers cancerrelated data from different projectsincluding TCGA To train DNNs we need as many data aswe can get Ergo our first criterion was to choose cancertypes that had the highest number of examples Additionally we decided to gice priority to cancer types withhigh mortality and high incidence rates We use the samethyroid skin and stomach datasets presented in []alongside the lung and breast datasets The data filteringprocess in the cBioPortal comprised searching with thekeywords PanCancer sorting the obtained results fromhighest to lowest RNASeq examples and finally selectingthe thyroid skin stomach breast and lung datasetsAll five datasets are composed of approximately thousand features Each column feature in these datasetsrepresents a specific gene and the cell values for each column are the expression of that gene in a particular sampleAll the RNASeq data were normalized according to thedistribution based on all samples The expression distribution of a gene is estimated by calculating the mean andvariance of all samples with expression values and discarding zero™s and nonnumeric values such as NA Nullor NaN which are substituted by NA [] With the fivedatasets we gathered examples of thyroid cancer of skin cancer of stomach cancer of breast cancer and of lung cancer We would like to emphasizethat this dataset is only a toy dataset since the data doesnot fairly reflect the immense difficulty associated withidentifying cancer in a real scenarioThe preprocessing pipeline was executed for each RNASeq dataset separately Firstly we removed the columnsthat had only one value throughout all samples Whena value is constant for all the examples there is noentropic value with no value variation one cannot inferany information In total and columns were removed on the thyroid skin stomach breast and lung datasets respectively By default weattributed the remaining missing values represented byNA in the dataset as observable in Table with the meanvalue of the column where the missing value is [] Further normalization was not applied in the data Finally weadded the Label column to link the instances to their typeof cancer when training the classifierSince we aim to distinguish several cancer variations wetest all cancers against each other assigning the positivevalue one to the class of interest and zero to the remainingones When detecting thyroid cancer all thyroid examplesare labeled as one and the skin stomach breast and lunginstances as zero and henceforwardAfter processing all the datasets it is improbable thatthe preprocessing phase removed the same columns in allof them To guarantee the same features describe all thesamples we intersect all the datasets and use the resultas our final dataset Also given that the breast cancerdatasets had almost the double of instances we applydownsampling and randomly select breast cancerexamples to keep the final dataset as evenly distributedfor all the cancers as possible In the end the resultingdataset has approximately instances and more than thousand genesData of features extracted from imagesWe use two datasets of two different diseases composedof features extracted from images malaria and breast cancer Since we aim to evaluate how well this methodologygeneralizes by using distinct types of data we are nowable to gather evidence supporting this premiseThe malaria dataset was created by the FraunhoferAICOS institution through the MalariaScope project[] Their main goal is to develop lowcost solutions thatcan provide fast reliable and accurate results on detecting such disease particularly in developing countries In[] the authors thoroughly describe the feature extraction process from thin blood smear images exclusivelyacquired with smartphones The resulting dataset is composed of samples and features These featureswere normalized between [ˆ’ ] via scaling and groupedinto three main groups geometry color and textureFrom all the examples approximately contain malariaparasites Due to the high unbalance between Malariaand NonMalaria labels we performed downsampling onthe NonMalaria class where we randomly selected examples We decided to choose instead of dueto a wide variety of nonparasite artifacts Once the samples were selected and similarly to the preprocessing stepof the RNASeq data we verify if there are features withconstant values and remove them if that is the case Ourworking malaria dataset has instances negativeand positive and feature columnsThe Wisconsin Breast Cancer dataset [] from the UCIMachine Learning Repository is composed of examples and features These features are computed from afine needle aspirate digitized image of a breast mass anddescribe the cell nuclei characteristics present in thoseimages such as texture area concavity and symmetryFrom the examples approximately are benignsamples and are malign ones No under or oversampling techniques were applied since we do not find it to beneeded As performed in the malaria data we checked ifthere were columns with constant values for which therewere not The data was used as is with the proportionsand characteristics described aboveAutoencodersAn autoencoder AE [] is an unsupervised featurelearning neural network that aims to copy its input based 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of on a lower dimensional representation This type of architecture is able to extract features by reducing the dimension of its hidden layer [] which helps the AE to focuson capturing the essential features that best represent thedataLet the encoding and decoding functions of the AE be fand g parameterized on θe and θd respectively where θ θeˆª θd L being the loss function and J the cost function tobe minimized When learning the AE aims to find value θthatargminθJθ X LX gθdfθe Xpenalizing the reconstruction of the input given by ˆX fθe X the more distinct ˆX is the bigger the appliedgθdpenalty When training an AE we use Mean Squared ErrorMSE as the loss function and the Rectified Linear Unitsactivation function ReLU [] for all its layers Currentlyusing ReLU as activation is the default recommendationwhen training neural networks [] Similarly using MSEas the loss function is a fairly common practice present inthe literature when training AEs [ “]We use the AEs as a weight initialization technique[] since evidence supports that using œunsupervised pretraining guides the learning towards basins of attractionof minima that support better generalization from thetraining dataset [] Thus we pretrained them beforeimporting the encoding part or all their layers to theclassification neural networkBasic autoencoder AEThe simplest AE has only one hidden layer This type ofAE learns through the optimization cost function presented in Eq With the combination of linear activationsReLU and the MSE loss function these AEs behave similarly to the Principle Component Analysis PCA method” when trained with an MSE an AE learns the principalsubspace of the training data consequentially []Denoising autoencoder DAEA Denoising AE DAE [] aims not just to reproduce theinput but also to keep its information intact to undo theeffect of an intentional corruption process applied to theoriginal data Its cost function can be described byFig Overall pipeline of our experiments This figure illustrates the chosen metodology for our work Firstly we pretrain the autoencoders AEsbefore embedding them to the top layers of the classification network fullfilling either Strategy import only the encoding layers from the AE orStrategy import the complete AE Each of the full assembled architectures is then trained to detect one of the cancer types in the input dataThe training process can follow two different approaches regarding the imported weights of the AEs A fixing them or B allowing subsequentfinetune I represents the input layer E the encoding layerˆI the output layer of the AE at the classification region of the network D represents thefully connected layer and O the output of the classifer 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of θfθe ˜XJθ X LX gθdargminwhere ˜X is a copy of the input X intentionally corruptedby a sort of noise [] To simulate a form of BernoulliNoise [] we apply a Dropout layer immediately after theinput layer where of the connections are randomly cutSparse autoencoderSimilarly to a DAE a Sparse AE SAE learning processalso has two main goals minimizing the reconstruction error when aiming to copy the input data and applying a sparsity penatly represented by 01 to theparameters involved in the encoding partJθ X LX gθdfθe X λ · 01θeargminθAlthough it also tries to reproduce X an SAE canaddress unique statistical features of the dataset it hasbeen trained on [ ] To deliver that sparsity elementwe use an L1 penalty with a λ of ˆ’MethodologyWe have adopted the methodology described in []which was also used in [] Our experiments consist ofan analysis of the performance of a DNN trained to classify different cancer types studying how three differentfactors may impact the network performance The top layers where we use three different AEs asweight initialization The dimension of the latent vector of the AEs thatmeans the encoding layer size The imputation technique to replace missing datawhen preprocessing the datasetsBesides the top layers imported from the AE the classification part of the full architecture is composed of aBatch Normalization layer [] followed by two FullyConnected layers with a ReLU [] activation Since weaim to detect one type of cancer at the time the last layer” the predictive one ” is a single neuron layer with aSigmoid nonlinearity [] This activation considers thatif the probability of the classification is lower than the sample is classified as negative that is not having thedisease otherwise the sample is classified as positiveTo assess the following experiments we decided to onlyuse the AE that achieved the best results in the firstexperiments For points and we try three different dimensions and For the data imputationstudy we use three strategies replacing the data witha the mean column value used as default a constantvalue in this case zero and b with the most frequentvalueFurthermore we want to study if when using Strategy importing the complete AE into the classification network the model yields better results just because it hasone more layer and therefore more parameters to trainTo observe if the classifier is better only by being deeperwe pretrained the AE and at the embedding step forStrategy we add a decoder layer with all its weightsrandomized guaranteeing that there are no discrepanciesconcerning the network™s topological complexity for bothstrategiesFinally we want to assess how the pipeline behaveswhen dealing with different data types besides RNAseq entries Hence we apply the same methodologyto the image extracted features datasets described inœThe data section to assess if the model can adapt andgeneralize well to these data characteristicsFor all these we follow the same pipeline see Fig Foreach experience we start by pretraining a different AE tominimize the reconstruction error before importing theminto the top of the classification architecture When doingso we choose one of the two strategies considered for thisstudy add just the encoding layers or add all thepretrained AE After the embedding of the AE to the toplayers we consider two different approaches in the training process A fixing the imported weights of the AElayers and B by allowing them to be finetuned duringthe model training for the classification taskWith the complete architectures AE as the top part ofthe classification network assembled we train each oneto distinguish¢ The RNASeq input data as one of cancers namely¢ The malaria input data as Malaria or NonMalaria¢ The breast masses input data as Malign or Benignthyroid skin stomach breast and lungEvaluationWe use stratified 10fold crossvalidation to ensure andprovide statistical evidence The AEs are trained during epochs and the classifier during with a batchsize of The classification model is trained with thebinary crossentropy loss function [] and with an Adamoptimizer [] Furthermore we assess the overall performance of the model in the training and validation setsby analyzing five more metrics Accuracy Matthews Correlation Coefficient MCC [] Precision Recall and F1score and provide the Receiving Operator Curve with therespective Area Under the Curve ROCAUC and thePrecisionRecall CurveFurthermore to study how the model generalizes tounseen data during the training phase we evaluate theperformance of the best architecture combination on aheldout test set for the Malaria and the Wisconsin BreastCancer datasets For both and separately we use a ratio ofone third to create two new splits Therefore 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of Table Baseline results for cancer detection using a Fully Connected Neural Network the classification architecture without the AEas top layersThyroidSkinStom

Thyroid_Cancer

Lasting and SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaMarissa R Keever1 Pan Zhang2 Courtni R Bolt1 Adrienne M Antonson1Haley E Rymut1 Megan P Caputo1 Alexandra K Houser1 Alvaro G Hernandez3Bruce R Southey1 Laurie A Rund1 Rodney W Johnson14 andSandra L RodriguezZas12456 Department of Animal Sciences University of Illinois at UrbanaChampaign Urbana IL United States Illinois InformaticsInstitute University of Illinois at UrbanaChampaign Urbana IL United States Highthroughput Sequencingand Genotyping Unit Roy J Carver Biotechnology Center University of Illinois at UrbanaChampaign Urbana ILUnited States Neuroscience Program University of Illinois at UrbanaChampaign Urbana IL United States Departmentof Statistics University of Illinois at UrbanaChampaign Urbana IL United States Carl R Woese Institute for GenomicBiology University of Illinois at UrbanaChampaign Urbana IL United StatesThe prolonged and sexdependent impact of maternal immune activation MIA duringgestation on the molecular pathways of the amygdala a brain region that ‚uencessocial emotional and other behaviors is only partially understood To address thisgap we investigated the effects of viralelicited MIA during gestation on the amygdalatranscriptome of pigs a species of high molecular and developmental homology tohumans Gene expression levels were measured using RNASeq on the amygdalafor 3weekold female and male offspring from MIA and control groups Amongthe genes that exhibited significant MIA effect a prevalence of differentiallyexpressed genes annotated to the neuroactive ligand“receptor pathway glutamatergicfunctions neuropeptide systems and cilium morphogenesis were uncovered Genesin these categories included corticotropinreleasing hormone receptor glutamatemetabotropic receptor glycoprotein hormones alpha polypeptide parathyroidhormone receptor vasointestinal peptide receptor neurotensin proenkephalinand gastrinreleasing peptide These categories and genes have been associatedwith the MIArelated human neurodevelopmental disorders including schizophreniaand autism spectrum disorders Gene network reconstruction highlighted differentialvulnerability to MIA effects between sexes Our results advance the understandingnecessary for the development of multifactorial therapies targeting immune modulationand neurochemical dysfunction that can ameliorate the effects of MIA on offspringbehavior later in lifeKeywords immune activation pigs RNAseq neuropeptides glutamatergic pathway GABAergic pathwayINTRODUCTIONThe maternal immune response triggered by pathogens and other environmental stressors duringgestation can also elicit an indirect response by the fetal immune cells Kroismayr Odorizzi and Feeney Prins Viral infection during gestation for exampleactivates a cytokinerelated signaling cascade and molecules from this process can cross theEdited byNo¨lia Fern ndezCastilloCentre for Biomedical NetworkResearch CIBER SpainReviewed bySilvia PellegriniUniversity of Pisa ItalyTewarit SarachanaChulalongkorn University ThailandCorrespondenceSandra L RodriguezZasrodrgzzsillinoiseduSpecialty sectionThis was submitted toNeurogenomicsa section of the journalFrontiers in NeuroscienceReceived May Accepted July Published August CitationKeever MR Zhang P Bolt CRAntonson AM Rymut HE Caputo MPHouser AK Hernandez AGSouthey BR Rund LA Johnson RWand RodriguezZas SL Lastingand SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaFront Neurosci 103389fnins202000774Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaplacenta and reach the fetal brain The resulting maternalimmune activation MIA can impactfetal developmentalprocesses and exert longterm postnatal eï¬ects in the oï¬springRutherford The relationship between MIAand neurodevelopmental disordersincluding schizophreniaspectrum disorders SSD and autism spectrum disorders ASDand neurodegenerative disorders such as Alzheimer™s diseaseAD in oï¬spring has been established Knuesel Canetta Mattei These diseases sharesome behavior symptoms comorbidities such as eating disordersand genetic and environmental ie MIA agents Canitanoand Pallagrosi The previous neurological disorders havebeen associated with abnormal structure and dysregulationof the amygdala Schumann FernandezIrigoyen and share genes and molecular mechanismsincluding histocompatibility complex MHC genes Andersand Kinney glutamatergic and GABAergicassociatedgenes Bourgeron Marin Li andmitochondrial activity processes Pieczenik and Neustadt Sragovich socialinteractionThe fetal amygdala is susceptible to ‚ammatory signals andthe plasticity of this brain structure to MIA can lead to alterationsof the developmental trajectory These disruptions may havelonglasting and maladaptive consequences for the oï¬springdue to the significant role that the amygdala plays in manyneurological pathways Located in the forebrain the amygdala‚uencescognition neuroendocrinebehavior learning memory emotion and autonomic systemsThe amygdala also modulates the response of these processesto stressors including pathogenic infections and those resultingfrom management practices such as weaning Tian The amygdala experiences high uptake of gonadalhormones and is anatomically connected to other sexuallydimorphic nuclei Therefore this brain region is involved inregulation of several dimorphic functions such as aggressionsexual behavior gonadotropin secretion and integration ofolfactory information Hines Evidence supports thediï¬erential activation of the amygdala to stimuli between malesand females Killgore and YurgelunTodd includingdiï¬erences in the sexual responses and emotional memoryHamann and diï¬erential vulnerability to insult Baird Due to the interconnected and multiregulatorynature of this brain structureinsults to the amygdala canimpact the individual™s social locomotor and feeding behaviorPetrovich and Gallagher growth and reproductivephysiology health status and immunological response tosecondary stressorsRecent studies lend support to the link between MIA andaltered amygdala function Carlezon In miceMIA elicited by polyinosinicpolycytidylic acid [PolyIC]increased the synaptic strength of glutamatergic projectionsfrom the prefrontal cortex to the amygdala Li In field tests mice exposed to MIA spentless timein the center and traveled a higher distanceindicativeof a higher anxiety behavior incidence than the controlcounterparts These findings suggest that the change in thebalance between excitation glutamatergic and inhibitiontherefore aï¬ecting brain circuitsspike output offeedforward GABAergic modified theamygdala neuronsthatcould regulate behavior in SSD and ASD A candidate genestudy of the eï¬ects of social stress during gestation reportedthatthe expression of a corticotropinreleasing hormonereceptor in the amygdala of 10weekold pigs was higherin females than in males Rutherford Thisstudy concluded that prenatal stress substantially increasedanxietyrelated behaviorstheimpact of maternal stressors during gestation on specificamygdala molecular profiles and associated neurological orbehavioral disorders in the oï¬spring later in life highlightthe complexity of the molecular mechanisms underlying thepathophysiology of MIAin female pigs Studies ofetalvirusadvantages ofrodents when consideringstudying a pig modelto pigsResearch on the lasting eï¬ects of MIA in pigs complementsAntonson et althe insights oï¬ered by rodent modelsstem Theratherfrom the greater homology of humansan physiologythan toin particular brain growth andsize development anddevelopment processesMurphy A pigmodel that has oï¬ered insights into MIA employs porcinereproductivePRRSVtothein the brain andmicroglia ie macrophagelike cellsisin neonatal pigsAntonson associated with behavioralelicit MIA Thisand respiratorysyndromechallengeactivatesimmunechangesThe study of MIA elicited by PRRSV allows for thecharacterization of the impact of a live viral pathogen thatselfreplicates in the host evoking extended activation ofimmune pathways PRRSV challenge during gestation is a wellcharacterized replicable and eï¬ective method for inducingMIA in pigs Antonson In additionPRRSV outbreaks impose a major economic burden to thelivestock industry PRRSV is an enveloped singlestranded RNAvirus thatinfects alveolar macrophages causing interstitialpneumonia and increased serum levels ofthe cytokinesinterleukin betainterleukin and tumor necrosis factoralpha Antonson The persistent repercussionsof MIA on the molecular pathways ofthe pig amygdalaare yet to be investigated Moreover the potentially distinctvulnerability to the prolonged eï¬ects of MIA between sexesremains unknownThe overarching goal of the present study is to advancethe understanding of the impact of MIA on the molecularmechanisms ofthe amygdala Three supporting objectivesare explored a characterization of prolonged transcriptomechanges elicited by viral MIA in pigs a species that hashigh neurodevelopmental homology with humansandfood production valueidentification of molecularpathwaysthat present diï¬erential vulnerability to MIAbetween sexes and c understanding the eï¬ect of MIA onmolecular interactions assisted by gene network inferenceThe findings from these complementary analyses supportthe use of multipleto amelioratethe potential detrimental eï¬ect of MIA on the oï¬springphysiology and behaviortherapeutictargetsbFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaMATERIALS AND METHODSAnimal ExperimentsAll experimental procedures used published protocols Antonson The animal studies were approved by theIllinois Institutional Animal Care and Use Committee IACUCat the University of Illinois and are in compliance with the USDAAnimal Welfare Act and the NIH Public Health Service Policy onthe Humane Care and Use of AnimalsCamborough gilts born and raised at the University of Illinoisat UrbanaChampaign herd were inseminated at days ofage using PIC boar sperm Antonson All gilts were PRRSV negative and were moved at gestationday GD into diseasecontainment chambers maintainedat —¦C and a h lightdark cycle with lights on at AM The gilts were fed daily kg of a gestational diet andhad ad libitum water access One week after acclimation fourgilts were intranasally inoculated with live PRRSV strain P129BV School of Veterinary Medicine at Purdue University WestLafayette IN United States using mL of — median tissueculture infectious dose TCID50 diluted in sterile Dulbecco™smodified Eagle medium DMEM mL total volume Thefour gilts in the Control group were intranasally inoculatedwith an equal volume of sterile DMEM PRRSV inoculationcorresponded to the last third of gestation in pigs and humansduring initiation of rapid fetal brain growth Antonson PRRSV and Control groups were housed in separatecontainment chambersThe rectal temperatures and diet consumption of the giltswere recorded daily until farrowing Antonson The PRRSVinoculated gilts were oï¬ered the maximumfed daily and feed refusal was measured The Control giltswere fed the same amount consumed by the PRRSVinoculatedgilts on the previous day The daily body temperature andfeed intake levels were compared using a mixedeï¬ects modelanalyzed with PROC MIXED SAS Institute Inc Cary NCUnited States The model included the eï¬ects of gilt treatmentand replicate while accommodating for heterogeneity of variancebetween MIA groupsFarrowing was induced with an intramuscular injection of mg of Lutalyse dinoprost tromethamine Pfizer New YorkNY United States on GD in consideration that the averagegestation length is approximately days Antonson Gilts farrowed in individual farrowing crates ofstandard dimensions — m After farrowing thegilts were fed twice a day up to kg of a nutritionallycomplete diet for the lactating period and water remainedavailable ad libitum Pigs received intramuscular injections ofiron dextran mgpig Butler Schein Animal Health DublinOH United States and Excede for Swine mgpig ZoetisParsippany NJ United States to control for respiratory diseasesThe pigs remained with their mothers until PD The bodyweight of pigs was measured daily and analyzed using the mixedeï¬ects model in SAS PROC MIXED SAS Institute Inc CaryNC United States The model included the eï¬ect of MIA andthe random eï¬ect of gilt accommodating for heteroscedasticitybetween pig treatment and sex groups The impact of MIA wasstudied at PD because this is a common age to wean pigsThe study of transcriptome profiles from older pigs could beconfounded with changes in diet and environment associatedwith weaning while profiles from younger pigs would hinder theassessment of the prolonged eï¬ects of MIARNA Extraction and SequencingA balanced experimental design was studiedincluding pigs evenly distributed between maternal PPRSV activatedMPA group of pigs and Control gilts CON group of pigseach group encompassing males and females denoted Maand Fe respectively At PD pigs were removed fromthe farrowing crate and anesthetized intramuscularly using atelazolketaminexylazine drug cocktail mg of tiletamine mg of zolazepam reconstituted with mL ketamine gL and mL xylazine gL Fort Dodge AnimalHealth Fort Dodge IA United States at a dose of mLkgbody weight following protocols Antonson Following anesthetization pigs were euthanized using anintracardiac injection of sodium pentobarbital mgkg bodyweight Fata Plus Vortech Pharmaceuticals Dearborn MIUnited States Pig brains were extracted the amygdalae wererecognized using the stereotaxic atlas of the pig brain Felix dissected out flash frozen on dry ice and stored at ˆ’—¦Cfollowing published protocols Antonson RNA wasisolated using EZNA isolation kit following the manufacturer™sinstructions Omega Biotek Norcross GA United States TheRNA integrity numbers of the samples were above indicatinglow RNA degradation The RNASeq libraries were preparedwith TruSeq Stranded mRNAseq Sample Prep kit Illumina IncSan Diego CA United States The libraries were quantitatedby qPCR and sequenced on one lane on a NovaSeq for cycles from each end of the fragments using NovaSeqS4 reagent kit FASTQ files were generated and demultiplexedwith the bcl2fastq v220 conversion software Pairedend reads nt long were obtained and the FASTQ files are availablein the National Center for Biotechnology Information GeneExpression Omnibus GEO database experiment accessionnumber GSE149695RNA Sequence Mapping and DifferentialExpression AnalysisThe average Phred quality score of the reads assessed usingFastQC Andrews was across all read positions andtherefore no reads were trimmed The pairedend reads fromthe individual samples were aligned to the Sus scrofa genomeversion Sscrofa Pruitt using kallisto v0430Bray with default settings The normalized trimmedmean of Mvalues gene expression values were described usinga generalized linear model encompassing the eï¬ects of the MIAgroup MPA or CON levels sex Fe or Ma levels and MIAbysex interaction and analyzed using edgeR version in the R v environment Robinson Genessupported by transcripts per million TPM by each MIA“sex combination were analyzed to ensure adequate representationacross comparisonsFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaOrthogonal pairwise contrasts between MIA and sex groupswere evaluated in addition to testing for the eï¬ects of MIAbysex interaction and main eï¬ects of MIA and sex Thefour groups compared in the contrasts identified by treatmentfollowed by the sex levels are MPA_Fe MPA_Ma CON_Fe andCON_Ma The Pvalues were adjusted for multiple testing usingthe Benjamini“Hochberg false discovery rate FDR approachBenjamini and Hochberg categoriesamongtheMFand KEGG pathways The GeneFunctional Enrichment and NetworkInferenceapproaches were used to identifyTwo complementaryoverrepresented functionalgenesexhibiting diï¬erential expression across MIA and sex groupsCaetanoAnoll©s GonzalezPena et al2016ab Functional categories investigated included GeneBPs GO molecularOntology GO biological processesfunctionsSetEnrichment Analysisapproach implemented inthesoftware package GSEAP Subramanian was used to identify category overrepresentationwith gene over and underexpressed while considering allgenes analyzed The normalized enrichmentscore NESofin the Molecular Signature DatabaseMSigDB was calculated using the maximum deviation ofthe cumulative sum based on the signed and standardizedfold change The statistical significance ofthe enrichmentwas assessed using the FDRadjusted Pvalue computed from permutationscategoriesGSEAtheThe overrepresentation of functional categories was alsoevaluated among genes that exhibited a significant MIAbysex interaction or main eï¬ect using the Database forAnnotation Visualization and Integrated Discovery DAVID Huang The enrichment of Direct GOcategories in the DAVID database was assessed The Susscrofa genome was used as the background for enrichmenttesting and enrichmentis reported using the ExpressionAnalysis Systematic Explorer EASE score that was computedusing a onetailed jackknifed Fisher hypergeometric exacttest Functional categories were clustered based on geneannotation and the statisticalissummarized as the geometric mean of the log10 EASE scoresofthe categories Delfino Serao Delfino and RodriguezZas significance of clustersWeighted Gene Coexpression NetworkAnalysis and Gene Network VisualizationAn approach complementary to the identification of diï¬erentiallyexpressed genes was used to uncover coexpression networksusing Weighted Gene Coexpression Network AnalysisWGCNA version Langfelder and Horvath The input data were voomtransformed read count valuesgenerated using the limma package version Ritchie in R version Genes were filtered to removethose with low expression levels or no variation across samplesper developer recommendations The number of genes usedfor network analysis was genes Considering potentialfor interaction patterns a sexdependent softthresholdingpower was used to call for network topology analysis Thelowest power values that support a scalefree topology powerused were for the CON_MaMPA_Ma contrast and for the MPA_FeMPA_Ma contrast The Pearson correlationcoefficient ofthe normalized expression values was usedto identify modules of connected genes The minimummodule size was set to with the deepSplit set to and themergeCutHeight set to Module profiles were identifiedusing the correlation between the eigengene of each moduleand pig group Enrichment of functional categories among thegenes in each module profile was explored with DAVID using theSus scrofa genome as background and testing included an FDRmultiple test adjustmentFurther understanding of the impact of the MIAbysexinteraction was gained through the reconstruction of genenetworks using the BisoGenet package Martin inthe Cytoscape platform Shannon Information fromgene and protein interactions annotated in databases includingBIOGRID HPRD DIP BIND INTACT and MINT was usedto visualize relationships between genes Salwinski Alfarano Mishra Stark Kerrien Licata Networks highlightingdiï¬erences in gene levels associated with MIA within sex iethe contrasts MPA_MaCON_Ma and MPA_FeCON_Fe werecompared The network framework includes genes that exhibiteda significant MIAbysex interaction eï¬ect FDRadjusted P and are annotated to enriched functional categoriesThe framework genes were identified by full nodes with sizereflecting the diï¬erential expression level between the MPAand CON groups The network edges depict known molecularrelationships curated in the BisoGenet databases The frameworkgenes were connected through correlated genes listed in theBisoGenet database of molecular interactions that did not reachsignificant MIAbysex interaction eï¬ect The comparison ofthese networks oï¬ered insights into the simultaneous eï¬ect ofMIA across interacting genes and enabled the detection of sharedand distinct coregulation patterns between MPA and CONpigs across sexesRESULTSMaternal Immune Activation andSequencing MetricsThe diï¬erences between MPA and CON giltsin rectaltemperatures and daily diet consumption indicated the activationof the maternal immune system in response to PRRSV Thediï¬erence in body temperature between CON and MPA giltson GD was —¦C standard error —¦C P Thediï¬erence in feed refusal between CON and MPA gilts on GD was g standard error g P A significantincrease in rectal temperatures and decrease in feed intakeP was observed within h of inoculation and returnedto baseline levels within days for body temperature and within days for feed intake At days of age CON pigs were kgFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaheavier than MPA pigs standard error P whileno significant sex or interaction eï¬ects were detectedThe sequencing of the RNA samples produced billionsequenced reads and million pairedend reads per sampleThe number of reads was consistent across MIA and sexgroups coefficient of variation and the eï¬ects ofMIA sex and MIAbysex interaction were tested on genes that surpassed the minimum number of reads per MIA“sex combinationTranscriptome Changes Associated WithMaternal Immune Activation That AreSexDependentOverall genes exhibited a significant FDRadjusted P MIAbysex interaction eï¬ect and among these genes hada significant eï¬ect at FDRadjusted P The profile ofthese genes indicated that the eï¬ect of MIA diï¬ered betweenfemales and males Fortysix genes that presented a MIAbysex interaction eï¬ect are listed in Table together with theirexpression pattern and Pvalue The majority of the genes inTable including neurotensin NTS displayed a reversal inthe expression level between CON and MPA groups across sexesie opposite Log2[fold change] sign across sexes An extendedlist including genes that exhibited a MIAbysex interactioneï¬ect at FDRadjusted P is provided in SupplementaryFile S1 Table AAnother frequent pattern among the genes that displayed aMIAbysex interaction eï¬ect was characterized by a consistentexpression profile between CON and MPA across sexes albeitthe magnitude diï¬ered between sexes Table For exampleglycoprotein hormones alpha polypeptide CGA was overexpressed in CON relative to MPA but the diï¬erential washigher in males than in females Other genes presentingthis pattern included guanylatebinding protein GBP1transthyretin TTR aldehyde dehydrogenase family memberA2 ALDH1A2 hemoglobin subunit beta HBB and basichelixloophelix family member e22 BHLHE22GRPNotable is the significant MIAbysex interaction eï¬ecton genes associated with neuropeptides and hormones andgenes that participate in glutamatergic processes Genes underexpressed in MPA relative to CON males while presentingthe opposite pattern in females Table included NTS theneuropeptide gene proenkephalin PENK the neuropeptidegene gastrinreleasing peptidethe neuropeptiderelated gene vasoactive intestinal peptide receptor VIPR2corticotropin releasing hormone receptor CRHR2 neuronderived neurotrophic factor NDNF reelin RELN glutamatemetabotropic receptor GRM4 solute carrier family member SLC17A6 calcium voltagegated channel auxiliarysubunit alpha delta CACNA2D3 EFhand domain familymember D1 EFHD1 glutathione peroxidase GPX3parathyroid hormone receptor PTH1R thyroid hormoneresponsive THRSP and CGA The CGA gene codes for thealpha subunit protein of the hormones chorionic gonadotropinCG luteinizing hormone LH folliclestimulating hormoneFSH and thyroidstimulating hormone TSHFunctional and Network Analysis ofGenes That Exhibit SexDependentAssociations With Maternal ImmuneActivationThe genes expressing significant MIAbysex interaction eï¬ectswere analyzed for functional enrichment Table presentsthe clusters of most enriched and informative categoriesfrom the DAVID analysis and the complete list of categoriesis in Supplementary File S1 Table B The categories inTable encompass genes presenting the mostfrequentinteraction profile characterized by underexpression in CONfemales relative to males but overexpression in MPA femalesrelative to males These genes include KEGG Autoimmunethyroid diseaseCluster and BP brain developmentGO0007420 Cluster Enrichment results from GSEA complemented the findingsfrom DAVID Highly enriched informative categories amonggenes that have a MIAbysex interaction eï¬ect are presentedin Table and the extended list of categories is presented inSupplementary File S1 Table C The categories in Table in Table including ion homeostasissupport pathwaysTable and regulation of voltagegated calcium channelactivity processesenrichmentinteraction pathwayofthe neuroactiveand the hormoneactivity processesinclude genes such as CGA and VIPR2 that were identifiedin Table ligand receptorand neuropeptideTable Notablythethe diï¬erentialNetwork visualization furthered the understanding ofthe impact of MIA on the relationships among genes thatexhibited a significant MIAbysex interaction eï¬ect Thenetworks in Figures depictthe relationships betweengenes in the enriched neuroactive ligand receptor pathwaythat highlightexpression between CONand MPA in males and females ie CON_MaMPA_Maand CON_FeMPA_Ferespectively Red andrectangular nodesblueframework genes andthe known associations between genesedgesrepresentbased on curated databases of molecularinteractionsRed and blue nodes denote over or underexpressionof the gene in CON relative to MPA and the size is anthe diï¬erential expressioninverse logarithmic function ofPvalue Thediï¬erentialexpression pattern and connectivity among genes highlightsthe discrepancyelicited by MIAbetween the sexesin network modulescontrastsrepresentsimultaneousstudytheofTranscriptome Changes Associated WithMaternal Immune ActivationOverall genes exhibited diï¬erential FDRadjusted P expression between MPA and CON pigs irrespective of sexTable lists notable highly diï¬erentially expressed genesis in Supplementary File S1 Tableand the complete listD The majority of these genes were overexpressed in MPArelative to CON pigs Among the genes overexpressed in MPAcompared to CON pigs were islet amyloid polypeptide IAPPFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaTABLE Genes exhibiting significant FDRadjusted Pvalue maternal immune activationbysex interaction effectGene symbolPvalueaCON FeCON Ma MPA FeMPA MaCON FeMPA FeCON MaMPA MaCON FeMPA MaCON MaMPA FeRGS16CGAPOMCGPX3RELNVIPR2ANKRD34CGBP1GRM4CCDC136SLC17A6BTBD11TTRCACNA2D3CRHR2NDNFCXCL12USP43CCDC17KCNIP4CAMK2N2ALDH1A2GRPPENKSYT12PTH1RHBBESYT1EFHD1BHLHE22ZFP37SLC2A2THRSPNR4A3LOC396781C1QTNF1RAB27ANTSGVIN1SSTR1CCDC9BCCDC33CCDC162PPTHSYNPO2LCHGB5E115E115E115E115E115E115E115E115E1153E0911E0844E0850E0848E0728E0628E0662E0664E0671E0672E0696E0614E0515E0516E0529E0537E0567E0585E0596E0510E0412E0415E0431E0433E0444E0445E0456E0479E0479E0486E0488E0414E0314E0314E0315E0318E03ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’aLog2[fold change] between two maternal immune activationsex groups MPA PRRSVinduced maternal immune activation CON control Fe females Ma malesankyrin repeat domain ANKRD24interferoninducedtransmembrane protein IFITM1 and IFITM3 cathepsinC CTSC mitogenactivated protein kinase kinase MAP2K7heparan sulfateglucosamine 3sulfotransferase HS3ST5secreted phosphoprotein SPP1 immunoglobulin heavy chainIGHG and transforming acidic coiledcoilcontaining protein TACC1 Among the genes underexpressed in MPA relative toCON pigs are insulinlike growth factor IGF2 cellular retinoicacidbinding protein CRABP2 and aldehyde dehydrogenase family member A1 ALDH1A1Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaFunctional Analysis of Genes AssociatedWith Maternal Immune ActivationTable presents the top significant clusters of informativeenriched categoriesfrom the DAVID analysis of genesTABLE Most enriched DAVID clusters and supporting functional categoriesenrichment score ES among the genes presenting significant maternalimmune activationbysex interaction effectaCategory Category identifier and namePvaluebFDRPvalueCluster KEGGBPKEGGKEGGKEGGCluster BPBPBPBPBPCluster BPCluster BPBPES ssc05320Autoimmune thyroid diseaseGO000250ˆ¼Antigen processing andpresentation of peptide orpolysaccharide antigen via MHC class IIssc04514Celladhesion moleculesCAMsssc05323Rheumatoid arthritisssc05164Influenza AES GO0051050ˆ¼Positive regulation oftransportGO0051049ˆ¼Regulation of transportGO0050801ˆ¼Ion homeostasisGO0048878ˆ¼Chemical homeostasisGO0030001ˆ¼Metal ion transportES GO0048871ˆ¼Multicellular anismalhomeostasisES GO0061564ˆ¼Axon developmentGO0007420ˆ¼Brain development290E06190E03450E04340E01230E03320E02480E03200E02560E02170E01440E03350E01450E03130E02280E02450E02320E01360E01480E01570E01200E04370E01640E05130E03170E01310E01aBP biological process KEGG KEGG pathway bFalse discovery rate adjustedPvalueTABLE Enriched informative categories NES using GSEA among thegenes based on the overall maternal immune activationbysex interactionaCategory Category identifier and namebNES PvalueKEGGKEGGMFBPBPBPKEGGBPˆ’ 10E10ˆ’ 10E10ˆ’ 10E10ˆ’ 10E10ssc04080Neuroactive ligand receptorinteractionssc04912GnRH signaling pathwayGO0005179ˆ¼Hormone activityGO0006970ˆ¼Response to osmoticstressGO0019221ˆ¼Cytokine mediatedsignaling pathwayGO1901385ˆ¼Regulation of voltagegated calcium channel activityssc04020Calcium signaling pathway ˆ’ 12E01GO0085029ˆ¼Extracellular matrixˆ’ 18E01assemblyˆ’ 91E02ˆ’ 12E01cFDRPvalue83E0299E0222E0135E0154E0154E0154E0155E01aMF molecularfunction KEGG KEGG pathway BP biological processbNormalized enrichment score negative values indicate genes underexpressionin CON females relative to males but overexpression in MPA females relative tomales cFalse discovery rate adjusted Pvaluediï¬erentially expressed between MPA and CON groupsacross sexes the extended list of categories is presented inSupplementary File S1 Table E Some categories identifiedby the DAVID analysis are consistent with the categoriesdetected at more significant levels among the genes presentingan MIAbysex interaction eï¬ect Table and include theBP angiogenesisand KEGG autoimmunethyroid disease and Epstein“Barr virus infection pathwaysTable Also enriched Supplementary File S1 Table Ewere the BP homeostatic GO0042592 MF ion bindingGO0043167 and BP anatomical structure formation inmorphogenesis GO0048646GO0001525The GSEA enrichment results within the gene expressionpatterns of CON relative to MPA groups complemented thefindings from DAVID The most informative enriched categoriesare presented in Table and the extended list of categories ispresented in Supplementary File S1 Table F Enriched clustersof genes overexpressed in CON relative to MPA detected byGSEA were the BP enrichment of microtubule bundle formationGO0001578 and cilium morphogenesis GO0060271Transcriptome Differences BetweenSexes Independent of Maternal ImmuneActivationOverall genes were diï¬erentially expressed between malesand females FDRadjusted P These genes exhibiteda consistent diï¬erential expression between sexes irrespectiveof the MIA group The complete list of genes diï¬erentiallyexpressed between sexes at FDRadjusted P is available inSupplementary File S1 Table G and the majority were overexpressed in males relative to females Among the previousgenes excluding those that presented MIAbysex interactioneï¬ect a selection of informative genes is listed in Table Genesoverexpressed in males relative to females included eukaryotictranslation initiation factor 1A Ylinked EIF1AYleptinreceptor LEPR luteinizing hormone beta polypeptide LHBLIM homeobox LHX9 luteinizing hormone beta polypeptideLHB and immunoglobulin family member IGSF1Informative categories among the DAVID clusters ofenriched categoriesthe genes diï¬erentially expressedbetween sexes are listed in Table a complete list i

Thyroid_Cancer

Paired box protein8 PAX8 immunohistochemical expression can be used as a diagnostic marker for epithelial cells tumors This study aimed at investigating the immunohistochemical expression of PAX8 among Sudanese females diagnosed with cervical endometrial and ovarian cancers between December and May by studying their Formalinfixed paraffin embedded blocksResults Sixty patients diagnosed with female reproductive tract cancers were included who aged ± years range ” Cervix was the most common cancer site in patients Regarding cancer stage there was and of the study population had stage 3B and 2B respectively The histopathological diagnosis included and poorly moderately and well differentiated cervical squamous cell carcinoma SCC as well as and endometrial adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma respectively PAX8 was positively expressed in endometrial adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma poorly and moderately differentiated SCC All patients diagnosed with well differentiated SCC and metastatic adenocarcinoma showed no expression of PAX8 A statistically significant was seen for PAX8 expression and the different histopathological diagnosis P value Keywords Female reproductive cancer Paired box protein8 Immunohistochemical expressionIntroductionPaired box protein8 PAX8 is a member of the family paired box proteins PAXs [ ] PAX8 consists of amino acids with a molecular weight of approximately kilo Dalton and its molecular properties are located on chromosome 2q13 [“] PAX8 is a transcription factor that regulates ans development during the embryonic period as well as to maintain normal cellular functions in some cells after birth [ ] During the embryonic period PAX8 also plays a significant role Correspondence nouh_saadoutlookcom Alfarrabi College for Science and Technology Khartoum SudanFull list of author information is available at the end of the in the development of genital ans derived from the mesonephric and the M¼llerian ducts [“] In a previous experiment the deletion of the PAX8 gene resulted in dysfunctional uterus absence of the endometrium and the vaginal ing Also resulted in poor development of the myometrial tissue [] Several studies have described the immunohistochemical utility of PAX8 as a diagnostic marker for epithelial cells neoplasms of many glands and ans such as thyroid thymus and kidney as well as some female reproductive tract tumors [ ]In a healthy female reproductive tract PAX8 shown to be overexpressed in the epithelial cells of the endocervix and the endometrium [“] PAX8 was found to be expressed among endometrioid carcinomas transitional The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cAli a0et a0al BMC Res Notes Page of undifferentiated cell carcinomas and the metastatic carcinomas at a range of “ “ and [ “] Whereas for the ovarian carcinomas PAX8 was under expressed [] Considering that few studies have investigated the immunohistochemical expression of PAX8 in carcinomas of the endometrium and uterine cervix in the different parts of the world but none from Sudan yet [ “] This study aimed at investigating the immunohistochemical expression of PAX8 among Sudanese females diagnosed with cervical endometrial and ovarian carcinomasMain textMaterials and a0methodsStudy design and a0population characteristicsThis is a descriptive retrospective hospital based study conducted at different histopathology laboratories during the period from December till May in Khartoum State Sudan We retrieved archived formalin fixed paraffin embedded blocks previously collected from female patients with cervical endometrial or ovarian carcinomas The retrieved formalin fixed paraffin blocks represent all the female population admitted at the hospitals for reproductive malignancies diagnosis The participants demographic data was collected including age place of residence The clinical data including site of cancer cancer grade and the histopathological diagnosis were also collectedSections Preparation for a0Immunohistochemistry StainingTwo sections were cut using Rotary microtome Leica Germany from each histopathological block Then one slide was stained by hematoxylin and eosin staining technique The other slide was mounted onto 3aminopropyltriethoxysilane coated slides for immunohistochemistry To retrieve PAX8 tissue™s antigen we treated the sections with citrate buffer at ° a0C for a0min in a waterbath Then the tissue sections were rinsed first in distilled water and later with Tris buffer saline TBS This was followed by treatment with peroxidase block hydrogen peroxide in methyl alcohol for a0min to quench endogenous peroxidase activity The slides were then placed in a humid chamber Then the slides were drained and rinsed in two successive changes of Tris buffer wash buffer for a0 min each Nonspecific protein“protein interactions were blocked by incubating and treating the tissue sections in a humid chamber with the power block casein in phosphate buffered saline for a0 min Then the remaining solution was drained from the slides The sections were then incubated in the primary antibody PAX8 antiPAX8 rabbit antihuman monoclonal antibody ab189249 Abcam United Kingdom at room temperature in the humid chamber according to the manufacture instructionsObserving the yellowishbrown or brown appearance of the nucleus was considered a positive result for the PAX8 For the negative control we omitted the incubation with the primary antibody step instead we incubated the section in the phosphate buffer saline PBSResults interpretationsFor the interpretation of the results we depended on the intensity as well as the number of the cells that expressed the marker and the expression was graded into categories Negative No staining less than of the cells were expressing the marker “ of the cells were expressing the marker more than “ of the cells were expressing the marker more than of the cells were expressing the marker The slides were interpreted and validated by two expert pathologists blindly of each other results Photomicrographs were taken using Olympus SP350 camera Olympus Imaging America Inc USAStatistical analysisThe statistical analysis of the results was done using IBM SPSS Statistics vs The ChiSquared test was performed to compare the frequencies of categorical variables Statistical significance level was defined as p value at confidence intervalResultsCharacteristics of a0the a0study participantsThe study included patients diagnosed with female genital tract cancer Patients aged ± a0years range “ a0years Patients were grouped into age groups Those aged “ a0 years constituted half of the study participants The remaining were and patients distributed across the remaining age groups of “ a0 years “ a0 years and “ a0 years respectively According to patients™ place of residence patients were originating from the four regions of Sudan Most of the patients were from western part of Sudan followed by from the central part of SudanRegarding the site of cancer the cervix was the most commonly involved patients There were and endometrial and ovarian cancer respectively Based on the International Federation of Gynecology and Obstetrics FIGO cancer grading the majority of the study population was diagnosed with stage 3B and 2B cancer and of the patients respectively The were and stage 4B 3A 2A 1B and 4A respectively 0cAli a0et a0al BMC Res Notes Page of No statistically significant association between FIGO staging and age group was found P value Histologically there were squamous cell carcinoma SCC all of which were cervical cancers and adenocarcinoma SCC and adenocarcinoma were further classified into poorly differentiated SCC moderately differentiated SCC and well differentiated SCC endometrium adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinomaBased on age groups age group showed no statistically significant relationship with either patients™ place of residence cancer site cancer histological type FIGO staging and cancer histopathological type Table a0Immunohistochemical Expression of a0PAX‘The immunohistochemical expression of PAX8 was shown as a yellowishbrown or brown staining of the nucleus Fig a0 Based on site of cancer all endometrium carcinoma showed positive expression of PAX8 with P value There were only patients who had positive expression of PAX8 including adenocarcinoma and SCC A statistically significant difference was noted for the PAX8 staining and cancer type with P value The analysis of PAX8 staining results based on the histopathological diagnosis showed that all patients who were diagnosed with well differentiated SCC and metastatic adenocarcinoma had negative results for the PAX8 expression While of the endometrium adenocarcinoma were found positive for the PAX8 expression A statistically significan was t seen for PAX8 expression and the different histopathological diagnosis P value Table a0Table Classification of a0Participants demographic and a0clinical diagnosis based on a0age groupAge group no Total no P value” a0years” a0years” a0years” a0yearsResidence of patient Central Sudan East Sudan West Sudan North SudanSite of cancer Cervix Endometrium OvaryCancer histological type SCC AdenocarcinomaFIGO staging Stage Stage 2A Stage 2B Stage 3A Stage 3B Stage 4A Stage 4BHistopathological cancer grades Well differentiated SCC Poorly differentiated SCC Moderately differentiated SCC Endometrium adenocarcinoma Endocervical adenocarcinoma Metastatic adenocarcinoma Ovarian mucinous cyst adenocarcinomaSCC Squamous Cell Carcinoma 0cAli a0et a0al BMC Res Notes Page of Fig Immunohistochemical expression of PAX8 among the different histopathological cancer types and grades The immunohistochemical expression of PAX8 is shown as a yellowishbrown or brown staining of the nucleus a Well differentiated SCC negative b Metastatic adenocarcinoma negative c Poorly differentiated SCC positive d Moderately differentiated SCC positive e Endometrium adenocarcinoma positive f Ovarian mucinous cystadenocarcinoma positive g Endocervical adenocarcinoma positive and h endometroid adenocarcinoma positive 0cAli a0et a0al BMC Res Notes Page of Table Association of a0clinical diagnosis and a0the a0immunohistochemical expression of a0PAX8PAX results no Total no P valuePositiveNegativeCancer histological type SCC AdenocarcinomaCancer site Cervix Endometrium OvaryFIGO staging Stage Stage 2A Stage 2B Stage 3A Stage 3B Stage 4A Stage 4BCancer histopathological grading Well differentiated SCC Poorly differentiated SCC Moderately differentiated SCC Endometrium adenocarcinoma Endocervical adenocarcinoma Metastatic adenocarcinoma Ovarian mucinous cyst adenocarcinomaSCC Squamous Cell Carcinoma DiscussionPrevious studies on the immunohistochemical expression of PAX8 in the normal female reproductive tract showed that PAX8 was expressed in the endometrial endocervical and ovarian epithelial cells as well as in nonciliated epithelial cells of the fallopian tubes [ ] This study investigated the immunohistochemical expression of PAX8 in Sudanese patients who were diagnosed with female reproductive tract cancers Patients on the 5th decade of life were constituting half of the study participants with no statistically significant association between age group and the type of cancer However previous studies had suggested other risk factors which could contribute in the development of certain gynecological cancer [ ]Regarding the place of residence the majority of patients coming from western Sudan This result is in contrary with a previous study in Sudan conducted by Saeed et a0al in which they showed that the percentage of patients suffering from different types of cancers residing in central and northern Sudan were higher compared to the other regions in Sudan [] Nevertheless these findings could suggest the involvement of environmental risk factors however the limited study samples size is insufficient to support this suggestion Therefore further research with a larger samples size investigating the potential environmental risk factors is essential for strategic prevention and protection measuresThe reported number of female patients with cervical cancer was high compared to ovarian and endometrium cancer Similar results were seen previously among Sudanese females [] Also the high frequency of stages 3B and 2B compared to the other stages were comparable to previous study conducted in Sudan [] This similarity underscores a delayed response among Sudanese females in seeking healthcare and urge the need for health promotion and education to encourage young Sudanese females for the early signs detection and seeking healthcare as early as possible for a better treatmentRegarding the classification based on the histopathological diagnosis most of the female diagnosed with SCC This result was also similar to previous study investigated the prevalence of the different gynecologic cancer in Sudan [] However the expression of PAX8 among the studied samples was relatively low compared to previous studies [ ] this could be attributed 0cAli a0et a0al BMC Res Notes Page of to the site of cancer development While agrees with another study where PAX8 was expressed only in patient []Interestingly a high frequency of PAX8 expression was noted among females diagnosed with endometrium cancer compared to SCC this finding is in contrary with a previous report where PAX8 was expressed among only of the studied samples [] Also the result was strongly in accordance with other studies [ ] Besides that the lack of PAX8 expression among those who were diagnosed with well differentiated SCC and metastatic adenocarcinoma could play a significant role in either gynecologic cancer differentiation or in detection of endometrium adenocarcinoma progression to metastatic adenocarcinoma [ ]ConclusionAlthough PAX8 showed a significant expression among adenocarcinomas lesions and negative expression was noted among those with well differentiated SCC and metastatic adenocarcinoma PAX8 might not be beneficial when used alone as a diagnostic marker for the tumors that occur in the female reproductive tractLimitations¢ The small sample size investigated in this study reduced the ability of using the expression of PAX8 as a diagnostic marker Therefore a largescale study is needed and it should include other types of malignant tumors encountered in the female reproductive systemAcknowledgementsThe authors would like to acknowledge the medical staff for their interest and cooperation during the study and thanks to all who participated in completing this studyAuthors™ contributionsETA NSM and EES provided conceptual framework for the study guidance for interpretation of the data and performed data analysis ETA EES IRS LAH and AMM performed laboratory work NSM EES MSM AAY and AA performed the statistical analysis NSM MSM EES and AA participated in the manuscript preparation revision and coordination All authors read and approved the final manuscriptFundingNot ApplicableAvailability of data and materialsThe datasets used andor analyzed during the current study are available from the corresponding author on reasonable requestEthics approval and consent to participateEthical approval was obtained from the Research Ethics Committee of the Faculty of Medical Laboratory Sciences University of Khartoum Sudan Ethical Approval No FMLSREC002042 All participant approved to participate by signing an informed consentConsent for publicationNot ApplicableCompeting interestsNo competing interests to discloseAuthor details Department of Histopathology and Cytology Faculty of Medical Laboratory Sciences University of Khartoum Khartoum Sudan Department of Histopathology and Cytology Faculty of Medical Laboratory Sciences National University Khartoum Sudan Alfarrabi College for Science and Technology Khartoum Sudan Faculty of Medicine Sinnar University Sennar Sudan Molecular Biology Department Faculty of Medical Laboratory Sciences Nile University Khartoum Sudan Faculty of Dentistry Ibn Sina University Khartoum Sudan Department of Neurology Mayo Clinic Jacksonville FL USA Department of Radiology Mayo Clinic Jacksonville FL USA Institute of Endemic Diseases University of Khartoum Khartoum Sudan Mycetoma Research Center University of Khartoum Khartoum Sudan Faculty of Medicine Nile University Khartoum Sudan Received July Accepted August References Gruss P Walther C Pax in development Cell “ Mansouri A Hallonet M Gruss P Pax genes and their roles in cell differentiation and development Curr Opin Cell Biol “ Macchia PE Lapi P Krude H Pirro MT Missero C Chiovato L Souabni A Baserga M Tassi V Pinchera A PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis Nat Genet “ Vilain C Rydlewski C Duprez L Heinrichs C Abramowicz M Malvaux P Renneboog Bt Parma J Costagliola S Vassart G Autosomal dominant transmission of congenital thyroid hypoplasia due to lossoffunction mutation of PAX8 J Clin Endocrinol Metab “ Park S VK C Genetics of congenital hypothyroidism J Med Genet “ Dahl E Koseki H Balling R Pax genes and anogenesis BioEssays “Lang D Powell SK Plummer RS Young KP Ruggeri BA PAX genes roles in development pathophysiology and cancer Biochem Pharmacol “Stoykova A Gruss P Roles of Paxgenes in developing and adult brain as suggested by expression patterns J Neurosci “ Mittag J Winterhager E Bauer K Grummer R Congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy Endocrinology “ Bouchard M de Caprona D Busslinger M Xu P Fritzsch B Pax2 and Pax8 cooperate in mouse inner ear morphogenesis and innervation BMC Dev Biol Mittag J Winterhager E Bauer K Grummer RJE Congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy Endocrinolog “ Laury AR Perets R Piao H Krane JF Barletta JA French C Chirieac LR Lis R Loda M Hornick JL A comprehensive analysis of PAX8 expression in human epithelial tumors Am J Surg Pathol “ Wong S Hong W Hui P Buza N Comprehensive analysis of PAX8 expression in epithelial malignancies of the uterine cervix Int J Gynecol Pathol “ Ozcan A Shen SS Hamilton C Anjana K Coffey D Krishnan B Truong LD PAX expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study Mod Pathol “ Bowen NJ Logani S Dickerson EB Kapa LB Akhtar M Benigno BB McDonald JF Emerging roles for PAX8 in ovarian cancer and endosalpingeal development Gynecol Oncol “ 0cAli a0et a0al BMC Res Notes Page of Ozcan A Liles N Coffey D Shen SS Truong LD PAX2 and PAX8 expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison Am J Surg Pathol “distinguishing ovarian mucinous neoplasms from colonic and appendiceal mucinous neoplasm BMC Res Notes Nesrin R KILIC D Risk factors for cervical cancer results from a hospital Ozcan A Liles N Coffey D Shen SS Truong LDJTAjosp PAX2 and PAX8 based casecontrol study Int J Hematol Oncol “expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison Am J Surg Pathol “ Nonaka D Tang Y Chiriboga L Rivera M Ghossein R Diagnostic utility of thyroid transcription factors Pax8 and TTF2 FoxE1 in thyroid epithelial neoplasms Mod Pathol “ Tacha D Zhou D Cheng L Expression of PAX8 in normal and neoplastic tissues a comprehensive immunohistochemical study Appl Immunohistochem Mol Morphol “ Bowen NJ Logani S Dickerson EB Kapa LB Akhtar M Benigno BB McDonald JFJGo Emerging roles for PAX8 in ovarian cancer and endosalpingeal development Gynecol Oncol “ K¶bel M Kalloger SE Boyd N McKinney S Mehl E Palmer C Leung S Bowen NJ Ionescu DN Rajput A Ovarian carcinoma subtypes are different diseases implications for biomarker studies PLoS medicine 2008512e232 Nonaka D Chiriboga L Soslow RA Expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas Am J Surg Pathol “ Tong GX Devaraj K HameleBena D Yu WM Turk A Chen X Wright JD Greenebaum E Pax8 a marker for carcinoma of M¼llerian origin in serous effusions Diagn Cytopathol “ Laury AR Perets R Piao H Krane JF Barletta JA French C Chirieac LR Lis R Loda M Hornick JLJTAjosp A comprehensive analysis of PAX8 expression in human epithelial tumors Am J Surg Pathol “ Tong GX Devaraj K HameleBena D Yu WM Turk A Chen X Wright JD Greenebaum EJDc Pax8 a marker for carcinoma of M¼llerian origin in serous effusions Diagn Cytopathol “ Chu PG Chung L Weiss LM Lau SK Determining the site of origin of mucinous adenocarcinoma an immunohistochemical study of cases Am J Surg Pathol “ Brunner AH Riss P Heinze G Meltzow E Brustmann H Immunoexpression of PAX in endometrial cancer relation to highgrade carcinoma and p53 Int J Gynecol Pathol “ Ozcan A Shen SS Hamilton C Anjana K Coffey D Krishnan B Truong LDJMP PAX expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study Mod Pathol “ Aldaoud N Erashdi M AlKhatib S Abdo N AlMohtaseb A GraboskiBauer A The utility of PAX8 and SATB2 immunohistochemical stains in Saeed ME Cao J Fadul B Kadioglu O Khalid HE Yassin Z Mustafa SM Saeed E Efferth T A fiveyear survey of cancer prevalence in Sudan Anticancer Res “ Saeed ME Cao J Fadul B Kadioglu O Khalid HE Yassin Z Mustafa SM Saeed E Efferth TJAr A fiveyear survey of cancer prevalence in Sudan Anticancer Res “ Mohamed KEH Ashmeig AAA Cervical cancer our experience in Sudan Philadelphia AACR Elhasan LME Bansal D Osman OF Enan K Abd Farag EAB Prevalence of human papillomavirus type in Sudanese women diagnosed with cervical carcinoma J Cancer Res Ther Tacha D Zhou D Cheng LJAI Morphology M Expression of PAX8 in normal and neoplastic tissues a comprehensive immunohistochemical study Appl Immunohistochem Mol Morphol “ Ord³±ez NG Value of PAX immunostaining in tumor diagnosis a review and update Adv Anat Pathol “ Gailey MP Bellizzi AM Immunohistochemistry for the novel markers glypican PAX8 and p40 ΔNp63 in squamous cell and urothelial carcinoma Am J Clin Pathol “ Yemelyanova A Gown AM Holmes BJ Ronnett BM Vang R PAX8 expression in uterine adenocarcinomas and mesonephric proliferations Int J Gynecol Pathol “ Liang L Zheng W Liu J Liang SX Assessment of the utility of PAX8 immunohistochemical stain in diagnosing endocervical glandular lesions Arch Pathol Lab Med “ Wong S Hong W Hui P Buza NJIJoGP Comprehensive analysis of PAX8 expression in epithelial malignancies of the uterine cervix Int J Gynecol Pathol “ De Andrade DAP Da Silva VD de Macedo MG De Lima MA de Andrade VM Andrade CEMC Schmidt RL Reis RM Dos Reis R Squamous differentiation portends poor prognosis in low and intermediaterisk endometrioid endometrial cancer PLoS ONE 20191410e0220086Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations ¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your field¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold Access which fosters wider collaboration and increased citations maximum visibility for your research over 100M website views per year ¢ At BMC research is always in progressLearn more biomedcentralcomsubmissionsReady to submit your research Choose BMC and benefit from 0c'

Thyroid_Cancer

edible fungus with a high nutritional value and medicinal effect theBachu mushroom has a broad market To distinguish among Bachu mushrooms with highvalue and other fungi effectively and accurately as well as to explore a universal identification method this study proposed a method to identify Bachu mushrooms by Fourier Transform Infrared Spectroscopy FTIR combined with machine learning In this experimenttwo kinds of common edible mushrooms Lentinus edodes and club fungi were selectedand classified with Bachu mushrooms Due to the different distribution of nutrients in thecaps and stalks the caps and stalks were studied in this experiment By comparing the average normalized infrared spectra of the caps and stalks of the three types of fungi we founddifferences in their infrared spectra indicating that the latter can be used to classify andidentify the three types of fungi We also used machine learning to process the spectraldata The overall steps of data processing are as follows use partial least squares PLS toextract spectral features select the appropriate characteristic number use different classification algorithms for classification and finally determine the best algorithm according to theclassification results Among them the basis of selecting the characteristic number was thecumulative variance interpretation rate To improve the reliability of the experimental resultsthis study also used the classification results to verify the feasibility The classification algorithms used in this study were the support vector machine SVM backpropagation neuralnetwork BPNN and knearest neighbors KNN algorithm The results showed that thethree algorithms achieved good results in the multivariate classification of the caps andstalks data Moreover the cumulative variance explanation rate could be used to select thecharacteristic number Finally by comparing the classification results of the three algorithms the classification effect of KNN was found to be the best Additionally the classification results were as follows according to the caps data classification the accuracy was according to the stalks data classification the accuracy was This studyshowed that infrared spectroscopy combined with a machine learning algorithm has thepotential to be applied to identify Bachu mushrooms and the cumulative variancea1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Gao R Chen C Wang H Chen C Yan ZHan H Classification of multicategoryedible fungi based on the infrared spectra of capsand stalks e0238149 101371journalpone0238149Editor Jie Zhang Newcastle University UNITEDKINGDOMReceived April Accepted August Published August Peer Review History PLOS recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s Theeditorial history of this is available here101371journalpone0238149Copyright Gao This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscript and its SupportingInformation filesFunding This work has been supported by theScience and Technology Project on aid to XinjiangPLOS ONE 101371journalpone0238149 August PLOS ONE 0cUygur Autonomous Region No2018E02058 theNational Science Foundation of ChinaNo61765014 Xinjiang Uygur AutonomousRegion Graduate Innovation Project of ChinaNational Innovation Program for College studentsNo201910755039 the Urumqi Science andTechnology Project No P161310002 theReserve Talents Project of the National HighlevelPersonnel of Special Support ProgramQN2016YX0324Competing interests The authors have declaredthat no competing interests existClassification of multicategory edible fungi based on the infrared spectra of caps and stalksexplanation rate can be used to select the characteristic number This method can also beused to identify other types of edible fungi and has a broad application prospect IntroductionThe Bachu mushroom is a characteristic edible fungus in Xinjiang China It belongs to the Saddle fungus genus and is produced in the natural Populus euphratica forest region of the Yeerqiang River Basin in Xinjiang [] The Bachu mushroom not only has high nutritional valuerich in various amino acids and proteins but also has high medicinal value [] Studies haveshown that the Bachu mushroom has antitumor antioxidation and cholesterollowering effectsand is used to treat gastric cancer cerebral arteriosclerosis cardiovascular disease hypertensionand other diseases [] Its nutritional value is much higher than that of general edible fungithus it is of great research value However because the Bachu mushroom cannot be cultivatedartificially the market has been in short supply increasing its price Presently the processingtechnology of the Bachu mushroom is developing and relatively mature such as polysaccharideextraction of the Bachu mushroom and preparation of compound beverages and these processing technologies have a wide application prospect [“] In the future after the derivatives ofthe Bachu mushroom are mass produced and the treatment process is widely used controllingthe quality of raw materials will be very significant to ensure product quality Therefore to prevent businesses from choosing other lowpriced mushrooms as raw materials for high profits itis necessary to identify a simple and rapid way to distinguish Bachu mushrooms from othertypes of mushrooms However current methods to identify Bachu mushrooms and other ediblefungi depend on appearance This method can distinguish mushroom species to a large extentbut it also has great limitations”that is it is limited to individual intact edible bacteria”but liquid extract and powdered mushroom powder cannot be distinguished Thus to overcome thelimitations of conventional methods and explore a more universal mushroom classificationmethod the spectral data of Bachu mushroom powder and two other types of mushroom powder were measured and the classification of Bachu mushroom powder and other types of mushroom powder was identified by a machine learning algorithm in this studyDue to the differences in the types and contents of nutrients in the stalks and caps of ediblefungi the caps and stalks were analyzed in this study [] Previous studies have shown somedifferences in the contents of proteins and amino acids between caps and stalks in edible fungi[] Additionally the difference in the distribution of nutrients is related to the species of edible fungi [] Therefore to make the experimental results more accurate and persuasive aswell as to avoid the uneven distribution of the substance content in the samples this studyadopted the classification analysis method of grouping according to the attribute index tostudy the caps and stalks []The research method used in this experiment classified the Bachu mushroom and two othertypes of edible fungi by infrared spectra combined with a machine learning algorithm Infraredspectra have the characteristics of wide applicability high efficiency convenience repeatabilityand high sensitivity thus it has been widely used in physics remote sensing biology foodmedical and other research fields [ ] Infrared spectroscopy has great application value infood research [ ] Additionally infrared spectroscopy combined with a machine learningalgorithm has been applied to the classification of mushroom as food the effect of the producing area on the nutritional value of fungi and the fine classification of rare edible fungi [“] The purpose of this study was to explore a universal identification method to identifyBachu mushroom using infrared spectra combined with a machine learning algorithm andPLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksverify the feasibility of the application of infrared spectra combined with an algorithm in theidentification of edible mushroom species Experimental methods Sample preparationIn this study Lentinus edodes and club fungi were selected and classified with Bachu mushrooms Among them Lentinus edodes are produced in Fujian Province of China and purchased from Fuchang Food Limited Company Fujian Province of China club fungi areproduced in Yunnan Province of China purchased from Wuweijin Store and Bachu mushrooms are produced in Bachu County Xinjiang Province and purchased from the mostfamous wholesale market in Urumqi”Six Markets The three kinds of mushrooms purchasedare all dried The caps of the three kinds of mushrooms are umbrellashaped and dark brownThe stalks of club fungi are longer and those of Lentinus edodes and Bachu mushrooms areshorter The three kinds of mushrooms are similar in appearance The samples of the threetypes of edible fungi were purchased from the market After incubating the prepared sample inan ˚C electric steam oven for dehydration treatment for hour the stalks were separatedfrom the caps Next every three complete caps were crushed together and the powder waspassed through a 200mesh sieve as a sample and named according to the mushroom speciesThe stalks were treated in the same way Finally samples of Lentinus edodes powder samples of club fungi powder and samples of Bachu mushroom powder were obtained Measurement of NIF spectraThe sample powder was placed into a 4ml sample tube and its infrared spectrum was measured The spectrum acquisition instrument was a VERTEX infrared spectrometer fromBRUKER Germany Before each measurement of the FTIR spectrum the atmospheric background data were measured using OPUS65 software The selected resolution was cm1 thenumber of scans was the scan range was “ cm1 and the atmospheric compensation parameter was CO2 To reduce the influence of human error and other factors each sample was scanned times Finally the data obtained for the caps were for Lentinus edodes for club fungi for Bachu mushrooms and the same number of stalks data Statistical algorithm analysisStatistical algorithms have been widely used to manage infrared spectral data [] In thisstudy PLS SVM KNN and BPNN were used to process and analyze the spectral data Thecaps data were reduced by PLS to extract features and then the appropriate characteristicnumber was selected as the input of the three classification algorithms namely SVM KNNand BPNN and then the accuracy was obtained Additionally the stalk data were processed inthe same way All algorithms in this study are implemented on MATLAB 2018aThe partial least squares PLS method is a mathematical optimization supervised learningmethod that can identify the best matching function for a set of data by minimizing the sum ofthe squares of errors Based on the advantages of the PLS model which is easy to identify noiseand allows regression modeling with a small sample number the PLS algorithm is widely usedin various research fields [ ] In food research PLS has been used in food nutrition testingfood quality research and food industry research [“] PLS is often used in combinationwith spectra for feature extraction and further spectral data analysis [] In this study toimprove the classification efficiency and filter out the worthless spectral information PLS wasused to reduce the dimensionality of the original spectral dataPLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksAfter dimensionality reduction of the original data an appropriate characteristic numbershould be selected as the basis for classification In this study the characteristic number wasselected based on the cumulative variance interpretation rate of the characteristic number ThePLS program used in this study is the plsregress function The variance explanation rates of thefactors extracted from the first and second columns of the PCTVAR matrix correspond to thevariances of x and y respectively this study chose the variance explanation rate of y [] Thevariance interpretation rate is the degree of interpretation of the data characteristics of thedependent variables by a single factor and the cumulative interpretation rate of n factors is thedegree of interpretation of the data characteristics of the dependent variables by n factors”that is the influence of n factors on the dependent variables Therefore in theory we can selectthe appropriate number of factors according to the cumulative variance interpretation rateand select as few factors as possible to improve the classification efficiency to ensure the integrity of the extracted features To explore the applicability of the theory this study will furtheruse the classification results to verify the theory After selecting an appropriate characteristicnumber it can be used as the input of the classification algorithms SVM KNN and BPNNSupport vector machine SVM is a commonly used generalized linear classifier in whichthe core idea is to apply the principle of risk minimization to the field of classification Regarding pattern classification it has good generalization performance robustness versatility andsimple calculation [] Therefore in food science SVM is widely applied to food classificationand food quality testing [ ] Based on the advantages of SVM and characteristics that canbe used for multiple classifications in this study SVM was used to classify the spectral data ofmushroom powder directly after three features were extractedThe knearest neighbors KNN classification algorithm is one of the most practical algorithms in data mining classification technology It is easy to understand and powerful at thesame time [] Different from other classification algorithms KNN does not need training Itdirectly finds the k samples nearest to the sample and divides them into categories with thelargest number of samples among the k samples thus KNN is suitable for multivariate classification and has high classification accuracy when the category boundary is obvious [] Additionally KNN has been widely used in food classification and quality inspection [“]Therefore we chose the KNN algorithm as the second algorithm of multivariate classificationThe backpropagation neural network BPNN is a multilayer feedforward neural networktrained according to the error backpropagation algorithm The BP neural network has strongnonlinear mapping ability parallel information processing ability and excellent selflearningability thus it has been widely used in food research biomedical fields and other researchfields [“] Additionally the BP neural network can achieve good classification resultswhen it is used in multivariate classification [] In summary we chose the BPNN as the thirdclassification algorithm Results and discussion Spectral analysisAfter the obtained spectral data were averaged normalized and smoothed the obtained spectrogram is shown in Fig Fig shows the FTIR spectra of the Bachu mushroom and Lentinusedodes stalks were similar both with characteristic peaks at cm1 and cm1 and thespectral intensity of Lentinus edodes was higher than that of the Bachu mushroom Additionally the spectrum of the Lentinus edodes stalk has a characteristic peak at cm1 and thespectral intensity in the range of cm1 was significantly lower than that of Lentinusedodes and the Bachu mushroom Comparing the spectra of the three caps the spectra of clubfungi are quite different from those of the other two types of fungi Fig shows that the averagePLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig The average normalized spectrum of the caps of the three types of edible fungi101371journalpone0238149g001normalized spectra of Lentinus edodes club fungi and Bachu mushroom stalks all had peaks at cm1 and cm1 and the spectral intensity of Lentinus edodes was the highest in thesetwo places At cm1 the peak intensity of club fungi was higher than that of the Bachumushroom however at cm1 the peak intensity of club fungi was slightly lower than thatof the Bachu mushroom Although the three spectral lines changed roughly the same howeverin the range of cm1 the spectral intensity of the Bachu mushroom was significantlylower than that of the other two kinds of fungi and the spectral intensity of the Bachu mushroom was significantly lower than that of the other two kinds of fungiThrough comparative analysis of the infrared spectra of the caps and stalks of the threetypes of fungi their infrared spectral images showed the same trend but many peak intensitieswere different Therefore we can classify them according to the spectral data based on thePLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig The average normalized spectrum of the stalks of the three types of edible fungi101371journalpone0238149g002infrared spectral differences among the three types However it is difficult to distinguishamong the three types of edible fungi directly and accurately only by spectroscopy Thus toclassify them efficiently and accurately we used the combined infrared spectrum analysis withmachine learning Data analysis Dimensionality reduction with PLSIn the PLS algorithm features wereselected to obtain the cumulative variance explanation rate curve Figs and The cumulative variance explanation rate of the first five features of the caps reached while the variance explanation rate of the first five features of the stalks also reached more than and thePLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig The principal component cumulative variance explanation rate curve of the caps101371journalpone0238149g003cumulative variance explanation rate of the first features of both was close to Figs and Thus the extracted features can fully express the features of the original data [] Thestalks data and caps data were classified by the SVM BPNN and KNN algorithms Classification by algorithmIn this experiment three classification algorithmswere used”SVM BPNN and KNN”to classify the caps data according to different characteristic numbers Additionally the stalk data were processed in the same way The parameter setting and classification results of the algorithm were as followsThe main ideas of the SVM model in this experiment were as follows Select the test set andtraining set preprocess the data select the best C and g parameters and then use the bestparameters for network training and prediction and obtain the accuracy Among them thetraining set and test set are randomly selected according to the proportion Preprocessingwas used to normalize all the sample data [] In the SVM algorithm the selection of C and gPLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig The principal component cumulative variance explanation rate curve of the stalks101371journalpone0238149g004parameters will directly affect the classification results thus it is necessary to select the best Cand g parameters to achieve the best classification results In this study the variation range ofparameter C was [“] the range of parameter g was [“] and the method of parameteroptimization was grid optimization [] To classify the caps data and features were selected The stalks data were processed in the same way The multipleclassification results of the stalks and caps were then obtained as shown in Table In the KNN algorithm the k value was the proportion of the random selection of the testset was and the method to calculate the distance between data was the cosine distanceCosine KNN [] To classify the caps data and featureswere selected The stalks data were processed in the same way Each result was expressed as theaverage of five computational results The multiple classification results of the stalks and capswere then obtained as shown in Table PLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksTable Classification results of the SVM algorithmNumber of PLS componentsClassification accuracy of CapsClassification accuracy of Stalks101371journalpone0238149t001In the BPNN algorithm of this experiment the transfer function of the hidden layer wastamsig the output layer was purelin the learning training function was trainlm and the weightlearning function was learngdm The network parameters were set to training times thenetwork performance goal was and the learning rate was [] Thirty percent of allsamples were selected randomly as the test set To classify the caps data and features were selected The stalks data were processed in the same way Eachresult was expressed as the average of five computational results The multiple classificationresults of the stalks and caps were then obtained as shown in Table Verification of the feasibility of selecting the characteristic number with thecumulative variance explanation rate Figs and shows the line chart of the classificationresults of the three algorithms when selecting different characteristic numbers The accuracyof SVM in the classification of caps was when selecting and features decreasedslightly with the increase in the characteristic number and then stabilized at Accordingto the fungal stalks data the accuracy of SVM classification increased gradually when selecting features fluctuated slightly with the increase in the characteristic number andthen stabilized at Using KNN to classify the caps data the accuracy was stable between and but fluctuated slightly When the characteristic number tended to theaccuracy was stable at According to the classification of stalks data by KNN the accuracy varied greatly when selecting “ features fluctuated slightly with the increase in thecharacteristic number and then stabilized at For the classification of caps by BPNNthe accuracy varied greatly when the characteristic number was and the classificationTable Classification results of the KNN algorithmNumber of PLS componentsClassification accuracy of CapsClassification accuracy of Stalks101371journalpone0238149t002PLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksTable Classification results of the BPNN algorithmNumber of PLS componentsClassification accuracy of CapsClassification accuracy of Stalks 101371journalpone0238149t003Fig Accuracy of the three algorithms to select different characteristic numbers according to the data of the caps101371journalpone0238149g005PLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksFig Accuracy of the three algorithms to select different characteristic numbers according to the data of the stalks101371journalpone0238149g006effect was unstable When the characteristic number was more than the accuracy decreasedslightly and finally stabilized at When BPNN classified stalks its accuracy variedgreatly when the characteristic number was less than when the characteristic number wasmore than the accuracy was stabilized at Combined with the cumulative variance explanation rate with the increase in the characteristic number the extraction degree of the extracted features to the original data informationgradually increased Thus the classification results become more reliable and the accuracywill gradually tend to a certain value”that is in the ideal state the accuracy of all featuresextracted for classification However in this process not all information is conducive toimproving accuracy and some information will interfere with the classification results thusthe accuracy will fluctuate slightly [] Therefore when we select the characteristic numberaccording to the variance interpretation rate we should consider the degree of featurePLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksextraction accuracy and classification efficiency comprehensively In summary when thecumulative variance interpretation rate of features reaches the classification results aresufficiently reliable and the accuracy is high confirming that it is feasible to select the characteristic number according to the cumulative variance interpretation rate Selection of the best algorithm According to the caps data classification thePLSSVM algorithm has the best classification effect when selecting features with anaccuracy of The PLSKNN algorithm has the best classification effect when selecting features with an accuracy of When selecting features the PLSBPNN algorithmhas the best classification effect with an accuracy of Fig According to the stalksdata classification the classification effect of the PLSSVM algorithm was the best when selecting features with an accuracy of The classification effect of the PLSKNN algorithm was the best when selecting features with an accuracy of When featureswere selected the classification effect of the PLSBPNN algorithm was the best reaching Fig Combined with the selection of the characteristic number to analyze the three algorithmsthe PLSKNN algorithm has a better classification effect on stalks and caps and the accuracy ismore stable when selecting different characteristic numbers Thus the PLSKNN algorithmwas chosen as the optimal algorithm Using comprehensive analysis of the accuracy ofPLSKNN classification when selecting different characteristic numbers a characteristic number of reveals a higher accuracy for both caps and stalks and a high classification efficiencyTherefore in this experiment the PLSKNN algorithm was finally selected and the characteristic number was The final classification accuracy was for the caps and forthe stalks ConclusionsThis study verified the feasibility of infrared spectroscopy combined with the PLSSVMPLSKNN and PLSBPNN algorithms in the classification of the Bachu mushroom and otheredible mushrooms We compared the classification results and selected the optimal algorithmand best feature number to reveal an efficient rapid and universal method to identify theBachu mushroom overcoming the limitation that the current identification of Bachu mushroom only depends on its appearance Moreover the method is universal and can be applied tothe classification and identification of other types of food Additionally this study proposed toselect the characteristic number according to the cumulative variance interpretation rate andused the classification results of the three algorithms to verify its feasibility This method ofselecting the characteristic number can also be extended to other research fields of factor analysis and the appropriate characteristic number can be selected intuitively and quicklySupporting informationS1 DataZIPAuthor ContributionsConceptualization Cheng Chen Zhiao WangData curation Rui Gao Cheng Chen Hang Wang Chen Chen Ziwei Yan Yan WuFormal analysis Hang WangFunding acquisition Cheng Chen Chen Chen Fangfang Chen Zhiao WangPLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalksInvestigation Rui Gao Fangfang ChenMethodology Rui Gao Cheng Chen Chen Chen Yuxiu ZhouProject administration Rui Gao Cheng Chen Rumeng Si Xiaoyi LvResources Rui Gao Ziwei Yan Fangfang Chen Yan WuSoftware Ziwei YanSupervision Cheng ChenValidation Rui Gao Rumeng SiWriting “ original draft Rui Gao Hang Wang Zhiao Wang Yuxiu ZhouWriting “ review editing Rui Gao Cheng Chen Huijie Han Xiaoyi LvReferencesZhao Q Species clarification of the culinary Bachu mushroom in western China Mycologia p “ 10385216002 PMID Chen X Wang J and Li H Basic Ingredientsand Nutritional Assessment Of Baehu Mushroom FarmProducts Processing p “Zeng D and Zhu S Purification characterization antioxidant and anticancer activities of novel polysaccharides extracted from Bachu mushroom International journal of biological macromolecules p “ 101016jijbiomac201709088 PMID XuJie H Extraction of BaChu mushroom polysaccharides and preparation of a compound beverage Carbohydrate Polymers p “ XuJie H and Wei C Optimization of extraction process of crude polysaccharides from wild edibleBaChu mushroom by response surface methodology Carbohydrate Polymers p “ Wang CY A Review on the Potential Reuse of Functional Polysaccharides Extracted from the ByProducts of Mushroom Processing Food and Bioprocess Technology p “ Akindahunsi AA and Oyetayo FL Nutrient and antinutrient distribution of edible mushroom Pleurotustuberregium fries singer LWT”Food Science and Technology p “Zhu Y and Tan ATL Chemometric Feature Selection and Classification of ltigtGanoderma lucidumltigt Spores and Fruiting Body Using ATRFTIR Spectroscopy American Journal of AnalyticalChemistry p “ Oboh G and Shodehinde S Distribution of nutrients polyphenols and antioxidant activities in the pileiand stipes of some commonly consumed edible mushrooms in Nigeria Bulletin of the Chemical Societyof Ethiopia Ball GH Classification Analysis Ferrari M and Quaresima V A brief review on the history of human functional nearinfrared spectroscopy fNIRS development and fields of application Neuroimage p “ 101016jneuroimage201203049 PMID Chen C Exploration research on the fusion of multimodal spectrum technology to improve performance of rapid diagnosis scheme for Thyroid Dysfunction Journal of biophotonics pe201900099 101002jbio201900099 PMID Van de Voort F Fourier transform infrared spectroscopy applied to food analysis Food Research International p “ Chen C Application of near infrared spectroscopy combined with SVR algorithm in rapid detection of cAMP content in red jujube Optik p Meenu M and Xu B Application of vibrational spectroscopy for classification authentication and quality analysis of mushroom A concise review Food chemistry Li Y Geographical traceability of wild Boletus edulis based on data fusion of FTMIR and ICPAES coupled with data mining methods SVM Spectrochim Acta A Mol Biomol Spectrosc p “ 101016jsaa201701029 PMID Fu H A comprehensive quality evaluation method by FTNIR spectroscopy and chemometricFine classification and untargeted authentication against multiple frauds for Chinese Ganoderma lucidum Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy p “PLOS ONE 101371journalpone0238149 August PLOS ONE 0cClassification of multicategory edible fungi based on the infrared spectra of caps and stalks Geladi P and DÃ¥bakk E An overview of chemometrics applications in near infrared spectrometryJournal of Near Infrared Spectroscopy p “ Rosipal R and Kra¨mer N Overview and recent advances in partial least squares in International Statistical and Optimization Perspectives Workshop Subspace Latent Structure and Feature Selection Springer Xie L Quantification of glucose fructose and sucrose in bayberry juice by NIR and PLS FoodChemistry p “Ivorra E Detection of expired vacuumpacked smoked salmon based on PLSDA method usinghyperspectral images Journal of food engineering p “ Heyder M Theuvsen L and HollmannHespos T Investments in tracking and tracing systems in thefood industry a PLS analysis Food Policy p “ Mehmood T et a

Thyroid_Cancer

CD146 was originally identified as a melanoma cell adhesion molecule MCAM and highly expressed in many tumors andendothelial cells However the evidence that CD146 acts as an adhesion molecule to mediate a homophilic adhesion through thedirect interactions between CD146 and itself is still lacking Recent evidence revealed that CD146 is not merely an adhesionmolecule but also a cellular surface receptor of miscellaneous ligands including some growth factors and extracellular matrixesThrough the bidirectional interactions with its ligands CD146 is actively involved in numerous physiological and pathologicalprocesses of cells Overexpression of CD146 can be observed in most of malignancies and is implicated in nearly every step of thedevelopment and progression of cancers especially vascular and lymphatic metastasis Thus immunotherapy against CD146 wouldprovide a promising strategy to inhibit metastasis which accounts for the majority of cancerassociated deaths Therefore todeepen the understanding of CD146 we review the reports describing the newly identified ligands of CD146 and discuss theimplications of these findings in establishing novel strategies for cancer therapySignal Transduction and Targeted Therapy 101038s41392020002598INTRODUCTIONIn Johnson first found that a tumor antigen MUC18was expressed most strongly on metastatic lesions and advancedprimary melanoma with rare detection in benign lesions Due tothe high sequence homology between MUC18 with cell adhesionmolecules CAMs this melanoma antigen was given an officialname melanoma CAM MCAM1 With an increasing number ofdiscoveries about MCAM by various research groups more aliasnames were given to this protein including P1H12 MUC18 A32antigen SEndo1 MelCAM METCAM HEMCAM or CD1461“There are three forms of CD146 proteins in human mouse andchicken The two membraneanchored forms of CD146 areencoded by cd146 gene and soluble form of CD146 sCD146is generated by the proteolytic cleavage ofthe membraneforms11“ Soluble CD146 can be detected in cell culturesupernatants serum and interstitial fluids from either healthyor unhealthy subjects14“ Because sCD146 does not have eitherto cell or cellCAM is a kind of proteins located on the cell surface andmediates contacting and binding of celltoextracellular matrix ECM4 These dynamic interactions providesignals input into the cellular decisionmaking process such as cellgrowth survival migration and differentiation5 essentialforembryonic development and for maintaining the integrity oftissue architecture in adults67 Dependent on adhesion someCAMs can initiate the formation of complexes composed ofextracellular ligands kinases and cytoskeletal proteins8 Abnormalexpression of CAMs can cause various diseases such as cancer and‚ammatory disorders910transmembrane or cytoplasmic regionsit is not competent incellular adhesion1718 Therefore we will not describe sCD146 itsligands and its functions in this review although it is a potentialtarget in tumor microenvironment of CD146positive invasivetumors19Recent evidence has revealed that membranebound CD146may act as a cellsurface receptor to bind with various ligandsinvolved in cellular signaling transduction independent of theadhesion properties In order to deepen the understanding of thefunctions of CD146 in physiological and pathological processeswe summarize the various newly identified ligands of CD146and the ligandelicited roles in signal transduction and discussthe implications of CD146 in remodeling interactions betweenthe cancerous cells with the elements oftheir surroundingmicroenvironmentsTHE CD146 PROTEINMembrane CD146 protein has two isoforms long form CD146lhas a long cytoplasmic tail short form CD146s has a shortcytoplasmic tail1718 These two CD146 isoforms are produced fromdifferent exon splicing strategies and the premature moleculeshave a signal peptide located on the anterior region of the aminoterminal20 In human mature CD146 protein is composed of anextracellular sections with five distinct Iglike domains that exist ina V“V“C2“C2“C2 structural motif a hydrophobic transmembraneregion and a short cytoplasmic tail21 The cytoplasmic domain inboth isoforms contains two potential recognition sites for protein1Key Laboratory of Protein and Peptide Pharmaceuticals Institute of Biophysics Chinese Academy of Sciences Beijing China 2College of Life Science University ofChinese Academy of Sciences Beijing China 3Department of Gastrointestinal Hepatobiliary Tumor Surgery Beijing Shijitan Hospital Capital Medical University Beijing China 4Departments of Pathology Beijing Shijitan Hospital Capital Medical University Beijing China and 5Nanozyme Medical Center School of Basic MedicalSciences Zhengzhou University Zhengzhou ChinaCorrespondence Zhaoqing Wang clairezqwanghotmailcom or Xiyun Yan yanxyibpaccnThese authors contributed equally Zhaoqing Wang Qingji XuReceived March Revised June Accepted June The Authors 0cCD146 from a melanoma cell adhesion molecule to a signaling receptorWang et alkinases C PKC an ERM protein complex of ezrin radixin andmoesin binding site a motif with microvilli extension and adouble leucine motif for basolateral targeting21 The two isoformscoexist as monomers and dimers and the dimerization ismediated through a disulfide bond between cysteine residues inthe C2 domain most proximal to the membrane2022 However theinformation about CD146 crystal structureincluding secondaryand tertiary is still lackingCD146 is a highly glycosylated type I transmembrane proteinand belongs to the immunoglobulin superfamily Based onbioinformation analysis eight putative Nglycosylation sites arepresent in the extracellular fragment across species23 In clear cellrenal cell carcinoma and prostate cancer CD146 glycosylationlevels were upregulated2425 In it was reported that CD146glycosylation is favorably carried out by b13galactosylOglycosylglycoprotein b16Nacetylglucosaminyltransferase3 whichwas overexpressed in highly metastatic melanomas Suchglycosylations can extend CD146 protein stability upregulateCD146 protein levels and lead to elevation of CD146mediatedcellular motility in melanoma cells26 These observations suggestthat the degree of CD146 glycosylation may be directly relatedto malignant progression of tumors especially CD146positiveneoplasmsTHE EXPRESSION PROFILE OF CD146 PROTEINBased on literature metazoan CD146 has been detected inmajority of cell types including vessel constituting cells endothelium pericyte and smooth muscle cell epithelia fibroblastsmesenchymal stem cells and lymphocytes except erythrocytes21Under physiological conditions CD146 expression is restricted tolimited adult normal tissues and its adhesive strength is relativelyweakto most other CAMs which show wideexpression patterns in normal adult tissues and strong adhesionstrength2123 However CD146 expression is broadly and highlydetected in embryonic tissues compared to its abundance innormal adult tissues21 In quickly proliferating cellsincreasedexpression of CD146 may allow cells to actively interact with eachother and with the elements of the cellular microenvironmentpromoting cell proliferation and migrationin contrastUnder pathological conditions such as ‚ammation andtumorigenesis CD146 was upregulated in the related cells andhas been identified as a reliable marker for numerous types ofcancers Accumulating evidence shows that CD146 overexpressionhas been linked to either the initial development of the primarylesion or progression to metastases of most of cancer typesprimarily including melanoma127“ breast63031 ovarian32“lung3637 prostate38“ glioma41 kidney42 hepatic4344 and gastriccancers2145 In Nollet reported that TsCD146 mAb fortumor specific antiCD146 monoclonal antibody can specificallyrecognize CD146 expressed in cancer cells but not CD146 inphysiological vessels suggesting that structural features of cancerCD146 differ from those of physiological CD14628RECOGNITION OF CD146 LIGANDS IN HISTORYThe recognition of CD146 ligands and analysis of their functionswas undertaken over a prolonged period in history Because CD146is highly expressed in vessel cells and cancer cells it is likely thatCD146 within these cells contributes to cancer metastasis throughthe mediation of a homophilic adhesion between cancerous cellsand vascular endothelia a key part of the metastatic processHowever evidence of the direct interactions between CD146 anditself is stillit is possible that CD146mediated adhesion between cancerous cells with vascularendothelia as well as with their surrounding elements occursthrough the bidirectional heterophilic interactions between CD146with its ligands but not the homophilic interaction with itselflacking46“ AccordinglyIn the first CD146™s ligand was found using chickensmooth muscle cells Taniura discovered that neuriteoutgrowth factor NOF was a ligand of chicken CD146 Gicerinand that binding of NOF to CD146 is essentialthedevelopment of the chicken retina4950 However at that timedue to technological limitations the molecular characteristics ofNOF were not determined In Laminin was revealed asthe ligand of CD146 facilitating the entry of blood lymphocytesinto the central nervous system CNS In this report the authorsclaimed that Laminin is a major tissue ligand for CD146 onlymphocytes51 In Ishikawa finally determined theidentity of NOF Laminin which has the same α4 subunit asLaminin forIn our laboratory identified that CD146 can bind withvascular endothelial growth factor receptor VEGFR2 as a coreceptor required for the activation by vascular endothelial growthfactorA VEGFA53 Because VEGFA is a wellknown growth factorwith strong proangiogenesis effects this finding provided themechanism underlying the roles of CD146 in tumor angiogenesisespecially in sprouting stage Subsequently ourlaboratoryidentified an array of proangiogenetic growth factors includingWinglessintegrase Wnt5a54 Netrin155 fibroblast growth factorFGF456 VEGFC57 and Wnt158 as the ligands of CD146 In we further identified that CD146 on endothelia can directly bindreceptorβ PDGFRβ onwith plateletderived growth factorrequired for PDGFBinduced PDGFRβ activation59pericyteBecause PDGFBPDGFRβ plays crucial roles in recruiting adjacentpericytes to the endothelia this finding indicates that CD146 isrequired for vessel integrityUntil now there had been a total of molecules or complexesidentified as the CD146 ligands Table According to thecharacteristics of these ligands they can be categorized into threegroups components of the ECM proangiogenic factor receptorsand growth factors All these ligands have been sown to directlyinteract with CD146 in physiological and pathological processesare involved in the promotion of CD146mediated angiogenesisand tumor metastasis Here we will review the various CD146²heterophilic ligands and discuss the implications of these findingsin tumoral contextCD146 IS THE RECEPTOR OF PROTEINS IN RELATION TO THEECMOne of the critical features of malignant proliferation is cancermetastatic plasticity affected by its microenvironment Thisplasticity is a major reason for the failure of inhibition of cancermetastasis The metastatic process involves epithelial mesenchymaltransition EMT attachment of metastatic cells to theendothelium of the vascular or lymphatic vessels and invasioninto distant metastatic tissues60 It is well known that the aberranthigh expression of CD146 is involved in nearly every step ofdevelopment and progression in almost all types of malignantcancers21 The findings that several ECMrelated proteins including Laminin and Galectin1 and S100A8A9 andmatriptase are specific ligands of CD146 may elucidate themechanism underlying the function of CD146 in remodelingtumor microenvironments during tumor development especiallymetastasis via vascular and lymphatic vessels Fig Laminins and Laminins are a family of large heterotrimeric αβγ proteins with over different isoforms Five laminin α chains α1“α5 four laminin βchains β1“β4 and three laminin γ chains γ1“γ3 constitute αβγheterotrimers They are denominated according to their chaincomposition for example laminin α4β1γ1 is designated as Laminin Laminins are predominantly found in basement membranesthat compartmentalize different tissues and surround blood vesselsnerves and adipocytes6263 They play a crucial role in physiologicalSignal Transduction and Targeted Therapy 0cCD146 from a melanoma cell adhesion molecule to a signaling receptorWang et alTable CD146 ligandsLigandsLaminin Laminin Galectin1Galectin3S100A8A9MatriptaseVEGFR2PDGFRβWnt5aWnt1Netrin1FGF4VEGFCFunctionTime of discoveryReferencesFacilitates lymphocytes entry into CNSImproves cancer metastasis via vascular andor lymphatic vesselsInhibits cell apoptosisEnhances cell migration and secretion of prometastasis cytokinesHelps lung tropic metastasisPromotes neuron differentiationProangiogenesisControl of vascular vessel integrityEnables cell migrationPromotes fibroblast activationProangiogenesisPromotes cell polarity establishmentMediates sprouting during lymphangiogenesisand pathological remodeling of the ECM during angiogenesiswound healing embryogenesis and tumor metastasis Remodelingof the ECM during metastasis allows tumor cells to invade theirsurrounding ECM spread via the vascular or lymphatic circulationand extravasate into distant ansLaminin isoforms particularly the laminin α chain are expressedin a cell and tissue specific manner and are distinctly bound byalmost ten different integrins and other cellsurface receptors6263The α4laminins are mesenchymal laminins expressed by the cells ofmesenchymal origin such as vascular and lymphatic endothelialcells pericytes and leukocytes and are required for normaldevelopment of the cardiovascular and neurological system inmice64“ Under pathological conditions α4laminins are expressedand secreted by various tumor cells such as melanoma andglioma67“ orlymphatic and vascular vesselsnervous system697374tumor stromaLaminin Laminin is expressed along the vascular endothelium687375This laminin isoform is recognized by various integrins includingα6β1 α3β1 α6β4 and αVβ3 which promote the migration ofseveral cell types along vascular or nervous system tracks76“In Laminin on the vascular endothelia was discovered asa specific ligand for CD146 on a subset of human CD4 T helper Thcells51 This subset of human T cells expresses CD146 and can entertissues to promote pathogenic autoimmune responses To determinethe CAMs involved in the migratory capacity of Th17 cells into tissuesresearchers used purified Laminin to identify its receptor In thisstudy the authors demonstrated that purified CD146Fc binds toLaminin with high affinity nM and thatthis bindingdisappeared when the endogenous Laminin was specificallydeleted Correspondingly blocking this binding by CD146 antibodyin vivo also reduced Th17 lymphocyte ltration into the CNSTherefore the authors concluded that Laminin is a major tissueligand for CD146 lymphocyteHoweverthe role of Th17 cells in the pathogenesis ofmalignant tumors is still remains controversial Some studiesrevealed that increased percentage of Th17 lymphocytes amongcells ltrating ovarian cancer cells stimulate tumor progression81 whereas other studies showed that Th17 lymphocytes haveanticancer activity and can reduce tumor growth and metastasis82Therefore the roles of CD146 Th17 cells in cancer developmentmay be worthy of further investigationsLaminin CD146 is a reliable biomarker of endothelia and is concentrated atthe intercellular junctions of endothelial cells of vessel system21Signal Transduction and Targeted Therapy Most cancer cellsincluding melanoma migrate along theabluminal sides of vascular andor lymphatic vessels as theydisseminate throughout the body83 Laminin is major lamininsof along the tumordissemination tracks blood and lymphaticvessels nerves and tumor stroma84“To determine the mechanism of CD146 roles in metastasisresearchers used melanoma cells to test what laminin isoformsother than Laminin can bind with the melanoma marker ofCD146 Therefore they used all laminin α chains to examine thebinding affinity with human CD146 in a solidphase ligand bindingassay87 Finally they found that only Laminin of severallaminin isoformsreadily bound to CD146 suggesting thatLaminin is a primary ligand for CD146 in melanomaAccordingly a functionblocking mAb to CD146 inhibited tumorcell migration on Laminin but not on laminins or Inaddition this investigation determined that the identity of NOFpreviously identified as a ligand for chicken CD146 gicerin isactually Laminin In this study the authors also determined that Laminin andespecially Laminin are capable of stimulating migration of abroad panel of cancer cell lines through a filter This investigationis consistent with the observation that the α4laminins includingLaminins and expressed and secreted by variouscarcinoma cells have already emerged as œoncolaminins“Melanoma CD146 binds with Laminin but not whereaslymphocyte CD146 only binds with Laminin suggesting thatthe epitopes of CD146 on somatic cancer cells are different fromthose of CD146 on blood lymphocytes Therefore the ltrationof CD146 invasive cancers into tumordissemination tracks islikely dependent on the interaction between CD146 and Laminin and blocking their binding may affect the efficacy of cell“cellinteractions and interfere metastasisGalectin1 and CD146 is a highly glycosylated junctional CAM involved in thecontrol of vascular vessel integrity Sequence analysis predicts thepresence of eight putative Nglycosylation sites atresiduepositions and It has beenestimated that of the CD146 molecular mass is attributed toglycans88 The galactose residues in glycans can bind withgalectins and such binding can be inhibited by lactoseGalectins are a family of soluble carbohydratebinding lectinsthat modulate celltocell and celltoECM adhesions89 Up to now galectins have been identified in mammals and are foundin humans Among them Galectin1 and are three bestinvestigated galectins and Galectin1 and promote tumordevelopment progression and immune escape90 Galectin1 and 0cCD146 from a melanoma cell adhesion molecule to a signaling receptorWang can hamper antitumor responses and are considered multifunctional targets for cancer therapy9192 The underlying mechanisms include interfering with drug efficacydelivery or reducingthe antitumor effect of immune cells For instance Galectin1confers drug resistance via inducing the expression of multidrugresistance protein which in turn helps tumor cells to pump outcytotoxic drugs facilitating cancer cells to combat anticancerdrugs93 Regarding the immunosuppressive effects of Galectin1and on T cells in a mouse melanoma model targeted inhibitionof Galectin1 enhanced T cellmediated tumor clearance94Galectin3 can neutralize glycosylated IFNγ in tumor matricesablating the immune response to tumors95 To increase overallresponsiveness of tumors to chemo or immunetherapy inhibitors of Galectin19697 and have been used in combinationwith antiCTLA4 or antiPD1 to treat cancer patients in clinicaltrialsBecause both CD146 and galectins are involved in themodulation of angiogenesis researchers hypothesized that somegalectins may be the ligands of CD146 and the interactionsbetween them are required for functional CD146 in angiogenesisas well as in cancer metastasis To date two galectins and have been identified as the ligands of CD146It has been reported thatGalectin1Galectin1 prefers to bind with the branched Nglycans of cellsurface glycoproteins and mediates a glycosylationdependentangiogenesis91100“increasedsecretion of Galectin1 in the ECM facilitates cancer cellproliferation and resistance to cancertherapy in prostatecancer104 and Kaposi™s sarcoma105 Mechanistic investigation hasrevealed that Galectin1 can bind to Nglycans on VEGFR2 toactivate VEGFlike signaling in antiVEGFA refractory tumorspromoting tumor progression Accordingly disruption oftheGalectin1Nglycan axis inhibits tumor growth by promotingvascular remodeling101 This research highlights the importance ofGalectin1 in tumor angiogenesis and cancer metastasis Howeverthese studies cannot exclude the fact that other cellsurfaceproteins with branched Nglycans are also involved in thisglycosylationdependent proangiogenesis pathwayEarly in it was reported that the coexpression of Galectin and CD146 is required for tumor vascularization in a humanmesenchymal stem cell strain with significant angiogenic potential106 In Jouve reported that Galectin1 binds toCD146 on endothelial cells facilitating cell survival107 In thisthey explained that CD146 glycosylation is mainlyreportcomposed of branched Nglycans They showed thattheinteraction of CD146 with Galectin1 is carbohydratemediatedusing both an enzymelinked immunosorbent assay and surfaceplasmon resonance assays In addition they demonstrated thatthe interaction between Galectin1 and CD146 protects endothelial cells against apoptosis induced by Galectin1 Thusit istempting to speculate that CD146 could be a decoy receptor falectin1 preventing the Galectin1 from binding to proapoptotic receptors107 However whether this interaction affectstumor cell survival remains unknown In Yazawa thusfurther analyzed the functions of this interaction on melanomaand found that when Galectin1 binds to CD146 it helps maintainintrinsic malignant features108 The authors examined the expressionidentity and function of Galectin1 ligands in melanomaprogression and demonstrated that CD146 is the major Galectin1ligand on melanoma cellsThese findings provide a perspective on the interactionsbetween CD146 and its ligands such as Galectin1 as contributorsto cancer malignancy Indeed various membrane glycoconjugateshave been identified as binding partners of Galectin1 such as β1integrins CD2 CD3 CD4 CD43 CD45 and GM1 ganglioside Inaddition Galectin1 can bind to a number of ECM components ina dosedependent and βgalactosidedependent manner Forinstance laminin and fibronectin which are highly Nglycosylatedinteract with Galectin1109 Because it has been reported thatCD146 can interact with Laminin Laminin and β1integrin it is reasonable to speculate that CD146 may also interactwith all of those Galectin1 interactors within cancerous cellsSince the tumor vasculature is an easily accessible target forcancer therapy understanding how galectins ‚uence cancerangiogenesis is important for the translational development oftherapies intended to prevent tumor progression Based on thefact that VEGFtargeted therapies often fail when tumors receivecontinuedglycosylationdependentGalectin1receptorsuch as Galectin1CD146VEGFR2 may increase the efficacy of antiVEGF treatmenttreatment110interactionstargetingGalectin3Like Galectin1 Galectin3 can also bind to various galactoseterminated glycans of cellsurface receptors and proteins of ECMand is involved in many physiological and pathological processesfrom cell adhesion and migration to cell activation111112 In cancercells it modulates cell“cell and cell“microenvironment communications contributing to cancer development progression andmetastasis113“ Patients with metastatic diseases tend to havehigher concentrations of circulating Galectin3 than those withonly localized tumors121 Increased circulating Galectin3 promotes bloodborne metastasis due to the interaction of Galectin3with receptors on vascular endothelial cellsfurther causingendothelial secretion of several metastasispromoting cytokinesTo identify the Galectin3binding molecules on the endothelialcell surface using the Galectin3 affinity purification methodfound that CD146 was the major cellsurfaceColomb et alreceptorto strongly bind and colocalize with Galectin3compared with other glycosylated receptors CD31 CD144 andCD106 They also showed that Galectin3 bound to Nlinkedglycans on CD146 and induced CD146 dimerization andsubsequent activation of protein kinase B AKTsignalingCorrespondingly suppression of CD146 expression abolishesGalectin3induced secretion of metastasispromoting cytokinefrom the endothelial cells Thus they concluded that CD146 is thefunctional Galectin3binding receptor on the endothelial cellsurface responsible for Galectin3induced secretion of cytokinesand therefore ‚uences cancer progression and metastasis122Subsequently the binding moieties of CD146 by Galectin3have been further identified The authors demonstrated thatGalectin3 interacts with the highly glycosylated Domain in theCD146 extracellularthe presence orabsence of lactose These findings provide a better understandingof how Galectin3 interacts with cellsurface receptors to mediateendothelial cell migration and the secretion of cytokines123124regardless offragmentThe endothelial galectins are confined to four family membersie Galectin1 and which contribute to tumorangiogenesis92 Tumorinduced angiogenesis is a pathologiccondition in which tumor cells secrete growth factors such asVEGFs to promote the growth of new blood vessels125126 Thesegrowth factors activate quiescent endothelial cells in host tissue tofacilitate them to invade into the tumor stroma for growth of newcapillaries127 Endothelial galectins binding with glycoconjugateson tumors are involved in different processes during tumorinduced angiogenesis Because Galectin1 and binding ofglycoconjugates on tumor cells mediates many key processes inangiogenesis and elevated levels of Galectin1 and in theendothelium are correlated with tumor vascularization105128“the promotion of tumor vascular remodeling by tumor CD146 maybe due to the interactions between CD146 with Galectin1 and S100A8A9S100 proteinsIn humans there are at least members of theS100 protein family132 which have EFhand calciumbindingmotifs and are soluble in saturated ammonium sulfate133Signal Transduction and Targeted Therapy 0cS100 family members typically form homodimers as well asheterodimers trimers and tetramers etc132134 S100 proteins aretypically cytoplasmic proteins but several family members aresecreted by cells as extracellulartheycontribute to a broad array ofintracellular and extracellularfunctions134135 Upregulation of S100 proteins promotes pro‚ammatory responses that contribute to the development andprogression of cancer and autoimmune and chronic ‚ammatorydiseases138“factors134“ ThusincludingadvancedThe secreted S100 proteins bind with several cellsurfacereceptorsproductsRAGE142“ tolllike receptor TLR4147 CD36148 FGFR1149ALCAM150 CD68151 and ErbB4152 However how the cellsurfacereceptors mediate extracellular S100 signaling is lacking and howS100 protein secretion is dynamically regulated in biologicalprocesses also still remains unknownglycationendseem to require the release ofThe secreted S100A8A9 proteins are theS100A8A9 heterodimer‚ammatorybest characterized soluble S100 proteins Mostthe S100A8A9processesinto the ECM153“ Significant upregulation ofheterodimerS100A8A9 has been observed in many tumors including lunggastric esophageal colon pancreatic bladder ovarian thyroidbreast and skin cancers156157 The upregulation of S100A8A9 iscaused either by the ltrating immune cells oftumormicroenvironment158 or by the tumor itself156157 contributingto the establishment of a premetastatic niche in the tumormicroenvironment159Mechanistic investigations demonstrated that upregulatedS100A8A9 induces the expression of serum amyloid which inturn recruits myeloidderived suppressor cell MDSC producing apro‚ammatory environment during metastasis of aggressivedisease160“ In addition enhanced expression of S100A8A9 isalso associated with poor prognosis168S100A8A9 proteins mediate these effects by binding to plasmamembrane elementsincluding heparan sulfate proteoglycanHSPG169 Nglycans170 TLR4171 and RAGE172173 In a melanomalung metastasis model Hiratsuka clearly demonstrated thatlung S100A8A9 as a strong chemokine interacts with TLR4 onmelanoma to attract distant cancer cells to the lungs174 Recentlyit has also been shown that CD146 on melanoma and breastcancer can respond to lung S100A8A9 to induce lungspecificmetastasis of melanoma175176 and breast cancer177S100A8A9 as the ligand of CD146The expression levels ofS100A8 and S100A9 were higher in the lungs than in other ansand the higher expression levels were induced by the primarytumor itself162 In lungassociated MDSC and endothelial cellstumorderived transforming growth factorbeta TGFβ and VEGFA can upregulate the expression and secretion of S100A8A9162Thus it has been recognized that S100A8A9 plays a critical role inlung tropic metastasis and the subsequent growth of cancer cellsin the lungs26178 During metastasis lung S100A8A9 might act asa guiding protein for cancer cells that possess high expressionlevels of CD146162In Ruma revealed that S100A8A9 uses CD146 as areceptor during lungspecific metastasis of melanoma cells175 In thisstudythey demonstrated that S100A8A9 binding to CD146activates nuclear factorkappa B NFκB and induces reactive oxygenspecies formation significantly increasing cell adhesion growth andinvasion Notably this study proposed that CD146 governs cancerinvasion toward the lungs by sensing the cancer microenvironmentas a soil sensor receptor of lung S100A8A9175 Therefore the authorsconclude that S100A8A9 plays a crucial role in lung tropic cancermetastasis by helping to establish an immunosuppressive metastaticniche to which it then attracts remote cancer cells by interactingwith CD146 on the cancer cell surfacesIn Chen further determined the importance of theSignal Transduction and Targeted Therapy CD146 from a melanoma cell adhesion molecule to a signaling receptorWang et alS100A8A9CD146 axis in melanoma dissemination in a skinlesion a critical early step for metastasis of melanoma Thismechanistic study revealed that S100A8A9CD146 bindingactivates a cascade of functions it leads to significant activationof the transcription factor ETS translocation variant ETV4 andthe subsequent induction of matrix metalloproteinase25 Theactivation of MAP3K8ETV4 by S100A8A9CD146 binding finallyresults in lung tropic metastasis of melanoma176Breast cancer cells prefer the lung liver bone and brain astheir metastatic sites This antropic metastasis is known asthe œseed and soil theory179 This conclusion was reachedbecause CD146 was remarkably overexpressed in metastaticbreast cancer cells180“ In in breast cancer cells theS100A8A9CD146 axiselicited downstream signals that producethe driving force for distant metastasis were identified This studyrevealed how S100A8A9 binding to CD146 accelerates breastcancer growth and metastasis They found that S100A8A9 actsas an extracellular cytokine to activate the CD146ETV4 axiswhich upregulates a very high level of ZEB1 a strong EMTinducer ZEB1 in turn induced a mobile phenotype ie EMT incellsthe downregulation of CD146ETV4 axisrepressed S100A8A9induced EMT resulting in greatly weakened tumor growth and lung metastasis Thus this reportsuggested that S100A8A9 contributesto these signalingprocesses through CD146177In contrastSince metastasis accounts for the majority of cancerassociateddeaths studies on metastasis mechanisms are needed to establishinnovative strategies for cancer treatments These findings thatCD146 as a novel receptor for S100A8A9 mediates the transitionof malignant cancers to metastatic sites suggest that strategiesmodulating the interaction between CD146 and S100A8A9 maybe useful for interference with cancer metastasis especially in theprogression of premetastatic tumors to the lungsMatriptaseMatriptase is an epithelialspecific membraneanchored serineprotease that proteolytically degrades targets such as ECMcomponents and the proforms of growth factors183“ Becausemost of solid tumors are originated from epithelia matriptase isthus critically involved in cancerinvasive growth throughdegradation events related to breaching the basement membrane reanization of the ECM and activation of oncogenicsignaling pathways187During neurogenesis matriptase expressed on neural stemprogenitor NSP plays a critical role in cellcontact signalingbetween NSP and brain endothelial cells188 In the directbinding between brain endothelial CD146 and NSP matriptase wasidentified to be involved in the direct endotheliaNSP contact189Such binding can activate the downstream signaling cascades fromincluding p38 and canonical Wntβcatenin pathways inCD146endothelia leading to secretio

Thyroid_Cancer

"Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunityNatasha D Sheybani1 Alexandra R Witter2 Eric A Thim1 Hideo Yagita3 Timothy N J Bullock Richard J Price To cite Sheybani a0ND Witter a0AR Thim a0EA et a0al Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunity Journal for ImmunoTherapy of Cancer 20208e001008 101136jitc2020001008 –º Additional material is published online only To view please visit the journal online http dx jitc NDS and ARW contributed equallyAccepted July Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Biomedical Engineering University of Virginia Charlottesville Virginia USA2Pathology University of Virginia Charlottesville Virginia USA3Department of Immunology Juntendo University Graduate School of Medicine Bunkyo ku Tokyo Japan4Radiology Medical Imaging University of Virginia Charlottesville Virginia USACorrespondence toDr Richard J Price rprice virginia eduDr Timothy N J Bullock tb5v virginia eduBackground Triple negative breast cancer TNBC remains recalcitrant to most targeted therapy approaches However recent clinical studies suggest that inducing tumor damage can render TNBC responsive to immunotherapy We therefore tested a strategy for immune sensitization of murine TNBC 4T1 tumors through combination of focused ultrasound FUS thermal ablation and a chemotherapy gemcitabine GEM known to attenuate myeloid derived suppressor cells MDSCsMethods We applied a sparse scan thermally ablative FUS regimen at the tumor site in combination with systemically administered GEM We used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity We also tested this combination in Rag1ˆ’ˆ’ mice or T cell depleted wild type mice to determine the essentiality of adaptive immunity Further we layered Programmed cell death protein PD1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalResults The immune modulatory effect of FUS monotherapy was insufficient to promote a robust T cell response against 4T1 consistent with the dominant MDSC driven immunosuppression evident in this model The combination of FUSGEM significantly constrained primary TNBC tumor outgrowth and extended overall survival of mice Tumor control correlated with increased circulating antigen experienced T cells and was entirely dependent on T cell mediated immunity The ability of FUSGEM to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti PD1Conclusion Thermally ablative FUS in combination with GEM restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic TNBC model This treatment strategy promises a novel option for potentiating the role of FUS in immunotherapy of metastatic TNBC and is worthy of future clinical evaluationTrial registration numbers NCT03237572 and NCT04116320BACKGROUNDMetastatic breast cancer BrCa particularly the triple negative breast cancer TNBC phenotype is resistant to most chemical and molecularly targeted therapeutic approaches Interestingly TNBC is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 Early studies in the use of immunotherapies targeting the PD1Programmed death ligand PD L1 checkpoint inhibitory axis showed some efficacy2“ in TNBC compared with other BrCa subtypes which are generally recalcitrant to checkpoint blockade Activity in the TNBC subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in TNBC Greater immunotherapy efficacy in TNBC has been recently observed with the use of antibodies targeting the PD1PD L1 checkpoint inhibitory axis in combination with Nab paclitaxel5 This outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment TME found in TNBCAmong the potential networks in TNBC that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets These have the capacity to impair adaptive immunity and promote tumor growth and metastasis Among these cell types myeloid derived suppressor cells MDSCs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both T cell activation and effector functions6 Increased levels of this cell type have been demonstrated in tumor tissues of patients with primary BrCa while those with metastatic disease bear the highest abundance of circulating MDSCs8 Studies have Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate MDSC can improve antitumor immunity9“To this end the central premise put forth in this study is that focused ultrasound FUS”a safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissues”can synergize with immunotherapy in a murine model of metastatic TNBC FUS is capable of rapidly heating tumors to thermally ablative temperatures Its extracorporeal application obviates the need for catheterization injection or implantation FUS can be targeted with millimeter precision under MRI or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues The bioeffects of FUS hold distinct implications for tumor antigenicity immune cell activation and trafficking13 Thermally active FUS regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17“ colon20 kidney21 and BrCa23 Pertaining to the challenge of myeloid cell immunosuppression in TNBC thermally ablative FUS has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin IL12 interferonÎ IFNÎ and tumor necrosis factorα TNFα from a variety of cancer cell lines and after in vivo treatment of tumors26 Whether the ability of FUS to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of BrCa is currently under debate with some studies showing activation of antigen presenting cells and T cell recruitment in patients with BrCa treated with thermally ablative FUS28 while others show that additional innate stimuli are needed to support antitumor immunity23 Notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells DCs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31Based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation FUS regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine TNBC with extensive granulocytic MDSC involvement that is recalcitrant to anti PD1 While some activity is evident with the partial ablation approach significantly greater control was achieved by targeting MDSC inhibition in combination with thermally ablative FUS This control was completely dependent on the adaptive immune responseMoreover we demonstrate that layering anti PD1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction These data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative FUS Once immunosuppressive myeloid cells are accounted for FUS treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockadeMETHODSCell line maintenance4T1 and E0771 cell lines were maintained in RPMI L glut or Dulbecco™s Modified Eagle™s Medium DMEM gL D glucose L glutamine respectively supplemented with Fetal Bovine Serum FBS at °C and CO2 Thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments Cells tested negative for mycoplasmaEight week old to week old female BALBc or C57Bl6 mice were obtained from NCI Charles River NCI CRL or The Jackson Laboratory Female BALBc Rag1ˆ’ˆ’ mice were obtained from The Jackson Laboratory 4T1 or E0771 cells × were subcutaneously implanted into the right flank of mice Mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum Tumor outgrowth was monitored via digital caliper measurements Tumor volume was calculated as follows volume length×width22 Approximately days 4T1 or days E0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsIn vivo ultrasoundguided FUS partial thermal ablationMice were treated with FUS either days 4T1 cohorts or days E0771 postimplantation On treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg Zoetis and dexdomitor mgkg Pfizer in sterilized saline Mouse flanks were shaved and depilated following which ultrasound guided FUS thermal ablation was performed using one of the two systems System and treatment details are provided in online supplementary materials and methods Mice that did not receive FUS treatment consistently underwent anesthesia and depilation of the flank Additionally these mice underwent a ˜sham™ treatment consisting of exposure to the °C degassed water bath exposure for min Following ˜sham™ or FUS treatment all mice were moved to a heating pad and given Antisedan for anesthesia reversal and recoveryGemcitabine therapyGemcitabine GEM mgmouse in µL volume Mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of FUS treatment following which administration was repeated for an additional weeks Administration of GEM doses was based on existing literature demonstrating the use of GEM for inhibition of MDSCs in 4T112 The initial dose of GEM was administered immediately prior to ˜sham™ or FUS treatment Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0cMice that did not receive GEM received an intraperitoneal injection of ˜vehicle™ treatment µL of sterile saline at the time points specifiedPD1 blockade therapyFor checkpoint inhibitor therapy the rat anti mouse PD1 antibody αPD1 RMP114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse Treatment was initiated on day ˜early αPD1™ or day ˜delayed αPD1™T cell depletionsT cell depletion antibodies”anti CD8 clone Bio X Cell and anti CD4 GK15 clone Bio X Cell”were diluted in sterilized saline and administered intraperitoneally every to days starting at day days post FUS for a total of seven doses µg of each antibody for a total µg per mouseImmunohistochemistryOn day sham or FUS exposed tumors were excised and fixed in neutral buffered formalin Sigma Fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin Digital images of stained slides were acquired using the Vectra Automated Quantitative Pathology Imaging System Akoya Biosciences Whole slide screening and image capture were subsequently performed using Phenochart Akoya BiosciencesFlow cytometryMice were bled at days and via tail vein and samples were RBC lysed Hybri Max Sigma and stained for flow cytometry analysis At days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment In order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse CD45 FITC clone F11 BD Biosciences min prior to euthanasia 4T1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor DLNs pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis Additional details are provided in online supplementary materials and methodsSamples were acquired on an Attune NxT flow cytometer ThermoFisher Scientific and data were analyzed with FlowJo TreeStar or FCS Express De Novo Software A representative gating strategy for granulocytic myeloid derived suppressor cell G MDSC and CD44 T cells is provided in online supplementary figure Statistical analysisAll statistical analyses were performed in GraphPad Prism GraphPad Software A detailed description of statistical methods for each experiment is provided in the corresponding figure legend accessAnimal study approvalAll animal work was performed under a protocol approved by the Animal Care and Use Committee at the University of Virginia and conformed to the National Institutes of Health guidelines for the use of animals in researchRESULTSPartial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsetsTo achieve partial thermal ablation of 4T1 tumors we used an ultrasound guided FUS system equipped with a single element therapeutic transducer driven at MHz figure 1A online supplementary figure A grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under B mode ultrasound guidance figure 1B“C The exceptionally small focus of this system rendered a low ablation fraction “ of total tumor volume Immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1D One week following FUS partial thermal ablation tumors and secondary lymphoid ans were excised for immunological characterization by flow cytometry figure 1B FUS partial thermal ablation of 4T1 tumors conferred a significant increase fold in the absolute number of CD11c hi DCs within the axillary tumor draining lymph node aDLN of mice figure 1E While this was accompanied by a nearly threefold elevation in the absolute number of CD86 DCs within the aDLN figure 1F the percentage of DCs expressing CD86 did not change figure 1G Increased numbers of DCs”and CD86 DCs in particular”suggest FUS is promoting the maturation or trafficking of these cells in the DLNs where they could encounter and activate T cells However this did not translate to tumor growth restriction data not shown We also did not observe significant differences in the absolute number of activated T cells in 4T1 tumors figure 1H or DLNs data not shown following FUS exposure suggesting limitations in the ability of FUS activated DC to further drive an antitumor T cell responseImmune profiling by flow cytometry revealed that irrespective of FUS exposure of the intratumoral CD45 immune cell population is comprised of CD11b myeloid cells figure 1I Similarly approximately of the circulating immune cell population in 4T1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure Notably Ly6G granulocytic myeloid derived suppressor cells G MDSCs significantly dominated the immune cell repertoire within 4T1 tumors relative to other myeloid including F480 macrophages Ly6C cell subsets monocytic myeloid derived suppressor cells M MDSCs and CD11c hi DCs figure 1J FUS partial thermal ablation did not significantly alter the absolute number per Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access Figure Partial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsets A Design overview of a custom ultrasound guided FUS system consisting of a MHz single element transducer orthogonally co registered to an MHz linear ultrasound imaging array The tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °C ˜Sham™ mice were similarly positioned but did not undergo sonications B Schematic illustration of FUS partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4T1 tumor bearing mice A grid of sonications was applied in a raster pattern onto the B mode ultrasound visible tumor In total two planes of sonication spaced mm apart were applied to each tumor Grid points were spaced mm apart within a single plane One week following thermal ablation tumors and secondary lymphoid ans were excised for sham n6 or FUS treated n5 mice and processed for flow cytometry C Representative B mode ultrasound images of ectopic 4T1 tumors either before top or during bottom FUS exposure Sonication grid depicting targets red points is superimposed on B mode image during treatment Subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed D Representative HE staining of either sham 4T1 tumors or those resected immediately following FUS partial thermal ablation Zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively E Absolute number of CD11c hi DCs in the axillary tumor draining lymph node aDLN of 4T1 tumor bearing mice p00136 vs sham F Absolute number of CD86 CD11c hi DCs in the aDLN p00063 vs sham G Percentage of CD86 subset out of total CD11c hi DCs within aDLN H Absolute number of intratumoral CD44 CD8 and CD44 CD4 T cells and regulatory T cells Tregs per gram tumor I Percentage of CD11b myeloid cells out of total CD45 immune cells across tumor spleen aDLN inguinal DLN iDLN and nontumor draining axillary and inguinal LNs nDLNs p005 vs all other groups irrespective of FUS exposure specifically tumor vs spleen p00226 tumor spleen vs all other ans p00001 J Absolute number of intratumoral myeloid cells CD11c hi DCs F480 macrophages Ly6C monocytic myeloid derived suppressor cells M MDSCs Ly6G granulocytic myeloid derived suppressor cells G MDSCs per gram 4T1 tumor p00001 vs all other cell types irrespective of FUS exposure All data represented as mean±SEM Significance assessed by unpaired t test F“H or two way analysis of variance followed by Tukey multiple comparison correction I“K ˜ns™not significant DCs dendritic cells FUS focused ultrasound HIFU high intensityfocused ultrasoundSheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets These observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4T1 TME must be addressed in order to facilitate the T cell response to FUSFUS partial thermal ablation in combination with GEM constrains primary TNBC tumor outgrowth and extends overall survivalOur observation of the overwhelming MDSC burden following 4T1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier To this end we tested a combinatorial paradigm incorporating GEM a myelosuppressive chemotherapy demonstrated to inhibit MDSCs transiently in the 4T1 model without consequence to T cell phenotype or function12To evaluate the efficacy of FUS and GEM in combination we used a preclinical ultrasound guided FUS system to achieve partial thermal ablation of established 4T1 tumors 14d after tumor implantation average tumor volume of mm3 In combination with the single session of FUS thermal ablation we initiated GEM therapy mgmouse which was then readministered weekly for a total of three GEM doses figure 2A Combinatorial therapy synergized to produce significant constraint of 4T1 tumor outgrowth compared with sham and monotherapy groups figure 2B“CBy termination of treatments at day 4T1 tumors exposed to FUSGEM combination saw nearly × and × reductions in average volume compared with sham or GEM exposed tumors respectively figure 2B Two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2D“E In a fraction of mice treated with FUSGEM we observed complete regression of 4T1 tumors although transient figure 2C tumor outgrowth eventually rebounded after termination of treatments 4T1 tumor bearing mice receiving FUSGEM treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and GEM groups respectively HRs and for FUSGEM relative to sham and GEM groups respectively figure 2F We additionally observed that FUSGEM significantly constrained outgrowth in a separate C57Bl6 metastatic mammary carcinoma model E0771 online supplementary figure To further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided FUS system Theraclion Echopulse that is already CE marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging FUS thermal ablation in combination with cancer immunotherapy We observed that partial thermal ablation using the Theraclion visualization and treatment unit MHz in combination accesswith GEM controlled 4T1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure These findings lend credence to the notion that the impact of combining GEM with FUS may be conserved across partial thermal ablation regimens Moreover they demonstrate that the efficacy of FUS partial thermal ablation in combination with GEM can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallyCombination of FUS partial thermal ablation with GEM increases the levels of circulating T cellsLymphocytes”in particular CD8 and CD4 T cells”play an important role in responding to tumor antigen and generating a durable antitumor response Based on the extended protective effect observed in mice treated with FUSGEM flow cytometry analysis was performed to evaluate the contribution of T cells in generating systemic and local tumor control We sampled the circulating immune cell repertoire in 4T1 tumor bearing mice via serial tail bleeds days and prior to readministration of GEM and a terminal cardiac bleed at the time of spleen harvest day figure 3A Combinatorial therapy significantly elevated absolute number of CD8 and CD4 T cells in the circulation at days and figure 3B“C and E“F Moreover a trend threefold to fivefold increase in circulating T cells was noted in the FUS group relative to sham figure 3B“C and E“F From days to systemic CD44 expressing antigen experienced T cell populations both CD8 and CD4 saw a steady significant increase after combinatorial therapy figure 3D and G A similar modest trend was noted for the FUS monotherapy group relative to sham and GEM figure 3D and G These changes were concordant with a decrease in circulating myeloid CD11b cells in GEM recipient groups demonstrating the ability of GEM to partially alleviate circulating myeloid burden figure 3HSplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4T1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression We observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6A“B Consistent with this observation immunological characterization of spleens revealed a significant decrease in CD11b myeloid cells”a “ reduction in FUSGEM spleens relative to sham or monotherapy figure 3I While there appeared to be a trend toward more CD11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute CD11b cell numbers within the spleen data not shown The decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following FUSGEM treatment Relative to these sham and GEM groups combination therapy elevated splenic CD8 T lymphocytes by fold and Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access Figure Combination of focused ultrasound FUS partial thermal ablation with gemcitabine GEM constrains primary triple negative breast cancer outgrowth and extends overall survival A Overview of experimental design for evaluation combination of FUS with serial GEM treatment in murine mammary carcinoma B Average 4T1 tumor outgrowth in sham n7 FUS monotherapy n5 GEM monotherapy n10 and combinatorial FUSGEM therapy groups n10 Data are represented up to select time points corresponding with mouse dropout due to humane endpoints All data represented as mean±SEM Significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by Tukey multiple comparison correction p005 vs all other groups specifically sham vs FUSGEM p00001 FUS vs FUSGEM p00001 shamGEM vs FUSGEM p00026 C 4T1 tumor outgrowth from individual mice in sham FUS shamGEM or FUSGEM groups Data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint D Representative images of 4T1 tumors excised at day Scale bar1 cm E Quantification of 2D tumor areas from images in previous panel F Kaplan Meier curve depicting overall survival of sham treatment n9 FUS monotherapy n6 GEM monotherapy n10 and combinatorial FUSGEM therapy n10 recipient mice Significance assessed by log rank Mantel Cox test p005 vs all other groups specifically sham vs FUS p02154 sham vs FUSGEM p00001 sham vs shamGEM p00050 FUS vs FUSGEM p00021 FUS vs shamGEM p00312 FUSGEM vs shamGEM p00041Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c accessFigure Combination of focused ultrasound FUS partial thermal ablation with gemcitabine GEM increases the levels of circulating T cells A Overview of experimental design to understand the impact of FUS andor GEM treatment on circulating immune cells B“C Absolute number of circulating CD8 T cells at day B and day C D Percentage of circulating CD8 T cells expressing CD44 from days to E“F Absolute number of circulating CD4 T cells at day E and day F G Percentage of circulating CD4 T cells expressing CD44 from days to H Percentage of CD11b myeloid cells out of total CD45 immune cell in circulation from days to I“K Percentage of myeloid cells I CD8 T cells J and CD4 T cells K out of total CD452 immune cells All data represented as mean±SEM All data representative of sham n6“ FUS monotherapy n4“ GEM monotherapy n9 and combinatorial FUSGEM therapy n6“ groups Significance assessed by analysis of variance followed by Tukey multiple comparison correction for B C E F or Fisher™s least significant difference LSD without multiple comparisons correction for I“K Significance for D G and H assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by Fisher™s LSD without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedSheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access fold figure 3J and CD4 T lymphocytes by fold and fold figure 3K These elevations were accompanied by a modest increase in percentage of Foxp3 regulatory T cells Tregs online supplementary figure 6E Additionally increases in percentage of NK and B cells were noted twofold to fivefold online supplementary figure 6C“D These findings indicate that combinatorial therapy with FUSGEM promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasesCombinatorial FUSGEM therapy does not promote robust local antitumor T cell responsesGiven the robust systemic immune signatures within the blood and spleen following FUSGEM we assayed 4T1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral T cell response figure 4A Approximately hours prior to euthanasia mice received intravenous brefeldin A injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry Immune characterization of tumors at days postimplantation”that is days subsequent to final GEM administration”revealed no significant changes in absolute number of antigen experienced CD44 CD8 or CD4 T lymphocytes figure 4B“C Moreover the polyfunctionality of these T cells as denoted by IFNÎ and granzyme B expression was not significantly altered figure 4D“E However intratumoral functional changes were noted in the myeloid compartment GEM monotherapy modestly increased IL 12p40 production by DCs fold but this was not conserved in the combinatorial therapy group figure 4F Moreover while FUS monotherapy generated a trend in elevated TNFα production by intratumoral G MDSCs GEM recipient groups saw a significant increase threefold relative to sham figure 4G These findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely Interestingly intratumoral T cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionProtection conferred by combination of FUS and GEM is dependent on adaptive immunitySince our findings revealed no obvious advantage or function of adaptive immunity in the local TME we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with FUSGEM To this end we utilized an Rag1ˆ’ˆ’ model that is deficient in T and B cells to address the hypothesis that mature T andor B cells play a role in the observed response Wild type WT or Rag1ˆ’ˆ’ mice bearing 4T1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes Mice were subsequently treated with either GEM monotherapy or the combination of FUSGEM The tumor growth inhibition offered by FUSGEM was entirely lost in Rag1ˆ’ˆ’ mice relative to their WT counterparts with average 4T1 tumor volume in Rag1ˆ’ˆ’ mice being over fivefold higher than that of WT mice on terminati

Thyroid_Cancer

"Pharyngitis Tonsillitis Deep learning Smartphone Automated diagnosis Telemedicine Purpose Severe pharyngitis is frequently associated with inflammations caused by streptococcal pharyngitis which can cause immunemediated and postinfectious complications The recent global pandemic of coronavirus disease COVID19 encourages the use of telemedicine for patients with respiratory symptoms This study therefore purposes automated detection of severe pharyngitis using a deep learning framework with selftaken throat images Methods A dataset composed of two classes of throat images with pharyngitis and normal throat images was collected Before the training classifier we constructed a cycle consistency generative adversarial network CycleGAN to augment the training dataset The ResNet50 Inceptionv3 and MobileNetv2 architectures were trained with transfer learning and validated using a randomly selected test dataset The performance of the models was evaluated based on the accuracy and area under the receiver operating characteristic curve ROC AUC Results The CycleGANbased synthetic images reflected the pragmatic characteristic features of pharyngitis Using the synthetic throat images the deep learning model demonstrated a significant improvement in the accuracy of the pharyngitis diagnosis ResNet50 with GANbased augmentation showed the best ROCAUC of for pharyngitis detection in the test dataset In the 4fold crossvalidation using the ResNet50 the highest detection accuracy and ROCAUC achieved were and respectively Conclusion The deep learning model for smartphonebased pharyngitis screening allows fast identification of severe pharyngitis with a potential of the timely diagnosis of pharyngitis In the recent pandemic of COVID19 this framework will help patients with upper respiratory symptoms to improve convenience in diagnosis and reduce transmission Introduction Diagnostic support in remote healthcare services has shown the ability to minimize the exposure of ill patients to healthcare providers and other patients [] The recent global pandemic of coronavirus disease COVID19 has encouraged the use of telemedicine for patients with upper respiratory symptoms [] Because smartphones have become ubiquitous many researchers are interested in utilizing them in telemedicine Deep learning technology can assist with patient examination using a smartphone when clinicians deal with limited information in a remote patient monitoring setting In particular a smartphone is useful to take a picture of the throat [] Therefore home monitoring using a smartphone will help in the diagnosis and treatment of patients with upper respiratory symptoms to improve convenience and to reduce Corresponding author Department of Ophthalmology Aerospace Medical Center Republic of Korea Air Force Danjaero Namilmyeon Cheongwongun Chungcheongbukdo Cheongju South Korea Corresponding author Epilepsy Center Neurological Institute Cleveland Clinic Euclid Ave Cleveland Ohio USA Email addresses eyetaekeunyoogmailcom TK Yoo jychoi717gmailcom JY Choi Tae Keun Yoo and Joon Yul Choi contributed equally to this work 101016jcompbiomed2020103980 Received June Received in revised form August Accepted August ComputersinBiologyandMedicine1252020103980Availableonline20August2020001048252020ElsevierLtdAllrightsreserved 0cimages were posted by users asking for medical advice via the social QA The search strategy was based on the key terms œsore throat  œpharyngitis œtonsillitis œexudative tonsillitis œtonsillopharyngitis  œthroat image and œsmartphone in Korean Japanese and English languages The most recent image from the database search was achieved on April Images that were not taken with smartphones were manually picked and excluded for this study Images with the characteristics of either pharyngitis or normal throat were manually classified by two clinicians and the ambiguous images were excluded to clarify the image domains Finally we collected the initial dataset with two classes including throat images with pharyngitis and normal throat images The dataset was randomly separated into training N validation N and test sets N to apply deep learning to an independent dataset Detailed data distribution and augmentation are described in Table Only the throat and tonsils images were used for the input data without further manipulation to reduce the intraclass variance Original images were extracted from the database in the PNG Portable Network Graphics format The images were resampled to a pixel resolution of — — in the PNG formatfor CycleGAN and deep learning models All procedures were performed in accordance with the ethical standards of the institutional and national research committee and the Helsinki declaration and its later amendments or comparable ethical standards This study did not require ethics committee approval instead the researchers used webbased and deidentified data All datasets for the development of the deep learning model are available at Mendeley Data repositories 10176328ynyhnj2kz TK Yoo transmission There have been several approaches adopting deep learning for automated diagnosis of several diseases using images captured by smartphones [] Pharyngitis which is diagnosed in more than million patients in the United States annually is a common condition associated with acute upper respiratory tract infection [] Pharyngitis is an inflammation of the back of the throat and tonsils Sore throat caused by pharyngitis is one of the main causes of medical visits for young patients [] The most common cause of acute pharyngitis is a selflimiting viral infection However Streptococcus pyogenes is the major bacterial infectious cause of pharyngitis and is responsible for an estimated “ of cases of sore throat [] Frequently severe pharyngitis with fever and exudative tonsillitis is associated with streptococcal pharyngitis which can cause immunemediated and postinfectious complications such as acute rheumatic fever [] Therefore timely diagnosis of pharyngitis for treatment is important to reduce symptoms fever and complications [] However many patients with upper respiratory infection ignore their symptoms in the early stage and medical visits do not routinely take place Moreover in recent days many patients hesitate to visit clinics because of the COVID19 outbreak The importance of a mobilebased monitoring system for patients with acute upper respiratory infections has been raised because of its applicability and effectiveness [] A previous study endeavored to collect throat images using additional equipment in conjunction with the smartphone and used the knearest neighbor algorithm in color distribution space to classify images with streptococcal tonsil [] However the need for additional equipment limited their effectiveness of this method in a realworld setting Moreover color distribution was unable to represent the characteristic features of pharyngitis Throat images exhibit variation in the size illumination and shape of the oral cavity Here we present a deep learning model with smartphonebased throat images facilitating the detection of severe pharyngitis using selftaken throat images Fig We performed automated detection of severe pharyngitis using a convolutional neural network CNN framework Methods Data collection The basic concept of our framework is throat examination using a selftaken smartphone image with computeraided diagnosis system which is similar to the previous dermatology study [] This study was performed using publicly accessible selftaken throat images on the web We collected throat images from webbased social QA systems including Naver Korea kinnavercom and Yahoo Japan chiebukuroyahoocojp The additional throat image datasets were extracted using the Google image search engine Most throat Data augmentation using GAN Because of the limited number of datasets and their imbalanced distribution data augmentation is required for deep learning training Basic data augmentation techniques such as flip translation rotation and brightness change have been applied to train deep learning models Several previous studies have attempted to train deep learning models using generative adversarial network GANbased synthetic images to increase the classification performance [] Inspired by previous works using a generative adversarial network we adopted the CycleGANbased data augmentation to increase the accuracy of diagnosis The cycle consistency in Fig allows CycleGAN to capture the characteristics of two image domains and automatically learn how these characteristics should be translated to transfer to domains without any paired datasets [] CycleGAN was developed to overcome the limitations of paired data when two generators and two discriminators are used It is considered to be a powerful technique that performs image domain transfer and face transfer [] Previous studies have demonstrated that CycleGAN can improve deep learning models by generating training situations to learn better decision boundaries between classes We built the CycleGAN augmentation model to increase the Fig Workflow of building a deep learning model for pharyngitis diagnosis using a smartphone ComputersinBiologyandMedicine12520201039802 0cTK Yoo Fig Schematics of the CycleGAN model generating new normal images and pathologic throat images with pharyngitis generalizability of the dataset and to improve the classification performance in the imbalanced dataset Before training the CycleGAN the throat images were augmented using linear transformation including left and right flip width and height translation from to random rotation from —¦ to —¦ zooming from to and random brightness change from to We defined these transformations as the basic augmentation step In this step normal throat and pharyngitis images were randomly sampled for the training set and normal throat and pharyngitis images were randomly sampled for the validation set Using the data with basic augmentation we trained the CycleGAN models to transform both normal to pharyngitis throat images and pharyngitis to normal images The trained CycleGAN model augmented the training set normal throat and pharyngitis images before augmentation and a total of throat images including normal and pharyngitis images were prepared to train the diagnostic classifier model after CycleGANbased augmentation It should be noted that supervised GAN techniques including conditional GAN and Pix2px were unable to be applied in this study because of a lack of paired normal and pharyngitis images To use a verified and predesigned image generator all the input images required resizing to a pixel resolution of — — which is the basic setup of a CycleGAN Therefore we used the default parameter settings that is the ADAM optimizer with a batch size of to optimize the GAN networks Development of CNN model We trained conventional deep learning models after data augmentation Because of a small image dataset in this study developing a custom deep learning method is challenging due to difficulty and time consuming eg a small training dataset can easily result in an over fitted model and low performance [] To overcome the problem of the small dataset transfer learning was widely used to train deep learning models using pretrained architectures [] This study also applied pretrained learning models to the classification task of throat images The last fully connected layer of the CNNs was only trained and the study used the pretrained conventional model as a feature extractor [] The CNN models including ResNet50 Inceptionv3 and MobileNet v2 were adopted to build binary classifiers [] These CNN models have been used successfully in many studies demonstrating stateoftheart performance with the saliency map [] The models were trained using the training set and the validation set was used to estimate how well the model had been trained We downloaded the CNN models which were pretrained on the ImageNet database and performed finetuning of the weights of the pretrained networks This process generally maintains the weights of some bottom layers to avoid overfitting and performs delicate modification of the highlevel features To use the images generated by CycleGAN the size of the input images for the deep learning models was set to a pixel resolution of — — and the images were resized for each pretrained model Most conventional deep learning models adopted a pixel resolution of — — or — — [] One deep learning research showed that the best performance was achieved at an image resolution of between — and — pixels for binary classification [] Therefore the resolution of our study was appropriate to detect pharyngitis in a binary decision The model was trained with epochs and a batch size of The ADAM optimizer with a learning rate of was also used with a crossentropy loss for all CNN models The crossentropy loss function is defined as Lcross entropy cid0ˆ‘Nipi logqi Where pi represents the ground truth value and qi represents predicted probability value from a classifier for the ith image The optimizer updated the network parameters to minimize the loss function In our experiments it tuned a fully connected layer of the CNN models For example the first layers of ResNet50 were left frozen and we trained the last fully connected layer using the training dataset which is described in Table the ADAM optimizer We chose the final classifier model which maximized the accuracy in the validation dataset To visualize the clusters to see if the classes are separable by a considerable margin the tdistributed stochastic neighbor embedding t SNE algorithm was executed using sampled instances The feature vectors from the last layer of the pretrained Inceptionv3 model were extracted to train the tSNE [] As there is a growing demand for explainable artificial intelligence methods in medicine [] we adopted githubcomjacobgilpytorch the GradCAM technique ComputersinBiologyandMedicine12520201039803 0cTK Yoo Table Throat image dataset and augmentation used in this study Number of validation set Number of training set Raw data Basic augmentation GANbased image synthesis augmentation Class Normal Pharyngitis Normal Pharyngitis Normal Pharyngitis Number of test set GAN generative adversarial network gradcam to generate the attention map [] The GradCAM visualizes the decisional areas of the CNN model using the gradients of any target flowing into the final convolutional network The heatmap has a low resolution and it was upsampled via bicubic interpolation Finally it produces heatmaps that highlight the area of interest and interprets the decision of the deep learning models The performance of the CNN models was evaluated based on the accuracy and area under the curve AUC of the receiver operating characteristic curve ROC and the precisionrecall curve PRC The Youden index which is an estimate of the optimal diagnostic threshold was adopted in this study [] After obtaining the sensitivity and specificity the Youden index was calculated at each cutoff point We selected the optimal value which maximized the Youden index These performance indexes are expressed as follows Accuracy TP TNTP TN FP FN Sensitivity Recall TPTP FNSpecificity Precision TNTN FPTPTP FP Youden index Sensitivity Specificity cid0 Where TP TN FP and FN denote true positives true negatives false positives and false negatives respectively We also performed a 4fold crossvalidation using the entire dataset to evaluate a generalized performance Google Colab Pro a cloud service for disseminating the deep learning research was adopted to implement the CycleGAN and CNN models [] Google Colab Pro provides a development environment using the Tensorflowbased deep learning libraries and a robust graphic processing unit GPU This enables the rapid processing of a heavy deep learning network without the need for a personal GPU [] The available hardware for each virtual machine varied by session but typically included top products of NVIDIA GPUs K80 T4 or P100 around “ GB of RAM and “ GB of free space on the virtual machine hard drive [] We used the Colab CycleGAN tutorial page to develop and validate the CycleGAN model and all of the code is available on the Tensorflow webpage wwwtensorflowtutorialsgene rativecyclegan We only modified the input pipeline to import our dataset The code of the CycleGAN and CNN models is presented in Supplementary Material Results We developed a deep learning model using GANbased augmentation in the challenging context of pharyngitis detection To build a balanced training dataset the CycleGAN models generated normal and pathologic throat images using the initial training dataset The color intensity distributions of the pharyngitis and normal throat images were not significantly different although most throat images had exudate regions Fig A The tSNE algorithm demonstrates that both groups are clustered and they are separable by a considerable margin Fig B The final CycleGAN model for the pharyngitis throat image was trained for epochs which required h After training normal throat images were translated into pathologic images and throat images with pharyngitis were translated into normal images Finally we constructed a balanced augmented training dataset including normal and pharyngitis images using CycleGAN The CycleGANbased synthetic images were realistic and reflected the characteristic features of pharyngitis Fig A The CycleGAN model synthesized white or gray patches and increased the redness on the throat wall and tonsils from normal images Fig B Generated throat images were reviewed by three clinicians including an otolaryngologist and an anesthesiologist All pharyngitis images generated were deemed by the three clinicians to show œmore pathologic and inflammatory results when compared to the original images This feature generation based on the CycleGAN model can be effective for generating a new sample to increase the intra class variation and generalizability The CNN models were trained using the final augmented dataset via the transfer learning technique Fig demonstrates the training process of the ResNet50 CNN model using the training and validation sets The training process for CNN took approximately h for epochs with finetuning for each training step After the 200th epoch the validation accuracy was not improved and the crossentropy of the validation result increased Therefore we considered that training for epochs was optimal in the ResNet50 model and selected the trained model at the 200th epoch Fig A shows the training validation and test datasets of CNN models with and without CycleGANbased augmentation The ROCAUCs of ResNet50 Inceptionv3 and MobileNetv2 without GAN based augmentation were and respectively Fig B We obtained the best ROCAUC of pharyngitis detection using ResNet50 with GANbased augmentation corresponding to Fig C At the optimal diagnostic cutoff value the ResNet50 model predicted pharyngitis with an accuracy of sensitivity of and specificity of The ROCAUC of Inceptionv3 and MobileNetv2 corresponded to and respectively The models with GAN based augmentation demonstrated superior performance in comparison with the models with only basic augmentation We also evaluated the performance of the models using PRCs Fig The PRCAUC also demonstrated that deep learning models with GANbased augmentation had better performance than those without GANbased augmentation ResNet50 with GANbased augmentation predicted pharyngitis with the highest PRCAUC of When a custom deep learning network without the transfer learning technique was trained the validation accuracy was lower than and was not improved during the training epochs Supplementary materials Table shows the performance of CNN models via 4fold cross validation in the entire dataset A similar tendency is observed for the average accuracy and AUC values in the crossvalidation The result shows that the highest detection accuracy and ROCAUC achieved were and respectively by using the ResNet50 Other CNN models showed lower performance than the ResNet50 with GANbased augmentation but there were no significant differences between CNN models in the 4fold crossvalidation A saliency map using the Grad CAM technique was generated to visualize the characteristic pathologic features of pharyngitis for the predicted evidence Fig A In normal throat images however some regions were highlighted Fig B When synthesized images were tested using the trained model synthesized exudates were highlighted correctly in the images as shown in Fig C Furthermore external clinical cases from previous literature were analyzed to investigate to show the capability of detecting pharyngitis of the framework developed in this study [] The representative cases with severe pharyngitis are shown in Fig One case presents an ComputersinBiologyandMedicine12520201039804 0cTK Yoo Fig Visual data exploration of pharyngitis and normal throat images A The mean redgreenblue RGB distributions B The feature space visualized using the tSNE technique For interpretation of the references to color in this figure legend the reader is referred to the Web version of this article Fig Data augmentation using CycleGAN to improve the diagnostic performance of pharyngitis A CycleGANbased augmentation for imbalanced data B Examples of pathologic throat images with pharyngitis generated by the CycleGAN Fig Training process of the ResNet50 CNN model for pharyngitis detection A Accuracy learning curves of the training and validation sets B Loss learning curves of the training and validation sets ComputersinBiologyandMedicine12520201039805 0cTK Yoo Fig Performance of pharyngitis detection deep learning models A Schematics of basic augmentation and GANbased augmentation B The receiver operating characteristic curves of deep learning models using basic augmentation C The receiver operating characteristic curves of deep learning models using GANbased augmentation Fig Precisionrecall curves of pharyngitis detection deep learning models A The deep learning models using basic augmentation B The deep learning models using GANbased augmentation Table Classification performance for severe pharyngitis detection in the 4fold crossvalidation using the developmental set AUC CI Accuracy CI Sensitivity CI Specificity CI Pvalue “ “ “ Basic augmentation ResNet50 Inceptionv3 MobileNetv2 GANbased image synthesis augmentation ResNet50 Inceptionv3 MobileNetv2 AUC area under the receiver operating characteristic curve CI confidence interval GAN generative adversarial network Comparison of receiver operating characteristics curves with the single best technique ResNet50 with GANbased image synthesis augmentation according to the Delong test “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ Reference 81yearold man with odynophagia Fig A and the other case presents a 41yearold diabetic man with throat pain Fig B The ResNet50 model was able to detect pharyngitis in both cases The GradCAM technique highlighted white patches on the throat wall as markers of severe pharyngitis in the deep learning model Discussion The current proofofconcept study investigated the possibility of deep learning using a smartphone for detecting pharyngitis A recentstudy demonstrated the ability of selftaken throat images to detect pharyngitis based on the knearest neighbor algorithm in a color space [] That study used additional equipment as well as a ComputersinBiologyandMedicine12520201039806 0cTK Yoo Fig Example of deep learning classification results with a saliency map using the GradCAM technique A Pharyngitis throat images B Normal throat images C Synthesized images smartphone to obtain throat images We utilized selftaken throat images without additional equipment using deep learning techniques for the detection of pharyngitis We showed that the trained GAN models were able to generate new realistic synthetic throat images which can improve the diagnostic accuracy The final deep learning model achieves high accuracy for automated diagnosis of pharyngitis using smartphone images Therefore this framework could be targeted toward patients with a sore throat who need screening for severe pharyngitis We believe that our study could be extended to computeraided diagnosis using images from an endoscope system in otolaryngology clinics similar to what has been done with colonoscope images using a deep learning model [] To the best of our knowledge no study has been performed to detect pharyngitis based on deep learning using smartphone images However it should be noted that this study is considered as only a preliminary and proofofconcept study because of its technical limitations due to using only Google Colab The current study framework is similar to that of Chamier which showed a deep learning framework using Google Drive and training and prediction on Google Colab [] Our proposed conceptual workflow is shown in Fig It needs the Flask servers and interfaces implemented using HTML and JavaScript to be applied in a real clinical setting This work could be part of a larger project to enable smartphonebased pharyngitis detection via a cloudbased applevel mobile data analysis We believe that a future appbased model can provide a robust solution for the costeffective and convenient screening of pharyngitis in a telemedicine setting We have highlighted the feasibility of a smartphonebased approach with deep learning to detect pharyngitis Our approach does not require Fig Classification results from the deep learning model applied to clinical cases with severe pharyngitis A An 81yearold man with odynophagia [] B A 41yearold diabetic man with throat pain [] Fig Example of a proposed smartphonebased system for pharyngitis detection ComputersinBiologyandMedicine12520201039807 0cexplored clinical datasets and webbased datasets and showed that the labeling of webbased images is often uncertain [] Therefore further clinical datasets with validation will be required to confirm the effectiveness of our framework Fourth because we collected only throat images via image search engines there was no metadata including gender race season or age According to a previous epidemiologic study these factors could affect the pharyngitis detection performance [] Fifth although the datasets were reviewed by authors images could be potentially duplicated in the dataset The duplicated images would affect the independence of the validation dataset We have shown the feasibility of deep learning for the detection of tonsil swelling and exudates in throat images The limitations of our study should be overcome by the availability of a sufficient number of throat images taken by a smartphone with a wellanized study protocol To validate the effectiveness of our framework a prospective study with many patients should be performed in a clinic once the app based framework is developed This will solve the problem of possible duplicated images in the dataset and the absence of metadata Conclusion TK Yoo additional equipment to collect throat images and we collected self taken images using a smartphone The use of webbased image capture of users from various devices and GANbased image augmentation may allow robust image processing While in the current study the implementation was performed on a desktop computer and the Colab™s remote server a smartphone application will also be possible to perform this identification for detecting pharyngitis The light deep learning models such as MobileNet can be executed on a smartphone without transmitting the images to a server [] Our result demonstrates that MobileNetv2 also has a high diagnostic performance similar to ResNet50 and Inceptionv3 In future research an increased amount of training data is needed to improve the detection accuracy using light models The utility of deep learning and smartphones may facilitate the widespread adoption of telemedicine and physical examination platforms via our approach Furthermore the presented framework enabled using a smartphone camera and deep learning techniques will help the patients in selfscreening for severe pharyngitis and may accelerate diagnostic support in remote healthcare services Because of the recent pandemic of COVID19 patients with respiratory symptoms need selfmonitoring to evaluate their pathologic status [] Advances in technology will require clinicians to embrace remote healthcare services The framework presented in this paper was designed for the timely diagnosis of pharyngitis for treatment Similar smartphoneimagebased diagnostic approaches have been introduced in several other medical image domains including skin diseases [] hematologic diseases [] oral diseases [] and eye diseases [] The main concern of deep learning models in this study

Thyroid_Cancer

"fundamental influences of sex and gender as modifiers of the major causes of death and morbidity We articulate how the genetic epigenetic and hormonal influences of biological sex influence physiology and disease and how the social constructs of gender affect the behaviour of the community clinicians and patients in the healthcare system and interact with pathobiology We aim to guide clinicians and researchers to consider sex and gender in their approach to diagnosis prevention and treatment of diseases as a necessary and fundamental step towards precision medicine which will benefit men™s and women™s healthIntroductionWhat clinicians know about the diagnosis treatment and prevention of disease originates from studies mostly done on male cells male mice and men1 Historically for multiple reasons including the purported safety of women and their offspring women of childbearing age were excluded from clinical trials As a result medical research and care have been centred on male physiology The assumption was that male and female cells and animals were biologically identical and evidencebased medicine was defined by clinical trials done predominantly in men1 In the US National Institutes of Health NIH mandated the inclusion of women in NIHfunded clinical trials but many investigators did not follow this mandate and many of those who did include women did not analyse the results by sex23 minimising the effectiveness of this policy Preclinical research and drug development studies have also predominantly used male animal models and cells4“ It is not surprising that a US Government Accountability Office report found that eight of the ten prescription drugs withdrawn from the market between and œposed greater health risks for women than for men Most funding agencies from Europe and North America have implemented policies to support and mandate researchers to consider sex and gender at all levels of medical research8 Still the field of sexbased biology and medicine is often viewed as a specialised area of interest rather than a central consideration in medical research Essential for the success of clinical care and translational science is awareness by clinicians and researchers that the diseases they are treating and studying are characterised by differences between women and men in epidemiology pathophysiology clinical manifestations psychological effects disease progression and response to treatmentThis Review explores the role of sex biological constructs and gender social constructs as modifiers of the most common causes of death and morbidity and articulates the genetic biological and environmental determinants that underlie these differences We aim to guide clinicians and researchers to better understand and harness the importance of sex and gender as genetic wwwthelancetcom Vol August biological and environmental modifiers of chronic disease Ultimately it is a necessary and fundamental step towards precision medicine that will benefit women and menSex as a genetic modifier of biology and diseaseSex differences in disease prevalence manifestation and response to treatment are rooted in the genetic differences between men and women Genetic sex differences start at conception when the ovum fuses with a sperm cell carrying an X or a Y chromosome resulting in an embryo carrying either XX or XY chromosomes This fundamental difference in chromosome complement eg genes outside the testisdetermining SRY gene generates ubiquitous sex differences in the molecular makeup of all male and female cells9 First the Y chromosome carries genes that exhibit subtle functional differences from their Xlinked homologues eg ZFY vs ZFX and UTY vs UTX and also carries genes with no homologue at all eg SRY In addition in men the X chromosome carries only maternal imprints ”ie epigenetic modifications made by the parent in generating the sex cells”which alter the expression of genes in the offspring As women have X chromosomes from both parents they carry maternal and paternal imprints which target a different set of genes Random inactivation of one of the X chromosomes in female cells which prevents sex differences in X chromosome gene dosage causes another degree of sex difference in gene expression As some of these Xlinked genes escape inactivation in women those genes are often expressed at higher levels in women than in men9 Sexspecific gene expression due to genomic Search strategy and selection criteriaWe searched PubMed for papers published in English between Jan and June using œsex or œgender and the name of the disease of interest as search terms Although we tried to cite seminal studies when necessary because of space limitation representative reviews were often selectedLancet “Diabetes Discovery SexBased Medicine Laboratory Section of Endocrinology John W Deming Department of Medicine Tulane University School of Medicine and Southeast Louisiana Veterans Health Care System Medical Center New Orleans LA USA Prof F MauvaisJarvis MD Barbra Streisand Women™s Heart Center CedarsSinai Smidt Heart Institute Los Angeles CA USA Prof N Bairey Merz MD National Heart Lung Institute Imperial College London London UK Prof P J Barnes MD Department of Pharmacology and Department of Neurology College of Medicine Center for Innovation in Brain Science University of Arizona Tucson AZ USA Prof R D Brinton PhD Department of Medical Epidemiology and Biostatistics and Center for Gender Medicine Karolinska Institutet Stockholm Sweden Prof JJ Carrero PhD Channing Division of Network Medicine and the Division of Pulmonary and Critical Care Medicine Department of Medicine Brigham and Women™s Hospital Harvard Medical School Boston MA USA D L DeMeo MD Neuroscience Institute and Department of Biology Geia State University Atlanta GA USA Prof G J DeVries PhD Department of Psychiatry University of Colorado School of Medicine Anschutz Medical Campus Aurora CO USA Prof C N Epperson MD Division of Oncology Department of Medicine Washington University School of Medicine St Louis MO USA Prof R Govindan MD W Harry Feinstone Department of Molecular Microbiology and Immunology The Johns Hopkins Bloomberg School of Public Health Baltimore MD USA Prof S L Klein PhD Department of Biomedical Metabolic and Neural Sciences University of Modena andReview 0cReggio Emilia Azienda OspedalieroUniversitaria di Modena Ospedale Civile di Baggiovara Modena Italy Prof A Lonardo MD Department of Psychiatry Department of Psychology and Department of Obstetrics Gynecology University of Illinois at Chicago Chicago IL USA Prof P M Maki PhD Department of Neurology McGovern Medical School University of Texas Health Science Center Houston TX USA Prof L D McCullough MD Berlin Institute of Gender Medicine Charit”Universittsmedizin Berlin Berlin Germany Prof V RegitzZagrosek MD Department of Cardiology University Hospital Z¼rich University of Z¼rich Switzerland Prof V RegitzZagrosek Center for Women™s Health Research Divisions of General Internal Medicine and Cardiology University of Colorado School of Medicine Aurora CO USAimprinting extends to autosomes as well and these imprinted genes exhibit sexspecific and tissuespecific expression in humans10 Thus fundamental sex differences deriving directly from genetic heterogeneity between the X and Y chromosome complements and parentoforigin inher itance exist at the molecular level in all human cells These sex differences persist throughout life and are independent of sex hormones figure Arguably the greatest source of differences between men and women comes from the Y chromosomal SRY gene which directs the development of a testis in men The ensuing developmental surge of testicular testosterone permanently masculinises the reproductive tract and the anisation of brain circuits affecting male behaviour at puberty1112 In humans the first surge occurs at the end of the first trimester of pregnancy Because it alters cellular gene expression and tissue structure in multiple ans of men via epigenetic mechanisms this testosterone surge is also paramount in programming sex differences in physiology and susceptibility to diseases that will manifest in adulthood After this initial testicular testosterone surge gonadal hormone concentrations remain low until puberty which triggers lasting sex differences in circulating oestrogens and testosterone concentrations After puberty cells with androgen or AFemale sexBRandom X chromosomeinactivation and escapeXXXXXXXXXXXXXXXXXXXY chromosome complementMale sexSRYCTesticular testosterone surgeFetal testisTestosteroneOHOGenetic diï¬erences of male and female cellsEpigenetic programming of male cellsFigure Genetic causes of sex differencesA Genetic sex differences start with cells carrying either XX or XY chromosome complement eg genes outside the testisdetermining SRY gene which generates ubiquitous sex differences in the molecular makeup of all male and female cells B Random inactivation of one X chromosome in female cells causes another level of sex differences in gene expression Some Xlinked genes escape inactivation in female individuals and have a higher expression in female than male individuals C The Y chromosomal SRY gene directs the development of a testis in male individuals which produces a surge of testicular testosterone at the end of pregnancy The testosterone surge programmes cellular gene expression and tissue structure in multiple ans of male individuals via epigenetic remodelling The combination of these genetic and developmental events programmes sex differences in physiology and susceptibility to diseases that will manifest in adulthoodoestrogen receptors will be affected differ ently in men and women The combination of all genetic and hormonal causes of sex differences aforementioned culminates in two different biological systems in men and women that translate into differences in disease predisposition manifestation and response to treatment Therefore sex is an important modifier of physiology and disease via genetic epigenetic and hormonal regulations figure Gender as a determinant of patients™ and doctors™ behaviour and as a modifier of health disease and medicineGender according to the Global Health definition refers to the socially constructed norms that impose and determine roles relationships and positional power for all people across their lifetime13 Gender interacts with sex the biological and physical characteristics that define women men and those with intersex identities13 Gender is not a binary term It includes the understanding that in many people traits of masculinity or femininity coexist and are expressed to different degrees Gender attributes are fluid more than two thirds of women and men report genderrelated characteristics traditionally attributed to the opposite sex1415 In transgender people gender identity differs with the sex they were assigned at birth So far transgender people have generally been underrepresented in clinical studies to date although this underrepresentation is changing Gender is an equally important variable as biological sex in human health and influences the behaviour of communities clinicians and patients1415 Gender roles represent the behavioural norms applied to men and women in society which influence individuals™ everyday actions expectations and experiences including diet perceived stress smoking and physical activity and affect health and disease susceptibility Gender identity describes the fluidity of how a person perceives oneself as a woman or a man which affects feelings and behaviours Gender relations refer to how we interact with or are treated by people on the basis of our ascribed gender Institutionalised gender reflects the distribution of power between men and women in the political educational and social institutions in society and shapes social norms that define perpetuate and often justify different expectations and opportunities for women and men1617 As such the distribution of genderrelated charac teristics within populations of men and women can influence health differently than biological sex Together these gender constructs determine access to health care helpseeking behaviours and individual use of the healthcare system Being perceived as a man or a woman triggers different responses from clinicians who might diagnose and suggest interventions differently according to gender As such gender largely determines the use of preventive measures and referral for or acceptance of invasive therapeutic strategies Genderrelated behaviours contribute to risk exposure and preventive behaviour in several wwwthelancetcom Vol August Review 0cdiseases This postulation is well exemplified in the cardiovascular field in which women often underestimate their risk compared with men and seek consultation later than men in the clinic for treatment of myocardial infarction1819 In the GENESISPRAXY prospective study mortality year after an acute coronary event was more strongly associated with gender than with biological sex1415 Similarly control of cardio vascular risk factors hypertension diabetes depres sive symptoms was better predicted by gender than by biological sex1415 Therefore including a gender dimension in clinical studies and practice will contribute to the understanding of different clinical manifestations and outcomes of diseases in women and men1617 Although beyond the scope of this Review it is also important to consider that regarding health and disease gender intersect with race or ethnicity and age20“ Sex and gender are fundamentally and frequently reciprocally interrelated in biology and disease24 Sex influences behaviours eg towards more aggressive or caring phenotypes On the other hand genderrelated behaviours eg smoking lifestyle perceived stress and pain and nutritional habits might produce epigenetic modifications that modulate gene expression and biological phenotypes Figure summarises how sex and gender are interrelated in biology and diseaseSex and gender differences in major chronic diseasesHaving established the importance of sex and gender in disease we will summarise their influences on the most common causes of death and debilitating diseases in the USA as an example figure Note that these sex and gender disparities are relevant to other highincome countries as well as lowincome and middleincome countries where the burden of these diseases becomes increasingly like those in highincome countries26 In most diseases efforts to separate the effects of sex and gender are still incomplete so that we just refer to the differences among women and men Because current knowledge about pathophysiology diagnosis and treatment of disease is primarily based on men as representative of the human species this Review focuses on how women differ from men We discuss some key aspects regarding the dimensions of men in a dedicated sectionHeart diseaseEpidemiology pathogenesis manifestations and diagnosisHeart disease is the leading cause of death in the USA In heart disease accounted for · of all deaths for men and for · of all deaths for women figure Ischaemic heart disease and heart failure are major contributors to heart disease mortality and have important sex and gender differences For example heart failure disproportionately contributes to coronary heart disease mortality in women27 potentially due to undiagnosed ischaemic heart disease in women The strength of the association with cardiovascular risk factors differ by sex Biological sexSex chromosomesEpigeneticeï¬ectsSex hormonesOHOHOHOBehaviour of patients and doctorsSocietyGender constructsLifestyleNutritional habitsExercisePerceived stressSmokingDiseasePathophysiologyManifestationResponse to treatmentDisease perceptionHelpseeking behaviourUse of health careDecision makingTherapeutic responseSex and gender diï¬erences in health disease and medicineFigure Interrelation between sex and gender in health diseases and medicineBiological sex causes sex differences through genetic and hormonal influences in disease pathophysiology clinical manifestations and response to treatment Sex also influences behaviours towards more aggressive or caring phenotypes On the other hand genderrelated behaviours eg smoking lifestyle perceived stress and nutritional habits produce epigenetic modifications that modulate the expression of biological sex Gender constructs determine patients™ perception of disease helpseeking behaviour and individual use of health care Gender constructs also influence decision making and trigger different therapeutic responses from providers biased by genderSystolic blood pressure and hypertension smoking and diabetes are associated with higher hazard ratios for myocardial infarction in women than in men28Ischaemic heart disease is the most recognised example for integrating the concept of gender and sex which shape divergent or distinct disease outcomes Compared with men women suffering from ischaemic heart disease are older this difference is historically believed to be due to the protection of endogenous oestrogens29 although contemporary study refutes this simplistic explanation30 and associations cannot be inferred to be causation Still women suffering from ischaemic heart disease are underdiagnosed3132 and less likely to have a prehospital diagnosis of myocardial infarction33“ The reasons for this disparity reflect the intersection between sex and gender First biological sex differences exist in the pathogenesis of ischaemic heart disease Whereas men are more likely to be affected by obstructive coronary artery disease of large vessels than women coronary microvascular dysfunction36 leading to chronic myocardial ischaemia without obstructive coronary artery disease has a higher prevalence in women than men37 A metaanalysis reported that following acute myocardial infarction both sexes most often presented with chest pain but compared with men women were more likely to present with pain between the shoulder blades nausea or vomiting and shortness of breathProf J G Regensteiner PhD Department of Medicine Department of Paediatrics and Department of Neuroscience Washington University School of Medicine St Louis MO USA Prof J B Rubin MD Center for the Study of Sex Differences in Health Aging and Disease Geetown University Washington DC USA Prof K Sandberg PhD Division of Gastroenterology Duke University Medical Center Durham NC USA A Suzuki MD and Durham VA Medical Center Durham NC USA A SuzukiCorrespondence to Prof Franck MauvaisJarvis Diabetes Discovery SexBased Medicine Laboratory Section of Endocrinology John W Deming Department of Medicine Tulane University School of Medicine New Orleans LA USA fmauvaistulaneeduwwwthelancetcom Vol August Review 0cMale individualsOther·Heart disease·protection might disappear after menopause45 By contrast testosterone induces adverse cardiac remod elling in the male heart44Chronic liver disease ·Influenza and pneumonia ·Suicide ·Alzheimer™s disease ·Type diabetes ·Stroke ·CPD ·Injuries ·Female individualsOther·Septicaemia ·Chronic kidney disease ·Influenza and pneumonia ·Type diabetes ·Injuries ·Alzheimer™s disease ·Stroke ·CPD ·Cancer·Heart disease·Cancer·Figure Percent distribution of the ten leading causes of death by sex USA Adapted from Heron25 CPDchronic pulmonary diseaseSecond a gender bias appears to be responsible for the absence of recognition of ischaemic heart disease presentation in women38 Men and women with ischaemic heart disease who score high on feminine roles and personality traits on questionnaires designed to ascertain aspects of gender are at an increased risk of recurrent ischaemic heart disease independent of female sex39Heart failure affects of adults aged years and older and more women than men in absolute numbers4041 Heart failure occurs at an older age and with less ischaemic causes in women than in men However hypertension and diabetes predispose older women to heart failure to a greater extent than men Heart failure with preserved ejection fraction a form of heart failure with normal systolic function is twice as prevalent in women as in men By contrast heart failure with reduced ejection fraction affects more men than women Women who have heart failure with preserved ejection fraction have smaller and stiffer hearts than men Inflammation and the resulting fibrosis play a sexspecific role in the pathogenesis of heart failure with preserved ejection fraction Under stress premenopausal women™s hearts develop less inflammation resulting in less fibrosis than men™s hearts4243 This difference is partially driven by sex hormones as oestrogens produce antiinflammatory actions on endothelial and immune cells and promote cardioprotective effects in premenopausal women44 This Response to treatmentCompared with men women suffering from ischaemic heart disease are less likely to receive evidencebased treatment3132 and when suffering from acute myocardial infarction they are less likely to receive reperfusion33“ An STelevation myocardial infarction registry revealed that compared with men women exhibit delayed reperfusion leading to higher mortality46 Women suffering from acute myocardial infarction treated by male emergency physicians have a higher mortality rate than those treated by female physicians38 Additionally male physicians are more effective at treating female patients with acute myocardial infarction when they work with female colleagues and when they have experience in treating female patients38 This treatment disparity between women and men can be corrected by improving emergency recognition of STelevation myocardial infarction in women and acceleration of percutaneous coronary intervention which equilibrates gender mortality47Guidelines for the treatment of heart failure are similar for women and men24 However evidence suggests that optimal survival in women occurs with lower doses of β blockers angiotensin receptor blockers and angiotensin converting enzyme inhibitors than in men48 Finally fewer women undergo heart transplantation than men although women are more frequently donors suggesting a referral bias could exist41CancersEpidemiology pathogenesis manifestations and diagnosisCancers are the second leading cause of death dominated by lung cancer accounting for · of deaths in men and · of deaths in women figure More men develop cancer than women49 With few exceptions eg meningioma thyroid cancer lung cancer in nonsmokers nonreproductive cancers exhibit a male predominance though for some cancers oropharynx larynx oesophagus and bladder the male versus female incidence ratios can be higher than A male predominance in cancers that affect both sexes is evident around the world in all races and at all ages50 Survival is also shorter for men than women across multiple cancer types The higher cancer risk in men is partially explained by gender constructs like dietary habits or risk behaviours such as smoking and alcohol consump tion51 It is unlikely to be the only cause After appropriate adjustment for these risk factors adult men still have a higher cancer risk than women52 Moreover a similar male bias in incidence and survival is seen in paediatric cancers before puberty and the adoption of highrisk cancerpromoting behaviours eg smoking53The universal male predominance in cancer incidence and differential outcomes argues for a fundamental role wwwthelancetcom Vol August Review 0cin inutero tumour suppressors of sex in addition to gender in cancer biology Sexspecific biology includes genetic differences XX vs XY chromosomes the incomplete Xinactivation in female individuals which results in biallelic expression of Xencoded female cells54 Y chromosomeencoded oncogenes such as the RNAbinding motif on Y chromosome in male cells55 and the chromatin remodelling effects of testicular testosterone in male cells56 These mechanisms have an influence on several of the hallmarks of cancer57 including metabolism growth regulation58 angiogenesis and immunity which all contribute to cancer predisposition59 A crucial example is the male predisposition to glioblastoma which is the most common form of brain cancer In glioblastoma there is a cellintrinsic predisposition of male astrocytes a subtype of glial cell to malignant transformation60 After puberty sex hormones produce additional epigenetic and acute effects on cells that further influence sex disparities in cancer For example the increased frequency and aggressive phenotype of hepatocellular carcinoma in male individuals has been linked to the stimulatory effects of androgens in male individuals and the protective effects of oestrogens in female individuals61 Importantly the biology of cancer is not the same across histological and genetic diagnostic groups or even within single histol ogical subtypes Thus the interaction between sex gender and cancer mechanisms cannot be expected to be constant Take colon cancer the second leading cause of cancerrelated death for example Although women have a lower overall incidence of colon cancer than men they have a higher incidence of rightsided colon cancers which have the worst outcomes62 Tumours from women with rightsided colon cancers exhibit a distinct molecular signature of energy metabolism compared with those of women with leftsided colon cancers63 This molecular signature is not observed when comparing tumours from men with rightsided colon cancers to men with leftsided colon cancers Thus overall the male predisposition to cancer is probably the consequence of genetic program ming of male cells and the effect of sex hormones after puberty interacting with genderspecific behaviours to establish cancer risksResponse to treatmentIn the future cancer prevention and treatment will be improved by sexspecific and genderspecific approaches For example immune checkpoint inhibitors can improve survival for men with advanced melanomas and nonsmallcell lung cancers more than for women64 The molecular subtyping of glioblastomas based on sexspecific transcriptomes has the potential to enhance chemotherapy in a sexspecific manner65 In colon cancer sex differences in xenobiotic metabolism regulatory networks might underlie greater treatment response in women than men and require modification of approaches for men with colon cancer66 Other elements of cancer metabolism are also ripe for novel sexspecific targeting in treatment Cellular nutrient partitioning is sexually dimorphic so approaches such as ketogenic diets or glutaminase inhibition might be associated with substantial sexspecific responses67 Furthermore data from models of development ageing and cancer all indicate that molecular pathways such as the enzyme phosphatidylinositol 3kinase and tumour suppressors such as p53 and retinoblastoma protein are sexually dimorphic and require sexspecific targeting545968Chronic pulmonary diseaseEpidemiology pathogenesis manifestations and diagnosisChronic pulmonary disease is the third leading cause of death for women · of deaths and the fourth for men · figure It is mostly accounted for by chronic obstructive pulmonary disease and to a lesser extent by asthma Chronic obstructive pulmonary disease is characterised by irreversible airflow limitation and is associated with previous exposure to smoking or air pollutants Women are overrepresented among individuals with chronic obstructive pulmonary disease especially among those with earlyonset disease or those who have never smoked69 Women are also twice as likely to have chronic obstructive pulmonary disease with chronic bronchitis and women with severe chronic obstructive pulmonary disease have as much emphysema as men countering the misconception of emphysema as a male form of chronic obstructive pulmonary disease70 The female lung is more susceptible to chronic obstructive pulmonary disease than the male lung and women develop symptoms of the disease at a younger age with less tobacco exposure than men71 The Genetic Epidemiology of Chronic Obstructive Pulmonary Disease COPDGene study72 suggests that earlyonset chronic obstructive pulmonary disease might originate in utero in susceptible women from alterations in lung development potentiated by maternal asthma and smoking genetic factors or hormonal influences Future studies should focus on the contribution of maternally inherited factors such as mitochondrial and X chromosome genes to understand disease pathogenesis It is important to consider gender constructs as well Smoking advertising campaigns targeting women rose in the 1960s and the resulting higher smoking rates influenced women™s risk for developing chronic obstructive pulmonary disease73 From a disease severity vantage point chronic obstructive pulmonary disease exacerbation rates are also higher in women than men especially at a younger age74 Additionally despite the burden of symptomatology and increased rates of hospitalisations and deaths women with chronic obstructive pulmonary disease are often misdiagnosed and disproportionally suffer from comorbid conditions including anxiety and depression Therefore physicians should consider chronic obstructive pulmonary disease in the differential diagnosis of women with pulmonary symptoms regardless of tobacco or pollutant exposure historieswwwthelancetcom Vol August Review 0cAsthma characterised by variable airflow obstruction and chronic airway inflammation also affects men and women differently Asthma is more prevalent in prepubertal boys than girls Regarding asthma both male biological sex lung development and atopy and male gender constructs related to outdoor play and indoor pet exposure are factors contributing to the devel opment of asthma and sex versus gender contributions could be difficult to separate75 F

Thyroid_Cancer

"Previous evidence has suggested that lower gestational vitamin D levels might increase the risks ofadverse pregnancy and birth outcomes The results remain inconsistent and require further explorationMethods A total of Chinese motherinfant pairs were included in this retrospective cohort study Serumconcentrations of 25OHD were reviewed in early pregnancy ± weeks Outcomes of maternal gestationaldiabetes mellitus GDM cesarean section fetal distress preterm birth low birth weight LBW and macrosomiawere extracted from the medical records Cox regression analysis was used to explore these associationsResults In total of mothers were pregnant at an advanced age ‰¥ years and of pregnant womenhad vitamin D deficiency nmolL After adjusting for potential covariates the hazard ratio HR CI perstandard deviation SD increase of serum 25OHD concentrations was for GDM for preterm birth and for LBW Similar protective associations were found for GDMcesarean section and preterm birth for a better vitamin D status when compared with vitamin D deficiencyConclusion Higher early pregnancy vitamin D was associated with a lower risk of GDM cesarean section pretermbirth and LBWKeywords 25ohd Vitamin D Pregnancy Maternal outcome Infant outcomeBackgroundVitamin D is a secosteroid hormone that is well knownfor its physiological function in maintaining bone metabolism and health A high prevalence of vitamin D deficiencyusually defined as serum 25OHD levels nmolL hasbeen found in pregnant women globally especially in developing countries [] including China [ ] Increasing evidence has suggested that vitamin D sufficiency is important Correspondence liuzphlk81outlookcom Gengdong Chen and Tingting Pang contributed equally to this work1Foshan Institute of Fetal Medicine Department of Obstetrics AffiliatedFoshan Maternity Child Healthcare Hospital Southern Medical UniversityFoshan Guangdong ChinaFull list of author information is available at the end of the for the prevention of pregnancy complications in mothersand adverse fetal birth outcomes [ ] although divergentresults have been reported in other studies [ “] Severalsystematic reviews based on randomized clinical trials orobservational studies have suggested that lower vitamin Dstatus contribute to adverse outcomes such as preeclampsia[] gestational diabetes mellitus GDM [ ] low birthweight LBW [] and preterm birth [] However increasing evidence shows different associations [ ]and no definitive has yet been made [] Severalproblems remain to be solved by further studies the heterogeneity of associations from diverse areas and populations with different vitamin D status serve as evidencethewhen makingguidelinesregionsforspecific The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Pregnancy and Childbirth Page of observation or supplementation of vitamin D in the thirdtrimester in many studies might present the problem ofcausal inference or miss the practical period for the intervention In addition the results from early trimesters mightbe helpful and more important for the early prevention ofadverse outcomesIn additionit has been suggested that the risk ofadverse complications or birth outcomes increases asmaternal age increases [“] With the change ofpopulation policy in China the percentage of womenpregnant at an advanced age years has increasedand proper strategies are urgently needed for the prevention of adverse complications [] However whetherthe influence of vitamin D on maternal and infant outcomes remained the same for women pregnant at young years and advanced ages remains unclear andmore studies are needed to better illustrate the problemWe investigated the relationship between early gestational serum 25OHD concentrations and several adverse maternal and infant outcomes in a retrospectivecohort study including Chinese motherinfantpairs Our results provide further evidence for clinicalrecommendations on the early prevention of related adverse outcomes in this fieldMethodsThe study used data that were gathered from a largecenter Affiliated Foshan Maternity Child HealthcareHospital Southern Medical University Foshan CityGuangdong Province China from September toJuly The hospital is the largest gynecology andobstetrics center in Foshan City and covers a large population of million people The included subjects werewomen who underwent early pregnancy serum vitaminD measurement ‰ gestational weeks and deliveredtheir infants at the hospital The exclusion criteria included twin or higherorder multiple pregnancies seriousdiseases such as type diabetes mellitus cardiovasculardiseases thyroid disorder and cancer that occurred before pregnancy Ultimately a total motherinfantpairs were included in this study The study was approvedby the ethics committee of Affiliated Foshan Maternity Child Healthcare Hospital Southern Medical UniversityData collectionVitamin D data from the clinicallaboratory werereviewed Blood samples were collected during the regular obstetric checkups and immediately measured by aclinical laboratory without being frozen Serum concentrations of 25OHD 25OHD2 and 25OHD3 weredetected using colloidal gold immunochromatographyCommercial kits were obtained from Mei Ning KangCheng Bio Tec Inc The intraassay and interassay coefficients of variation were less than Outcomes including GDM cesarean section fetal distress preterm birth low birth weight and macrosomiawere extracted from medical records and reexamined bytwo independent staff members The disease diagnoseswere made by professional doctors with the samestandardization criteria and were extracted from themedical records Gestational hypertension preeclampsiaor eclampsia was not included because of the lack ofavailable cases An oral glucose tolerance test was performed from to gestational weeks and GDM wasdiagnosed if the subjects met any of the following criteria fasting blood glucose ‰¥ mmolL onehour bloodglucose postoral sugar ‰¥ mmolL or twohour bloodglucose postoral sugar ‰¥ mmolL Preterm birth wasdefined as delivery at ‰¥ but gestational weeksLBW was diagnosed if the neonatal birth weight g while a neonatal birth weight ‰¥ g was defined asmacrosomia Other variablesincluding the maternalage body mass index BMI gestational age parity season of blood collection and time of delivery were alsoextracted from the medical recordsStatistical analysesContinuous variables were represented by the mean ±standard deviation SD or median interquartile rangeand tested by Student™s ttest Categorical variables wererepresented by frequencies percentage and tested bythe chisquare test Cox regression analysis was performed to explore the associations between vitamin Dand maternal or infant outcomes Serum concentrationsof 25OHD 25OHD2 and 25OHD3 were first Zstandardized before being included in the regressionVitamin D deficiency was defined as serum 25OHDlevels of nmolL Two different models were testedwith Model as a univariate model without adjustmentand Model adjusted for maternal age BMI parity andseason the blood was collected and measured Stratifiedanalyses were performed according to the gestationalage young years advanced ‰¥ years All the analyses were performed using SPSS software version SPSS Inc Chicago IL USA A twosided P value ofless than was considered statistically significantResultsA total motherinfant pairs were included in thisstudy A high prevalence of gestational vitamin D deficiency was discovered in the mothers Even insubjects without vitamin D deficiency the highest serum25OHD concentration was only nmolL Compared with women pregnant at a younger age the subjects with advanced age of pregnancy ‰¥ years tendedto have a higher BMI parity higher percentage of vitamin D deficiency higher incidence of GDM cesareansection preterm birth and LBW but a lower gestational 0cChen BMC Pregnancy and Childbirth Page of age lower serum 25OHD and OHD3 concentrations lower incidence of fetal distress and lower incidence of macrosomia Table As shown in Table although the 25OHD concentrations were statistically higher in spring and summerthan those in autumn and winter only small differenceof 25OHD values to nmolL was observedbetween different seasons especially for 25OHD2 Significant protective associations were found between thevitamin D levels and GDM and preterm birth Afteradjusting for potential covariatesTable higher25OHD concentrations per one SD increase were associated with a HR CI decrease in the GDM risk a HR CI decrease in the preterm birth risk and a HR CI decrease in theLBW risk Similar protective results were also found for25OHD2 and 25OHD3 but the associations tended tobe more pronounced for 25OHD2 Null associationsbetween vitamin D and cesarean section fetal distressand macrosomia were observed in all the subjects Maternal serum 25OHD levels ‰¥ nmolL were associated with a decrease in the GDM risk a decrease in the cesarean section risk and a decrease in the preterm birth risk but the trend did nothold with the other outcomes compared with those withvitamin D deficiency Table After stratification by gestational age Table higherlevels of vitamin D were associated with a lowerrisk of GDM in those years In contrast a protectiveassociation of vitamin D with low birth weight wasfound for women pregnant at ‰¥ years but not years However no significant interactions were discovered Pinteraction Table Characteristic of subjectsAge yearsBMI kgcm2Gestational age weeksParity timesNeonatal birth weight kg25OHD nmolL25OHD2 nmolL25OHD3 nmolLVitamin D deficiency N YesNoGestational diabetes mellitus N YesNoCaesarean section N YesNoFetal distress in uterus N YesNoPreterm birth N YesNoLow birth weight N YesNoMacrosomia N YesNoTotal N ± years N ± ‰¥ years N ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± P 0cChen BMC Pregnancy and Childbirth Page of Table Seasonal difference between serum vitamin D indicatorsDetected Seasonspring summer ± ± ± autumn winter ± ± ± P 25OHD nmolL25OHD2 nmolL25OHD3 nmolLDiscussionIn this retrospective cohort study including Chinese motherinfant pairs protective associations werefound for higher serum 25OHD concentrations withGDM cesarean section postpartum hemorrhage preterm birth and low birth weight but not for the otheroutcomes The results for 25OHD2 tended to be morepronounced than those for 25OHD3Interestinglyhigher serum vitamin D contributes to a higher risk ofpostpartum anemia in women pregnant at a young agebut not in advanced yearsIn this population with a high prevalence of vitamin Ddeficiency better serum vitamin D status contributed toa lower risk of GDM This result was consistent withseveral other studies A to higher risk of GDMwas observed for pregnant women with insufficient ordeficient vitamin D status in three systematic reviewsand metaanalyses based on observational studies orclinical trials [ ] In a large randomized controltrial RCT based on Iranian women the intervention of 25OHD3 was associated with lower risk ofGDM [] The results were further supported by severalother prospective cohort studies [ ] However nullassociations were found between the vitamin D status orsupplements and GDM in a systematic review based onfive randomized trials including subjects [] anested casecontrol study of women [] and alarge prospective study including motherchildpairs [] Additionally two studies found detrimentalassociations of higher vitamin D levels with GDM butthe effects were tiny [] or restricted to specificethnicities Hispanic []Consistent with our results the protective associationof maternal vitamin D status with a lower risk of preterm or low birth weight has also been reported in severalstudies An inverse doseresponse relation ofvitamin D status was found for preterm birth in a systematic review and metaanalysis based on longitudinal studies[] Rostami reported that a 25OHD3 interventionof monthly IU could be beneficial for the preventionof preterm delivery in an RCT of Iranian women []In a large prospective cohort including motheroffspring pairs the risk of low birth weight was CI and CI among subjects with vitamin D deficiency and insufficiency respectively [] Higher maternal serum 25OHD was positivelyTable Associations between early pregnant serum vitamin D concentrations and maternal infant outcomes25OHD aHR95CIPa25OHD2HR95CIPa25OHD3HR95CIPGestational diabetes mellitusModel Model Caesarean sectionModel Model Fetal distressModel Model Preterm birthModel Model Low birth weightModel Model MacrosomiaModel Model Cox regression analysis were operated for exploration of associations Model without adjustment Model adjusted for age BMI parity season of bloodcollected a Per one SD increase 0cChen BMC Pregnancy and Childbirth Page of Table Associations between vitamin D status and maternal infant outcomesGestational diabetes mellitusCaesarean sectionFetal distressPreterm birthLow birth weightMacrosomia25OHD nmolL‰¥ nmolL nmolL‰¥ nmolL nmolL‰¥ nmolL nmolL‰¥ nmolL nmolL‰¥ nmolL nmolL‰¥ nmolLHR95CI PCox regression analysis were operated for exploration of associations and adjusted for covariates including age BMI parity season of blood collectedassociated with higher birth weight in a study of UnitedArab Emirates [] and supplementation of vitamin D at adose of IUd was optimal and safe for mothers andtheir infants in United Arab Emirates [] However nullor detrimental associations were also reported in otherstudies Although vitamin D increased the mean birthweight it did not significantly reduce the risk of low birthweight or preterm birth in a metaanalysis based on randomized trials [] these results were further supported byanother update study [] In addition higher 25OHDconcentrations were found to increase the riskof preterm delivery in a prospective study of pregnant Chinese women []Gestational vitamin D deficiency was found to be associated with a 2fold increased risk of cesarean section ina cohort of lowincome pregnant women []Within a cohort of women from the United Statesfor women with 25OHD concentrations lower than nmolL the risk of a primary cesarean section wasalmost times higher [] These results were consistent with ours However a metaanalysis of trials subjects found null associations of vitamin D supplements with cesarean section []Much heterogeneity exists in the previous evidence inthis field and there may be several factors that partly explain these differences First most of the randomized trials included had a small sample size and were of lowquality Most subjects included in the trials had sufficient vitamin D status ‰¥ nmolL and a further doseof a or IUd supplement might have attenuatedTable Subclass analysis of relationship between serum vitamin D concentrations and related outcomes stratified by gestational ageP bP bP baa25OHD aHR95CIP25OHD2HR95CIP25OHD3HR95CIPGestational diabetesmellitusCaesarean sectionFetal distressPreterm birth y ‰¥ y y ‰¥ y y ‰¥ y y ‰¥ y Low birth weight y ‰¥ y Macrosomia y ‰¥ y Cox regression analysis were operated for exploration of associations and adjusted for covariates including age BMI parity season of blood collecteda Per one SD increase b P for interaction 0cChen BMC Pregnancy and Childbirth Page of the detrimental influence of vitamin D deficiency or insufficiency in the reference group [] Increasing benefitsmight not exist for higher doses Second the vitamin Dreceptor is important for the utilization of vitamin D[ ] The difference in vitamin D receptor gene polymorphism might help explain the ethnic heterogeneity[] This requires further confirmation in the futureThird the studies were conducted during different trimesters of gestation which might increase the difficultyof making comparisons those studies conducted duringan early gestational period might be advantageous forcausal inference and early preventionBecause 25OHD3 contributes most to the 25OHDconcentrations it has been used to represent 25OHDin many studies and only a few studies have focused on25OHD2 In our study the results were generally consistent between these two subcomponents The associations tend to be more pronounced in 25OHD2 than in25OHD3 and mightindicate the prominence of25OHD2 over 25OHD3 for the prevention of relateddiseases This theory merits further confirmation Nevertheless our results and others provide further evidencefor the importance of a better early vitamin D status forthe prevention of GDM cesarean section preterm birthand low birth weight in a population with a high prevalence of vitamin D deficiency Our results indicated thatmore consideration should be given to distinguishing between the vitamin D subcomponents women pregnantat different ages and women with more pregnancy complications in this field Considering the high prevalenceof vitamin D deficiency during pregnancy observed inChina [ ] it is a matter of urgency to call for the supplementation of vitamin D and more efforts should bemade to improve the gestational vitamin D status ofChinese populationVitamin D deficiency was found to decrease vasculardiameter within the labyrinth region and dysregulateplacental development during early pregnancy in an animal experiment [] Vitamin D supplementation duringearly pregnancy might rescue this situation while furthersupplementation might be futile after missing this timepoint [] Moreover vitamin D supplementation duringearly pregnancy is likely to affect genetic information ofsystemic inflammation and immune responses involvedin the development of gestational comorbidities egGDM preeclampsia and infection as reviewed by Hollis [] These mechanisms help to explain the beneficialinfluences of vitamin D for maternal and infanthealth and emphasize the importance of improving vitamin D status during early pregnancyOurstudy had several advantages Firstserum25OHD concentrations were measured during an earlygestational period and with the retrospective cohort design we assured the temporal sequence and avoided thepossibility of causal inversion Second we studied multiple outcomes and conducted further analysis of thesubcomponent of vitamin D and a stratified analysis ofgestational age which provided a more comprehensiveunderstanding of this field There are also several limitations of our study First our study was limited by thelack of information on dietary vitamin D intake including by supplement and sunlight exposure during thepregnancy period The obtained data were difficult touse for a retrospective study while the use of blood indicators might be more precise than a dietary survey sowe adjusted the association for different seasons to attenuate their influence Second the 25OHD concentration was measured only once we could not monitor thedynamic changes afterward or determine whether theywere normalized after receiving vitamin D supplementation However the associations tended to be underestimated instead of overestimated Third Wagner et al[] and Mirzakhani [] indicated a 25OHDconcentration ‰¥ nmolL in early pregnancy was optimal for the prevention of preterm birth and preeclampsia however the highest 25OHD concentration in ourstudy is only nmolL Therefore we were unable toperform the analyses using a threshold of nmolLand assess the influences of higher 25OHD concentrations Finally residual confounding might still existthough we tried to control several potential covariatesConclusionsThis retrospective cohort study showed the beneficial associations of early gestational vitamin D with outcomesof GDM cesarean section preterm birth and low birthweight More welldesigned randomized clinical trialsare needed for further exploration and confirmation ofthese resultsAbbreviationsGDM Gestational diabetes mellitus LBW Low birth weight BMI Body massindex SD Standard deviationAcknowledgementsWe would like to thank Ye Shaoxin Yang Xiaoming Liu Haojing Wangdong and Huang Shaobing for their generous help in this studyAuthors™ contributionsGDC and ZPL devised the idea and designed the study GDC TTP PSLZXZ DXL DZF contributed to the primary data collection GDC and TTP reexamined the data GDC TT P PS L ZX Z DXL DZF contributedto the analysis of the data GDC and TTP wrote the original draft whichwas revised by XLG and ZPL XLG and ZPL supervised the study andZPL administered the project All authors have read and approved themanuscriptFundingThis work was supported by the Basic and Applied Basic ResearchFoundation of Guangdong Province No 2019A1515110163 GDC and theFoundation of Bureau of Science and Technology of Foshan City No GDC The funding sponsors had no role in the design ofthe study in the collection analyses or interpretation of data in the writingof the manuscript and in the decision to publish the results 0cChen BMC Pregnancy and Childbirth Page of Availability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author upon reasonable requestEthics approval and consent to participateThe study was approved by the ethics committee of Affiliated FoshanMaternity Child Healthcare Hospital Southern Medical University TheAffiliated Foshan Maternity Child Healthcare Hospital providedadministrative permissions for the research team to access and use the dataincluded in this research Data were extracted from medical records and theconsent to participate was unavailable due to the retrospective design of thestudy and difficulty in reconnection however the private information waswell protectedConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Foshan Institute of Fetal Medicine Department of Obstetrics AffiliatedFoshan Maternity Child Healthcare Hospital Southern Medical UniversityFoshan Guangdong China 2Department of Medical RecordsAffiliated Foshan Maternity Child Healthcare Hospital Southern MedicalUniversity Foshan Guangdong ChinaReceived February Accepted August ReferencesSaraf R Morton SM Camargo CA Jr Grant CC Global summary of maternaland newborn vitamin D status a systematic review Matern Child Nutr“Yun C Chen J He Y Mao D Wang R Zhang Y Yang C Piao J Yang XVitamin D deficiency prevalence and risk factors among pregnant Chinesewomen Public Health Nutr “Zhou J Su L Liu M Liu Y Cao X Wang Z Xiao H Associations between hydroxyvitamin D levels and pregnancy outcomes a prospectiveobservational study in southern China Eur J Clin Nutr “Agarwal S Kovilam O Agrawal DK Vitamin D and its impact on maternalfetal outcomes in pregnancy a critical review Crit Rev Food Sci Nutr “von Websky K Hasan AA Reichetzeder C Tsuprykov O Hocher B Impact ofvitamin D on pregnancyrelated disorders and on offspring outcome JSteroid Biochem Mol Biol “Nobles CJ Markenson G ChasanTaber L Early pregnancy vitamin D statusand risk for adverse maternal and infant outcomes in a biethnic cohort thebehaviors affecting baby and you BaBY study Br J Nutr “Roth DE Leung M Mesfin E Qamar H Watterworth J Papp E Vitamin Dsupplementation during pregnancy state of the evidence from a systematicreview of randomised trials BMJ 2017359j5237HautaAlus HH Viljakainen HT HolmlundSuila EM EnlundCerullo MRosendahl J Valkama SM Helve OM Hytinantti TK Makitie OM AnderssonS Maternal vitamin D status gestational diabetes and infant birth size BMCPregnancy Childbirth Khaing W Vallibhakara SA Tantrakul V Vallibhakara O Rattanasiri S McEvoyM Attia J Thakkinstian A Calcium and vitamin D supplementation forprevention of preeclampsia a systematic review and network metaanalysisNutrients 2017910E1141 Zhang Y Gong Y Xue H Xiong J Cheng G Vitamin D and gestationaldiabetes mellitus a systematic review based on data free of Hawthorneeffect BJOG “ Zhang MX Pan GT Guo JF Li BY Qin LQ Zhang ZL Vitamin D deficiencyincreases the risk of gestational diabetes mellitus a metaanalysis ofobservational studies Nutrients “ Aghajafari F Nagulesapillai T Ronksley PE Tough SC O'Beirne M Rabi DMAssociation between maternal serum 25hydroxyvitamin D level andpregnancy and neonatal outcomes systematic review and metaanalysis ofobservational studies BMJ 2013346f1169 Qin LL Lu FG Yang SH Xu HL Luo BA Does Maternal Vitamin D DeficiencyIncrease the Risk of Preterm Birth A MetaAnalysis of Observational StudiesNutrients 201685E301 Rodriguez A GarciaEsteban R Basterretxea M Lertxundi A RodriguezBernalC Iniguez C RodriguezDehli C Tardon A Espada M Sunyer J et alAssociations of maternal circulating 25hydroxyvitamin D3 concentrationwith pregnancy and birth outcomes BJOG “ Ogawa K Urayama KY Tanigaki S Sago H Sato S Saito S Morisaki NAssociation between very advanced maternal age and adverse pregnancyoutcomes a cross sectional Japanese study BMC Pregnancy ChildbirthKhalil A Syngelaki A Maiz N Zinevich Y Nicolaides KH Maternal age andadverse pregnancy outcome a cohort study Ultrasound Obstet Gynecol“Jolly M Sebire N Harris J Robinson S Regan L The risks associated withpregnancy in women aged years or older Hum Reprod “Li Q Deng D New medical risks affecting obstetrics after implementation ofthe twochild policy in China Front Med “ Poel YH Hummel P Lips P Stam F van der Ploeg T Simsek S Vitamin Dand gestational diabetes a systematic review and metaanalysis Eur J InternMed “ Rostami M Tehrani FR Simbar M Bidhendi Yarandi R Minooee S Hollis BWHosseinpanah F Effectiveness of prenatal vitamin D deficiency screeningand treatment program a stratified randomized field trial J Clin EndocrinolMetab “ Xu C Ma HH Wang Y Maternal early pregnancy plasma concentration of25Hydroxyvitamin D and risk of gestational diabetes mellitus Calcif TissueInt “ Boyle VT Thorstensen EB Mourath D Jones MB McCowan LM Kenny LCBaker PN The relationship between 25hydroxyvitamin D concentration inearly pregnancy and pregnancy outcomes in a large prospective cohort BrJ Nutr “Schneuer FJ Roberts CL Guilbert C Simpson JM Algert CS Khambalia AZTasevski V Ashton AW Morris JM Nassar N Effects of maternal serum hydroxyvitamin D concentrations in the first trimester on subsequentpregnancy outcomes in an Australian population Am J Clin Nutr “ Amegah AK Klevor MK Wagner CL Maternal vitamin D insufficiency andrisk of adverse pregnancy and birth outcomes a systematic review andmetaanalysis of longitudinal studies PLoS One 2017123e0173605 Chen YH Fu L Hao JH Yu Z Zhu P Wang H Xu YY Zhang C Tao FB XuDX Maternal vitamin D deficiency during pregnancy elevates the risks ofsmall for gestational age and low birth weight infants in Chinesepopulation J Clin Endocrinol Metab “ Amirlak I Ezimokhai M Dawodu A Dawson KP Kochiyil J Thomas LAbdulle AM Current maternalinfant micronutrient status and the effects onbirth weight in the United Arab Emirates East Mediterr Health J “ Dawodu A Saadi HF Bekdache G Javed Y Altaye M Hollis BW Randomizedcontrolled trial RCT of vitamin D supplementation in pregnancy in apopulation with endemic vitamin D deficiency J Clin Endocrinol Metab“ PerezLopez FR Pasupuleti V MezonesHolguin E BenitesZapata VA Thota PDeshpande A Hernandez AV Effect of vitamin D supplementation duringpregnancy on maternal and neonatal outcomes a systematic review and metaanalysis of randomized controlled trials Fertil Steril “1288e1274Scholl TO Chen X Stein P Maternal vitamin D status and delivery bycesarean Nutrients “ Merewood A Mehta SD Chen TC Bauchner H Holick MF Associationbetween vitamin D deficiency and primary cesarean section J ClinEndocrinol Metab “ Chun RF Peercy BE Orwoll ES Nielson CM Adams JS Hewison M VitaminD and DBP the free hormone hypothesis revisited J Steroid Biochem MolBiol Pt A132“ Bikle DD Gee E Halloran B Kowalski MA Ryzen E Haddad JG Assessmentof the free fraction of 25hydroxyvitamin D in serum and its regulation byalbumin and the vitamin Dbinding protein J Clin Endocrinol Metab “ Powe CE Evans MK Wenger J Zonderman AB Berg AH Nalls M Tamez HZhang D Bhan I Karumanchi SA Vitamin Dbinding protein and 0cChen BMC Pregnancy and Childbirth Page of vitamin D status of black Americans and white Americans N Engl J Med“Liu NQ Ouyang Y Bulut Y Lagishetty V Chan SY Hollis BW Wagner CEquils O Hewison M Dietary vitamin D restriction in pregnant female miceis associated with maternal hypertension and altered placental and fetaldevelopment Endocrinology “ Mirzakhani H Litonjua AA McElrath TF O'Connor G LeeParritz A Iverson RMacones G Strunk RC Bacharier LB Zeiger R Early pregnancy vitaminD status and risk of preeclampsia J Clin Invest “ Hollis BW Wagner CL Vitamin D supplementation during pregnancyimprovements in birth outcomes and complications through directgenomic alteration Mol Cell Endocrinol “ Wagner CL Baggerly C McDonnell SL Baggerly L Hamilton SA Winkler JWarner G Rodriguez C Shary JR Smith PG Posthoc comparison ofvitamin D status at three timepoints during pregnancy demonstrates lowerrisk of preterm birth with higher vitamin D closer to delivery J SteroidBiochem Mol Biol “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

Neck Tissues A a0Systematic ReviewJerome a0R a0Lechien1234 Stphane a0Hans13 a0· Maria a0R a0Barillari18 a0· Giovanni a0Cammaroto19 a0· Graldine a0Descamps12 a0· Julien a0Hsieh110 a0· Luigi a0Vaira111 a0· Giacomo a0De a0Riu111 a0· Leigh a0Sowerby112 a0· Isabelle a0Gengler113 a0· Justin a0Michel15 a0· Sven a0Saussez124 a0· Thomas a0Radulesco15 a0· Christian a0Calvo‘Henriquez16 a0· Carlos a0M a0Chiesa‘Estomba17 a0· Received July Accepted August Springer ScienceBusiness Media LLC part of Springer Nature AbstractTo review the data regarding the expression of angiotensin converting enzyme2 ACE2 and transmembrane protease serine2 TMPRSS2 in head and neck tissue Scopus Cochrane Library Medrxiv Google Scholar and PubMEDMEDLINE were searched by four independent investigators for studies investigating ACE2 or TMPRSS2 expressions in head and neck tissues The following outcomes were considered sample origin animal versus human detection method anatomical location and cell types PRISMA checklist and modified population intervention comparison outcome timing and setting PICOTS framework were used to perform the review Of the identified studies met our inclusion criteria Thirteen studies were conducted during the severe acute respiratory syndrome a0coronavirus2 SARSCoV2 pandemic ACE2 and TMPRSS2 were expressed in oral pharyngeal sinusonasal human mucosa The following cell types expressed ACE2 basal apical goblet minor salivary and endothelial cells TMPRSS2 was found in goblet and apical respiratory cells ACE2 and TMPRSS2 were found in the olfactory region especially in sustentacular nonneural and neural stem cells Animal studies suggested that ACE2 expression may vary regarding age There was an important heterogeneity between studies in the methods used to detect ACE2 and TMPRSS2 leading to a potential identification bias The SARSCoV2 receptors ACE2 and TMPRSS2 are both expressed in many head and neck tissues enabling the viral entry into the host anismKeywords ACE2 a0· TMPRSS2 a0· SARSCoV2 a0· COVID a0· Coronavirus a0· Head NeckIntroductionThe renin angiotensin aldosterone system is one of the most important systems regulating the homeostasis of cardiovascular and pulmonary function this involves many molecules including angiotensin converting enzyme2 ACE2 [] ACE2 is also known to be the functional receptor of some coronavirus species as initially discovered in during the severe acute respiratory syndrome coronavirus SARSCoV epidemic [] The current pandemic of coronavirus disease Jerome R Lechien and Thomas Radulesco have contributed equally to the paper and are joint as cofirst authorsJustin Michel and Sven Saussez equally contributed to the paper and are cosenior authorsjeromelechienumonsacbe Jerome R Lechien Extended author information available on the last page of the COVID19 has brought to light the importance of ACE2 regarding development of infection viral spread and the development of the clinical COVID19 [] At the same time another SARSCoV2 receptor has been identified the transmembrane protease serine2 TMPRSS2 []ACE2 and TMPRSS2 tissue expressions are particularly important to identify viral entry pathways and to better understand the anrelated clinical presentation of the disease [ ] Further evaluation of ACE2 and TMPRSS2 expression in ear nose and throat mucosa is warranted to shed light on the pathophysiology of disease in the head and neck [“]The aim of this systematic review is to summarize the current data about the expression of ACE2 and TMPRSS2 in head neck tissueVol01234567891 0c MethodsThe review was conducted regarding the Preferred Reporting Items for a Systematic Review and Metaanalysis PRISMA checklist [] A modified population intervention comparison outcome timing and setting PICOTS framework was used to structure the review process [] For this review the PICOTS structure was kept but adapted to experimentalbasic research studies on human and animal tissuesStudiesAnimal and human experimental published studies in Englishlanguage peerreviewed journals were considered Preprint studies were also considered in light of the current pandemic and the significant wealth of knowledge derived over the last few months All studies where investigators assessed ACE2 or TMPRSS2 expressions in head neck tissues through immunochemistry IHC in a0situ hybridization Western Blot RNA sequencing RNAseq or reverse transcription polymerase chain reaction RTPCR were evaluatedParticipants and a0Inclusion CriteriaThe papers had to include either human or animal subjects The authors extracted substantial information about the sample characteristics including species involved and ACE2 and TMPRSS2 identification methodOutcomesThe primary outcome studied was tissue expression of ACE2 and TMPRSS2 The anatomical location the types of cells that expressed both receptors were recorded Particular attention was paid to the method used to detect ACE2TMPRSS2 in tissues Additional useful information such as viral impact on the functioning of the tissuecell that expressed the receptor or interindividual differences were also collectedIntervention and a0ComparisonBecause the aim of the study was to investigate the tissue ACE2 and TMPRSS2 expression we did not consider potential intervention on patient or animal modelsHead and Neck PathologyTiming and a0SettingWe included the studies where the receptor analysis was made on normal subjects andor infected patientsSearch StrategyThe PubMedMEDLINE Google Scholar Medrxiv Scopus and Cochrane search was conducted by independent authors JRL TR CCH GD CMCE to identify papers published between January and April The authors screened publications with database s and available full texts referring to the condition The following keywords were used for the search strategy ˜ACE2² ˜TMPRSS2² ˜COVID19² ˜COVID™ ˜SARS™ ˜coronav™ ˜coronavirus™ ˜salivary™ ˜gland™ ˜Receptor™ ˜Head™ ˜Neck™ ˜Nasal™ ˜ear nose throat ENT™ ˜Tissue™ and ˜Cell™ The authors investigated papers for number of samplesindividuals study type design inclusion criteria and ACE2TMPRSS2 detection outcomesResultsThe electronic search identified papers of which met our inclusion criteria Table a0 [“] A total of studies investigated the expression of ACE2 and TMPRSS2 in human head and neck tissues while five papers focused on mouse and two on monkey samples respectively Table a0 One study focused on ACE2 genetic analysis without reporting sitespecific anatomical expression [] The flow chart of the study process is available in Fig a0 Five studies were preprint [ “ ]Tissue Expression in a0HumanACE2 ExpressionACE2 was assessed in studies [“] The expression of ACE2 was found in all mucosa of the respiratory upper tract including trachea [ ] sinus and nasal cavities [ ] Among the respiratory mucosa ACE2 was expressed in several types of cells including epithelial goblet and endothelial cells [ ] One study reported that ACE2 was expressed on ciliated epithelial cells and not on nonciliated goblet cells [] Butowt et a0al compared the intensity of expression of ACE2 in the upper and lower respiratory tract [] They found that nasal epithelial cells had lower levels of ACE2 expression compared with epithelial cells of the lower respiratory tract [] Among the nasal region two studies investigated the ACE2 expression in the 0cHead and Neck Pathology Table Studies reporting ACE2 or TMPRSS2 head and neck expressionAuthorsDesignVaarala Mixed [] StudyHamming HumanSamples MethodsHuman TMPRSS2MouseHuman ACE2RNAseqACE2 TMPRSS2 expressionSalivary glandsTMPRSS2 humanOral Nasal Nasopharyngeal Epithelium endotheliumACE2 human all mucosa [] StudyIHCHumanJia [] StudyHuman ACE2Tracheal Epithelium endotheliumIHC biotinylation ACE2 humanLiuExperimenal Monkey ACE2 [] StudyIHCNaso Oro Hypopharyngeal Tracheal EpitheliumACE2 MonkeyVirion in SalivaBilinska Animal [] StudyBrannMixed [] StudyButowtMixed [] StudyMouse ACE2 TMPRSS2 Olfactory EpitheliumRNAseq RTPCRACE2 sustentacular cellsIn situ hybridization TMPRSS2 sustentacular cellsWB IHCMouse ACE2 TMPRSS2 Olfactory EpitheliumHuman RNAseqACE2 TMPRSS2 Mouse nonneuronal cellsACE2 TMPRSS2 Human glial and neuronal stem cellsMouse ACE2 TMPRSS2 Olfactory EpitheliumHuman RNAseqACE2 Mouse Human non neuronal cellsCaoHumanHuman ACE2 gene [] StudyGenetic AnalyzisChenHuman [] StudyHikmetHuman [] StudyHuman ACE2RNAseqHuman ACE2IHCLeeHumanHuman ACE2TMPRSS2 Mouse Human neuronal nonneuronal cellsRespiratory EpitheliumACE2 TMPRSS2 Human Lower Airway NasalNo localization providedSalivary glandsACE2 humanNasopharyngeal EpitheliumACE2 human no expression in nasopharynxTracheal Nasal Sinusal EpitheliumFindings TMPRSS2 is expressed in human salivary gland tissues ACE2 was found in endothelial arteries veins and epithelial cells of nasal rhinopharyngeal and oral mucosa Precisely the epithelium expression concerned the basal layer cells ACE2 was more expressed on the apical than the basolateral surface of polarized airway epithelia ACE2 is expressed in salivary gland ducts of the pharyngeal glands ACE2 was expressed in epithelial cells lamina propria respiratory tract Virus was found in saliva of infected monkeys ACE2 TMPRSS2 are expressed in sustentacular cells of the olfactory epithelium but notmuch less in most olfactory receptor neurons Expression of the entry proteins increases in animals of old age In human ACE2 TMPRSS2 were not identified in purified olfactory neurons ACE2 was identified in glial cells olfactory stem cells Nasal epithelial cells have lower levels of ACE2 TMPRSS2 compared with epithelial cells of the lower respiratory tract ACE2 has nonneuronal expression in olfactory epithelium The expression of ACE2 TMPRSS2 mouse were increased in elderly mouse unique expression quantitative trait loci variants were found for ACE2 The genotypes of ACE2 gene polymorphism may be characterized by higher expression levels of ACE2 in East Asian population There would be different susceptibility or response to SARSCoV2 in different populations ACE2 is expressed in human granular cells of salivary glands There was no ACE2 expression in nasopharyngeal cells ACE2 is expressed in ciliated epithelial cells cilia anelle 0c Table continuedAuthorsDesign [] StudyHumanLiHikmetHuman [] Study [] StudySungnak Human [] StudySamples MethodsIHCACE2 TMPRSS2 expressionACE2 humanHuman ACE2Human ACE2IHCRNAseqThyroidNasopharyngeal EpitheliumACE2 human no expression in nasopharynxACE2 humanHuman ACE2 TMPRSS2 Airway Nasal epitheliumRNAseqACE2 humanTMPRSS2 human subset of ACE2 cellsXuHumanHuman ACE2 TMPRSS2 Oral Epithelium [] StudyRNAseqHumanXu [] StudyWuHuman [] StudyHuman ACE2RNAseqHuman ACE2RNAseqACE2 humanACE2 humanOral T cells B cells fibroblastsACE2 humanMinor salivary glandsACE2 humanNasal Oral EpitheliumHead and Neck PathologyFindings2There was no ACE2 expression in the nonciliated goblet cells ACE2 expression is influenced by patient demographics clinical characteristics comorbidities or medication use The use of ACE inhibitor drugs did not increase ACE2 protein expression ACE2 is expressed by thyroid cells There was no ACE2 expression in nasopharyngeal cells ACE2 was expressed in airway epithelial cells ACE2 is more expressed in nasal epithelial cells compared with other respiratory cells goblet ciliated cells TMPRSS2 is only expressed in a subset of ACE2 cells ACE2 is expressed in the oral cavity epithelial cells ACE2 expression was higher in tongue than buccal and gingival tissues ACE2 is expressed in minor salivary glands ACE is expressed in nasal epithelial cells The was a higher virus concentration in the nasalswab comparing with throatswab which is attributed to ACE2expression in nasal epithelial cells ACE2 TMPRSS2 are coexpressed in nasal goblet secretory cellsMixedZiegler [] StudyACE2 Angiotensin Converting Enzyme2 IHC Immunohistochemistry RTPCR reverse transcription polymerase chain reaction SARSCoV2 severe acute respiratory syndrome a0coronavirus2 TMPRSS2 transmembrane protease serine2 WB Western BlottingHuman ACE2 TMPRSS2 Sinusal Nasal goblet epithelial cellsMonkey RNAseqACE2 TMPRSS2 Humanmucosa of the olfactory region including olfactory bulb [ ] The ACE2 receptor was identified in sustentacularnonneuronal cells of the olfactory tissues Moreover ACE2 was found in a low proportion of neuronal stem cells in the olfactory bulb [ ] The expression of ACE2 in olfactory neurons nonstem cells remains uncertain because Butowt et a0al and Brann et a0al observed that ACE2 has only nonneuronal expression pattern in olfactory epithelium [ ]Five studies investigated ACE2 expression in oral and pharyngeal regions including oral and hypo oro and nasopharyngeal spaces [ “] The study that explored ACE2 expression in human nasopharynx [] did not exhibit significant ACE2 immunostaining in nasopharyngeal cells [] ACE2 receptor was identified in oral endothelial [] epithelial [ ] and salivary [] cells Xu et a0al found that ACE2 was also expressed in T and B cells as well as fibroblasts of the oral cavity [] Moreover ACE2 was expressed in major salivary gland tissues [] and thyroid tissue [] In many publications authors reported the type of cells goblet versus epithelial versus stem cells that expressed ACE2 or TMPRSS2 Table a0 Interestingly Xu et a0al almost as much ACE2 expression in the thyroid as in the lungs []The genetic analysis of Cao et a0al reported that there are unique expression quantitative trait loci variants in the East Asian population supporting a gene polymorphism and tissuerelated differences between individuals [] 0cHead and Neck Pathology Fig Flow chart ACE2 Angiotensin Converting Enzyme2 TMPRSS2 transmembrane protease serine2TMPRSS2 ExpressionTMPRSS2 expression was investigated in studies [ ] Similarly to ACE2 TMPRSS2 was identified in nasal [ ] and respiratory mucosa cells [] including both epithelial and goblet cells with higher expression in lower airway compared with upper airway [] Moreover TMPRSS2 receptor was identified in sustentacular and neuronal olfactory cells [ ] but not in olfactory neurons [] TMPRSS2 was also identified in salivary major gland tissue []ACE2 TMPRSS2 Tissue Expression in a0Mouse and a0MonkeySix studies used animal models to assess ACE2 or TMPRSS2 expressions in head and neck tissues [ “ ] The mouse studies of Butowt et a0al and Bilinska et a0al revealed that elderly mice had higher expression of both ACE2 and TMPRSS2 in nasal mucosa compared with younger mice [ ] In olfactory tissue ACE2 was identified in sustentacularnonneuronal and neural stem cells of mice [“] Liu et a0al analyzed ACE2 expression in monkeys [] 0c Head and Neck PathologyTable Summary of Cell Expression of ACE2 and TMPRSS2AuthorsBilinska []Brann []SamplesMouseHuman MouseTissueOlfactoryOlfactoryButowt []Human MouseNasalOlfactoryChen []Hamming []Hikmet []Jia []Lee []Li []Liu []Sungnak []Vaarala []Xu []Xu []Wu []Ziegler []HumanHumanHumanHumanHumanHumanMonkeyHumanMajor Salivary GlandOral Nasal NasopharyngealNasopharyngealTracheal Nasal SinusalThyroidPharyngealTrachealTracheal NasalHuman MouseHumanMajor Salivary GlandOralHumanHumanHuman Mouse MonkeyMinor Salivary GlandNasalOralNasal SinusalCell typesSustentatorialSustentatorialNeuronalStem NeuronalEpithelialSustentatorialNeuronalGranularBasal layerEndothelialEpithelialApical EpithelialEndothelialApical EpithelialGobletUnspecifiedMinor salivary ductalBasal layerGobletApical EpithelialUnspecifiedApical EpithelialFibroblastT and BcellsUnspecifiedUnspecifiedBasal layerGobletACE2ˆ’ˆ’ˆ’NATMPRSS2ˆ’ˆ’NANANANANANANANANANANANANANANANANAACE2 Angiotensin Converting Enzyme2 NA not available TMPRSS2 transmembrane protease serine2reporting a higher ACE2 expression in tracheal naso oro and hypopharyngeal tissues as well as in the salivary ducts of the pharyngeal gland and consequently in saliva In this study the cell expression was mainly localized in the lamina propria In the same vein Vaarala et a0al reported TMPRSS2 expression in mouse salivary tissues []Cell Detection MethodsThe following methods have been used for detecting ACE2 and TMPRSS2 in cells of human and animal tissue RNAseq N IHC N RTPCR N in a0situ hybridization ISH N and WB N Different detection approaches were used in studies [ ] One study reported specific genetic analysis [] There were significant differences between studies regarding methods used While Ziegler et a0al and Sungnak et a0al detected ACE2 by RNAseq in goblet cells Lee et a0al did not find any immunohistochemical labeling [ ] However the results reported in sustentacular cells agree in the same direction whatever the technique used whether by RNAseq or by ISH and immunocytochemistry [“] The discrepancies are rather observed between studies having performed immunohistochemistry Indeed using two different antibodies Hikmet did not find ACE2 expression in nasopharynx epithelium whereas others demonstrated the staining of the apical surface of epithelia and ciliated epithelial cells [ ] Interestingly all the studies which carried out RNAseq found an expression of ACE2 or TMPRSS2 at the epithelial level which implies that the technique used could generate biases between the studies [ “ “] 0cHead and Neck Pathology DiscussionThe presentation of COVID19 infection may be in several clinical forms ranging from anosmia in isolation to severe multiple an failure and death The mechanisms underlying the COVID19 polymorphism are still unknown To infect tissues SARSCoV2 needs to entry into the cells which is allowed through ACE2 and TMPRSS2 receptors [] The identification of virus receptor expression in the tissues makes particularly sense to better understand the clinical expression of the disease This systematic review sheds light on many pointsFirst ACE2 and TMPRSS2 receptors are expressed in epithelial and nonepithelial cells throughout the head and neck The head and neck expression may support the otolaryngological clinical picture of the disease which was recently found in European and North American COVID patients [ ] By entering the body via the epithelial cells of the upper aerodigestive tract mucosa the SARSCoV2 virus leads to an inflammatory reaction and the development of otolaryngological symptoms Nasal entrance of the virus through high ACE2 expression was supported in the study of Wu et a0al who found a higher virus concentration in nasal swabs compared with throat swabs []The olfactory cleft is a nasal region that has drawn the attention of many researchers over the past few weeks Indeed recent data supported that more than of COVID19 patients developed subjective olfactory dysfunction especially when patients suffered from mildtomoderate forms of the disease [ ] Because ACE2 and TMPRSS2 are both expressed in the nasal mucosa of the olfactory cleft entrance into the olfactory bulb seems plausible Once in the bulb according to some human studies [ ] the virus could infect cells that express ACE2 or TMPRSS2 namely glial and neuronal stem cells Fig a0Integrating the molecular clinical and radiological characteristics of SARSCOV2 olfactory loss may shed light about its pathophysiological process Taking into account that the loss is often temporary SARSCOV2 may primarily infect the sustentacular cells supporting the olfactory sensory neurons This infection may cause rapid disruption of the olfactory epithelium structure and function with a possible inflammatory response inducing sudden onset smell loss This inflammation is observed in a minority of patient with congested olfactory cleft who underwent CT scan [ “]ACE2 and TMPRSS2 are also found in horizontal basal olfactory stem cells located in the basal layer They are less exposed to the external environment thus less likely to be infected in first line the loss would have been more Fig Epithelium of Olfactory Cleft The figure summarizes the olfactory cleft epithelium 0c Head and Neck Pathologyprogressive However once infected they might slow down recovery time because horizontal basal cells give rise to many cell type in the olfactory epithelium They may also contribute to virus spread to the olfactory bulb vascular pericytes Magnetic resonance imaging MRI studies of the olfactory bulb [ ] in which ACE2 are only expressed in vascular pericytes but not in neurons may show inflammatory signs suggesting that the infection process can extend more centrally and promote inflammatory response [] Inflammatory causes are often quickly reversible for example after a oneweek trial of high dose of corticosteroids or simply days after the resolution of the viral infection suggesting that the olfactory neurons and bulbs are still somewhat intact This seems to be the case for a majority of patients In contrast with more sustained destruction of neuronal olfactory structures the recovery time is much longer and may take “ a0years given the slow neuroregeneration process [ ]In this systematic review we found that three studies reported high ACE2 expression in major or minor salivary gland human tissues [ ] These data corroborate the literature findings that reported a salivary pattern of SARSCoV2 and related parotitis [ ] Moreover the virus spread into the salivary gland tissues allows us a better understanding the mechanisms underlying salivary transmission Interestingly in Liu et a0al observed that monkeys infected by SARSCoV had a salivary viral spread which was associated with a salivary virion excretion [] These data support the need to conduct future studies investigating the presence of SARSCoV2 in the saliva of infected human and to corroborate the saliva findings with the ACE2 salivary gland expressionThe head and neck expression of ACE2 and TMPRSS2 and the related otolaryngological symptom pattern seems obvious but could vary across individuals and populations In support of this Lee et a0al observed that ACE2 expression is influenced by patient demographics clinical characteristics comorbidities and medication [] As reported in the genetic analysis of Cao et a0al there would be different susceptibilities or responses to SARSCoV2 in different populations [] The polymorphism of ACE2 and TMPRSS2 expression could explain the clinical differences between individuals Indeed many physicians reported in clinical and epidemiological studies different clinical presentation of the disease [ ] which could be associated with virus mutations [ ] The virus mutations and the related impact on receptor binding and infectivity is another point that has to be considered in future studies Otherwise according to the Bgee database https bgee expression of ACE2 and TMPRSS2 evaluated in murine models may increase with age These findings have to be confirmed in humans but could explain more severe clinical pictures in the elderlyThe present study has several limitations First the heterogeneity between studies about the detection method may lead to detection bias as some approaches are more sensitive than others Some studies interrogate gene expression at mRNA level and others at protein levels both types of analysis having their advantages and limitations Compared to transcriptomic analyzes immunohistochemistry brings additional important spatial information in tissue samples but recently Sorokin et a0al demonstrated high and statistically significant correlations between the RNA sequencing and immunohistochemical measurements [] Interestingly they highlighted the complementarity of both techniques for measuring cancer biomarkers in FFPE samples However differences observed across IHC studies suggest the involvement of many parameters The antibody specificity is a big challenge to ensure reproducibility of antibodybased studies and given the high homology between ACE1 and ACE2 cautions must be taken regarding antibody selection Besides a report from the International Working Group for Antibody Validation IWGAV proposed five scientific approaches to validate antibody specificity [] then such strategies must be considered in future investigations to confirm the published observations In addition it seems essential to enlarge and diversify patient cohorts and to combine transcriptomic and proteomic strategies as well as colocalize different markers of SARSCoV2 such as ACE2 and TMPRSS2 to provide an accurate representation of ACE2 expression through all head and neck areas of the whole populationSecond the majority of studies that were conducted during the SARSCoV2 pandemic did not consider many demographic and clinical factors such as the age of patients from who the tissues were extracted or the use of ACE inhibitor medications among others Third some otolaryngological regions remain uninvestigated such as the vocal folds The investigation of these remaining regions may shed further light on some recently reported unusual clinical phenomena such as severe dysphonia []ConclusionACE2 and TMPRSS2 are both expressed in head and neck tissues which may explain the otolaryngological clinical pattern of the disease and the entry of SARSCoV2 into the host anism Future studies considering demographical and clinical characteristics of patients from who the tissues are extracted are needed to better understand the cell entry mechanisms of SARSCoV2Author contributions JR TR SS JM design acquisition of data data analysis interpretation drafting final approval and accountability 0cHead and Neck Pathology for the work final approval of the version to be published agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved CCH CMCE MRB IG design data analysis interpretation revising the manuscript for important intellectual content final approval of the version to be published final approval and accountability for the work final approval of the version to be published agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved LS SH GC GD JH LV GR design acquisition of data data analysis interpretation drafting some parts of the manuscript final approval of the version to be published final approval and accountability for the work final approval of the version to be published agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolvedCompliance with Ethical Standards Conflict of interest The authors declare that they have no conflict of interestReferences Crackower MA Sarao R Oudit GY et a0al Angiotensinconverting enzyme is an essential regulator of heart function Nature “ Li W Moore MJ Vasilieva N Sui J Wong SK Berne MA Somasundaran M Sullivan JL Luzuriaga K Greenough TC Choe H Farzan M Angiotensinconverting enzyme is a functional receptor for the SARS coronavirus Nature “ Wang Z Xu X scRNAseq profiling of human testes reveals the presence of the ACE2 receptor a target for SARSCoV2 infection in spermatogonia leydig and sertoli cells Cells https doi103390cells Shang J Wan Y Luo C Ye G Geng Q Auerbach A Li F Cell entry mechanisms of SARSCoV2 Proc Natl Acad Sci USA https doi101073pnas20031 Vaira LA Hopkins C Salzano G et a0al Olfactory and gustatory function impairment in COVID19 patients An Italian objective multicenterstudy Head Neck https doi101002hed26269 Lechien JR ChiesaEstomba CM Place S Van Laethem Y Cabaraux P Mat Q Huet K Plzak J Horoi M Hans S Barillari MR Cammaroto G Fakhry N Martiny D Ayad T Jouffe L Hopkins C Saussez S COVID19 task force of YOIFOS Clinical and epidemiological characteristics of European patients with mildtomoderate Coronavirus Disease J Intern Med https doi101111joim13089 Lechien JR ChiesaEstomba CM De Siati DR et a0al Olfactory and gustatory dysfunctions as a clinical presentation of mildtomoderate forms of the coronavirus disease COVID19 a multicenter European study Eur Arch Otorhinolaryngol https doi101007s0040 McInnes MDF Moher D Thombs BD et a0al Preferred reporting items for a systematic review and metaanalysis of diagnostic test accuracy studies the PRISMADTA statement JAMA “ https doi101001jama201719163 Thompson M Tiwari A Fu R Moe E Buckley DI A Framework To Facilitate the Use of Systematic Reviews and MetaAnalyses in the Design of Primary Research Studies Rockville Agency for Healthcare Research and Quality US Vaarala MH Porvari KS Kellokumpu S Kyll¶nen AP Vihko PT Expression of transmembrane serine protease TMPRSS2 in mouse and human tissues J Pathol “ https doi10100210969896200099999999 Hamming I Timens W Bulthuis ML Lely AT Navis G van Goor H Tissue distribution of ACE2 protein the functional receptor for SARS coronavirus A first step in understanding SARS pathogenesis J Pathol “ https doi101002path1570 Jia HP Look DC Shi L et a0al ACE2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia J Virol “ https doi101128JVI792314614 Liu L Wei Q Alvarez X et a0al Epithelial cells lining salivary gland ducts are early target cells of severe acute respiratory syndrome coronavirus infection in the upper respiratory tracts of rhesus macaques J Virol “ https doi101128JVI02292 Bilinska K Jakubowska P Von Bartheld CS Butowt R Expression of the SARSCoV2 entry proteins ACE2 and TMPRSS2 in Cells of the olfactory epithelium identification of cell types and trends with age ACS Chem Neurosci https doi101021acsch emneu ro0c002 Brann DH Tsukahara T Weinreb C et a0al Nonneuronal expression of SARSCoV2 entry genes in the olfactory system suggests mechanisms underlying COVID19associated anosmia Preprint https doi1011012020032500908 Butowt R Bilinska K SARSCoV2 olfaction brain infection and the urgent need for clinical samples allowing earlier virus detection ACS Chem Neurosci “ https doi101021acsch emneu ro0c001 Cao Y Li L Feng Z Wan S Huang P Sun X Wen F Huang X Ning G Wang W Comparative genetic analysis of the novel Coronavirus 2019nCoVSARSCoV2 receptor ACE2 in different populations Cell Discov https doi101038s4142 Chen R Wang K Yu J et a0al The spatial and celltype distribution of SARSCoV2 receptor ACE2 in human and mouse brain Preprint https doi1011012020040703065 Hikmet F Mar L Uhln M Lindskog C The protein expression profile of ACE2 in human tissues bioRxiv https doi1011012020033101604 Lee IT Nakayama T Wu CT et a0al Robust ACE2 protein expression localizes to the motile cilia of the respiratory tract epithelia and is not increased by ACE inhibitors or angiotensin receptor blockers Preprint https doi1011012020050820092 Li MY Li L Zhang Y Wang XS Expression of the SARSCoV2 cell receptor gene ACE2 in a wide variety of human tissues Infect Dis Poverty https doi101186s4024 x Sungnak W Huang N Bcavin C et a0al SARSCoV2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes Nat Med “ https doi101038s4159 Xu H Zhong L Deng J Peng J Dan H Zeng X Li T Chen Q High expression of ACE2 receptor of 2019nCoV on the epithelial cells of oral mucosa Int J Oral Sci https doi101038s4136 80200074x Xu J Li Y Gan F Du Y Yao Y Salivary glands potential reservoirs for COVID19 asymptomatic infection J Dent Res https doi10117700220 Wu C Zheng S Chen Y Zheng M Singlecell RNA expression profiling of ACE2 the putative receptor of Wuhan 2019nCoV in the nasal tissue https doi1011012020021120022 0c Head and Neck Pathology Ziegler CGK Allon SJ Nyquist SK et a0al SARSCoV2 receptor ACE2 is an interferonstimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues Cell 2020S0092““ https doi101016jcell202004035 Kaye R Chang CWD Kazahaya K Brereton J Denneny JC III COVID19 anosmia reporting tool initial findings Otolaryngol Head Neck Surg https doi10117701945 Lechien JR ChiesaEstomba CM Hans S Barillari MR Jouffe L Saussez S Loss of smell and taste in European patients with mild to moderate COVID19 Ann Intern Med https doi107326M202428 Vaira LA Salzano G Deiana G De Riu G Anosmia and ageusia common findings in COVID19 patients Laryngoscope https doi101002lary28692 Spinato G Fabbris C Polesel J Cazzador D Borsetto D

Thyroid_Cancer

Coronavirus disease is caused by severe acute respiratory syndrome coronavirus SARSCoV2 and represents the causative agent of a potentially fatal disease The spread of the infection and the severe clinical disease have led to the widespread adoption of social distancing measures Special attention and efforts to protect or reduce transmission have been applied at all social levels including health care operators Hence this reports focuses on the description of a new protocol for the safe management of cytological samples processed by liquidbased cytology LBC with an evaluation of the changes in terms of morphology and immunoreactivity METHODS From March to April cytological cases suspicious for SARSCoV2 were processed with a new virusinactivating method suggested by Hologic Inc for all LBC specimens RESULTS The samples showed an increased amount of fibrin in the background A slight decrease in cellular size was also observed in comparison with the standard method of preparation Nonetheless the nuclear details of the neoplastic cells were well identified and the immunoreactivity of the majority of those cells was maintained The cell blocks did not show significant differences in morphology immunoreactivity or nucleic acid stability S Despite some minor changes in the morphology of the cells the results of this study highlight that the adoption of the new protocol for the biosafety of LBCprocessed samples in pathology laboratories is important for minimizing the risk for personnel trainees and cytopathologists without impairing the diagnostic efficacy of the technique Cancer Cytopathol American Cancer Society KEY WORDS coronavirus disease COVID19 cytology diagnosis liquidbased cytologyINTRODUCTIONCoronaviruses are enveloped viruses with a positivesense singlestranded RNA genome They infect birds and mammals and cause a variety of lethal diseases and they can also infect humans and cause disease to varying degrees ranging from upper respiratory tract infections resembling the common cold to lower respiratory tract infections such as bronchitis pneumonia and even severe acute respiratory syndrome Hubei Province China In late December several health facilities reported clusters of patients with pneumonia of unknown cause that were epidemiologically linked to a seafood and wet animal wholesale market in Wuhan Hubei Province China7The causative agent of this unidentified pneumonia has been confirmed to be a novel coronavirus by sequencing and etiological investigations by several independent laboratories in China8 The new coronavirus Corresponding Author Patrizia Straccia BD PhD Unit of Anatomic Pathology Agostino Gemelli University Polyclinic Foundation IRCCS Largo Francesco Vito Rome Italy stracciapatrizialiberoit Unit of Anatomic Pathology Agostino Gemelli University Polyclinic Foundation IRCCS Rome Italy Anatomic Pathology Section Department of Life Sciences and Public Health Catholic University of the Sacred Heart Rome ItalyWe thank Mrs Elena Visca for her invaluable technical support and expertiseReceived May Revised June Accepted July Published online Month in Wiley Online Library wileyonlinelibrarycom 101002cncy22341 wileyonlinelibrarycomCancer Cytopathology Month 0cOriginal originally called novel coronavirus 2019nCoV and officially renamed severe acute respiratory syndrome coronavirus SARSCoV2 by the International Committee on Taxonomy of Viruses and the disease it causes namely coronavirus disease COVID19 have quickly become of tremendous concern worldwide There have been significant outbreaks in many regions of China as well as global expansion to Asia Europe North America South America Africa and Oceania Persontoperson transmission occurs mostly through contact and respiratory transmission droplets but also by the fecaloral route9 For this reason there is an international push to contain the virus and prevent its spread The response to the COVID19 pandemic can be regarded at all social levels eg social community hospital laboratory and individual levelsBecause it is possible that infected samples may be submitted to pathology and cytopathology laboratories for diagnostic purposes it is important for us to take adequate precautions to protect ourselves and our staff The World Health anization recommends that all specimens collected for laboratory investigations be regarded as potentially infectious Health care workers who collect handle or transport any clinical specimens should adhere rigorously to the standard precautionary measures and biosafety practicesThe role of the cytology laboratory for a patient with known SARSCoV2 is limited although it may involve the examination of samples from the oropharyngeal and respiratory tract which is likely to host a significant amount of viruses Because the laboratory personnel might be exposed to contamination during the preparation and handling of fresh specimens from such patients a new procedure for the sterilization of material to be processed by liquidbased cytology LBC has been applied10 This study is focused on a description of this new procedure and on an evaluation of the changes in terms of morphology and cell immunoreactivity that this technique produces in cellular materialMATERIALS AND METHODSFrom March to April cytological cases considered to be possibly infected by SARSCoV2 were sent to the Cytopathology Laboratory of the Agostino Gemelli University Hospital of Rome IRCCSThe cytological material was processed in a dedicated highlevel biosafe hood by specialized technicians wearing adequate personal protective equipment eg mask face or eyes protection disposable medical gloves a disposable waterrepellent gown or coveralls with sleeves fully covering the forearms and shoe covers or dedicated shoes To each vial is added an amount of alcohol ethanol for at least the same amount of its volume to the material this is considered the safest way of handling cytological samples infected by SARSCoV2 The following is the modified method adopted at the study institution for all LBC specimens processed under the protocol suggested by Hologic Inc Marlborough Massachusetts Collect the sample in a ethyl alcohol solution Centrifuge it at 600g for minutes or at 1200g for Pour off the supernatant fluid and resuspend the cell Add mL of CytoLyt solution to reduce biological minutespelletcontamination Centrifuge at 600g for minutes Pour off the supernatant fluid Resuspend the cell pellet Evaluate the cell pellet if it is necessary repeat from step Add an appropriate amount of the specimen depending on the size of the cell pellet to the PreservCyt solution vial Allow it to stand in PreservCyt for minutes Run on a ThinPrep processor or a ThinPrep processorImmunohistochemistry was performed on either LBC slides or formalinfixed paraffinembedded cell blocks obtained from stored ThinPrep material All molecular testing was performed on cell block material only All patients consented to their procedure We received institutional Catholic University of the Sacred Heart ethical approval for this studyMolecular AnalysisA mutational analysis of epidermal growth factor receptor EGFR was performed with the Therascreen EGFR Rotene Q RGQ polymerase chain reaction kit Qiagen in the RGQ 5plex high resolution melt analyzer instrument according to the manufacturer™s protocol sensitivity The mutation nomenclature used in this work follows the guidelines indicated by the Human Genome Variation Society11Cancer Cytopathology Month 0cNew Protocol for Cytological SamplesStraccia et alTABLE Summary of Cytological Samples and Distribution of Molecular and Immunohistochemical AnalysesCytological SampleNumber of CasesMolecular Analysis NumberImmunohistochemical Analysis NumberThyroidUrineliquor in cerebrospinal fluidLungmediastinal FNABronchoalveolar washingsPleural effusionsPeritoneal effusionsPericardial effusionsAbbreviation FNA fineneedle aspirationTABLE Comparison of the Morphological Features of the New Method and the Standard MethodNew MethodSlightly smallerPresentNo changeFibrin mucusDecreaseStandard MethodNormalPresentNormalClearNormalFeatureCellular sizeNucleoliCytoplasmBackgroundCellularityRESULTSThe material processed according to the modified method consisted of specimens In all thyroid specimens urine specimens cerebrospinal fluid specimens lung aspiration specimens bronchoalveolar washings pleural effusions peritoneal effusions and pericardial effusions were evaluated Table The series included men and women and the median patient age was years range years The morphological features of the modified and standard methods were compared Table All cytological samples particularly the fineneedle biopsies showed an increased amount of fibrin in the background A decrease in cellularity in comparison with the standard method of preparation was also noted Figs In all cytological samples we observed that the cells were smaller and more scattered in comparison with samples processed with the original technique Therefore the distinction between normal reactive and atypical cells was slightly more difficult in the samples treated with the modified preparation in comparison with the standard method Nonetheless the nuclear details of the neoplastic cells were generally well identified and the immunoreactivity of the majority of the cells was maintained The cell blocks taken from the material processed by LBC did not show significant differences in morphology immunoreactivity or nucleic acid stability in comparison with the standard LBC method Molecular test data were available for lung fineneedle aspiration specimens Approximately ng of genomic DNA was FIGURE Urothelial cells suspicious for high grade urothelial carcinoma SHGUC ThinPrep Papanicolaou ×FIGURE Cluster of neoplastic cells adenocarcinoma ThinPrep Papanicolaou ×from a lung isolated from the samples quantified and amplified by polymerase chain reaction Sanger sequencing A molecular analysis for EGFR exons and was ordered for all specimens Mutations were identified in of the cases We found EGFRmutated non“small cell lung Cancer Cytopathology Month 0cOriginal FIGURE Same cells identified in the cell block taken from the sample shown in Figure FIGURE Group of neoplastic cells from a nodal metastasis of oropharyngeal carcinoma showing strong positivity for pancytokeratins Avidin Biotin Complex ThinPrep ×suggests that the morphological details and quality of the cellular component can be preserved to achieve the diagnostic efficacy of the original method Our results show that this modified technique might increase the amount of fibrin in the background especially for fineneedle aspiration biopsies this is probably related to the sudden fixation of the hemorrhagic material in a large volume of ethanol When we analyzed the efficacy of the cytological diagnosis only minimal differences from the standard procedure mostly concerning some nuclear details were noted In fact the degrees of nuclear hyperchromasia and nuclear atypia are more difficult to assess only if the cells are less preserved or show artifactual changesDespite the difficulty in diagnosing atypical cells due to these overlapping cytomorphological features the results of our study show that the morphological details combined with the use of immunohistochemical techniques whose quality is not affected by the procedure can lead to a definitive diagnosis of malignancy in the large majority of casesAs recently reported in the literature1214 during the COVID19 pandemic the adoption of strict protocols and guidelines is important for establishing and maintaining a safe work environment Because the pandemic will probably last for months from this point the adoption of protocols for the biosafety of the laboratory and the staff will enable the processing of cytological material until the end of the danger and can be useful for future critical situations Although the modification of the original FIGURE Clusters of neoplastic follicular cells of a papillary thyroid carcinoma ThinPrep Papanicolaou ×cancers case with short inframe deletions of exon and case with a singlenucleotide substitution in exon characterized by the missense mutation pL858RDISCUSSIONGiven the extraordinarily fast spread of the disease and the pace of change in the information and procedures concerning how to deal with the various aspects of fighting this infection one can give only general suggestions for a cytology laboratory™s response12In this study we report a series of cytological samples processed with a modified protocol that ensures effective biosafety in handling the samples for the staff exposed to the viral load The use of this protocol Cancer Cytopathology Month 0cprotocol results in limited changes in the morphology of cells the benefits in terms of laboratory biosafety during this COVID19 pandemic have to be considered significantly more importantFUNDING SUPPORTNo specific funding was disclosedCONFLICT OF INTEREST DISCLOSURESThe authors made no disclosuresAUTHOR CONTRIBUTIONSPatrizia Straccia Conceptualization data curation formal analysis investigation methodology project administration resources software supervision validation visualization writing“original draft and writing“review and editing Esther Diana Rossi Data curation formal analysis investigation methodology resources validation visualization writing“original draft and writing“review and editing Maurizio Martini Data curation investigation and resources Antonino Mulè Data curation investigation and resources Federica Cianfrini Data curation investigation and resources Mariangela Curatolo Data curation investigation and resources Alessandra Cancellieri Data curation investigation and resources Chiara Brunelli Data curation investigation and resources Gian Franco Zannoni Data curation investigation and resources Guido Fadda Data curation formal analysis investigation methodology resources validation and writing“review and editingREFERENCES Owusu M Annan A Corman VM et al Human coronaviruses associated with upper respiratory tract infections in three rural areas of Ghana PLoS One 20149e99782New Protocol for Cytological SamplesStraccia et al Van der Hoek L Human coronaviruses what do they cause Antivir Ther pt B651 Cui J Li F Shi ZL Origin and evolution of pathogenic coronaviruses Nat Rev Microbiol Fehr AR Perlman S Coronaviruses an overview of their replication and pathogenesis Methods Mol Biol De Wit E van Doremalen N Falzarano D Munster VJ SARS and MERS recent insights into emerging coronaviruses Nat Rev Microbiol Woo PC Lau SK Huang Y Yuen KY Coronavirus diversity phylogeny and interspecies jumping Exp Biol Med Wuhan City Health Committee WCHC Wuhan Municipal Health and Health Commission™s briefing on the current pneumonia epidemic situation in our city Accessed January httpwjwwuhangovcnfront webshowD etail Zhou P Yang XL Wang XG et al A pneumonia outbreak associated with a new coronavirus of probable bat origin Nature Kang S Peng W Zhu Y et al Recent progress in understanding novel coronavirus SARSCoV2 associated with human respiratory disease detection mechanisms and treatment Int J Antimicrob Agents Rossi ED Fadda G Mule A Zannoni GF Rindi G Cytologic and histologic samples from patients infected by the novel coronavirus SARSCoV2 an Italian institutional experience focusing on biosafety procedures Cancer Cytopathol Taschner PEM den Dunnen JT Describing structural changes by extending HGVS sequence variation nomenclature Hum Mutat Pambuccian SE The COVID19 pandemic implications for the cytology laboratory J Am Soc Cytopathol Published online March 101016jjasc202003001 Barbareschi M Facchetti F Fraggetta F Sapino A What are the priorities of pathologists™ activities during COVID19 emergency Pathologica Published online April 1032074 951X1520 Phua J Weng L Ling L et al Intensive care management of coronavirus disease COVID19 challenges and recommendations Lancet Respir Med Published online April 101016S2213 Cancer Cytopathology Month 0c'

Thyroid_Cancer

"differentiation of human stromal mesenchymal stem cells hMSCs is a critical procedure for thedevelopment of osteoblast SNHG14 is a newly discovered lncRNA that has been barely studied Our preliminaryexperiments showed that SNHG14 may be dysregulated in the differentiation of hMSCs In this study we focusedon elucidating the relationships among SNGH14 miR2861 and osteoblastic differentiation of hMSCsMethod To investigate the roles of SNHG14 and miR2861 in hMSCs differentiation qRTPCR luciferase activity celltransfections the detections of ALP activity and Alizarin Red staining were performedResult We found that the expression of SNHG14 was enhanced while the expression of miR2861 was suppressedin serum and hMSCs from patients with osteoporosis SNHG14 could target miR2861 and shSNHG14 suppressedosteoblast differentiation of hMSC MiR2861 suppressed osteoblast differentiation of hMSC In addition the effectsof SNHG14 on osteoblast differentiation of hMSC were attenuated by miR2861Conclusion In our experimental data showed that the induction effects of SNHG14 on osteoblastdifferentiation of hMSC were attenuated by miR2861 SNHG14 could induce osteogenic differentiation of hMSCin vitro by targeting miR2861Keywords SNHG14 Osteogenic differentiation Human stromal mesenchymal stem cells miR2861BackgroundMesenchymal stem cells have the capabilities of selfrenewal and multilineage differentiation which are critical factors in the regeneration or repairment of bone tissues [ ] Human bone marrow mesenchymal stem cellhMSCs could fully differentiate to many cell types including osteoblasts chondrocytes and adipocytes [ ]The differentiation of hMSCs is thus critical for the development of osteoblast Studies have modulated the cell signaling pathways to control the differentiation of hMSCs to Correspondence vs4190163com Mingchang Du and Bo Wu contributed equally to this workThe Orthopedic Hospital of Shenyang No Dong bei da ma lu road Dadong district of Shenyang Shenyang City Liaoning Province PRChinaosteoblasts [ ] However the underlying mechanismsremain to be elusiveNoncoding RNAs have become the hotspot in severalresearch fields including long noncoding RNAs lncRNAs nt [] and microRNAs miRNAs nt [] Various lncRNAs have been reported to be involved in theosteoblastic differentiation of hMSCs For instance downregulation of lncRNAANCR promoted osteoblast differentiation by targeting EZH2 and regulating the expression ofRunx2 [] LncRNA H19 was reported to mediate BMP9induced osteogenic differentiation of MSCs through theNotch signaling [] LncRNA SNHG14 is a newly discovered lncRNA that has been barely demonstrated regardingits biological roles in human diseases It was reported thatSNHG14 promoted microglia activation by regulating miR The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cDu BMC Musculoskeletal Disorders Page of 1455pPLA2G4A in cerebral infarction [] Very limitedinformation has been revealed for its functions in hMSCsMiRNAs are another group of noncoding RNAs thathave been widely reported in human diseases ManymiRNAs exert essential roles in the differentiation ofhMSCs to osteoblast For example microRNA138 wasrevealed to regulate the osteogenic differentiation of human stromal mesenchymal stem cells in vivo [] Another study also reported thatthe microRNA320RUNX2 axis regulates adipocytic differentiation of human mesenchymal skeletal stem cells [] MoreovermiR2861 has been demonstrated to participate in theregulatory feedback loop during differentiation of mouseosteoblast []From our preliminary experiment we noticed thatSNHG14 may be dysregulated in hMSCs differentiationand miR2861 may share the common binding sequenceswith lncRNA SNHG14 In this study we aimed to clarifythe role of lncRNA SNHG14 in the formation of osteoblast from hMSCs focusing on elucidating the relationshipsand osteoblasticdifferentiation of hMSCsamong SNGH14 miR2861MethodsHuman samplesIn this study patients with hip fracture were recruited atThe Orthopedic Hospital of Shenyang Patient sampleswere divided into two groups patients in each groupincluding the treatment group osteoporosis patientswith a fracture and the control group nonosteoporosispatients with a fracture Serum and bone tissues werecollected during endoprosthesis and gamma nail wasimplanted into the proximal femur All patients enrolledin this study signed the informed consent This studywas approved by the Research Ethics Committee of TheOrthopedic Hospital of ShenyanghMSC preparationshMSCs were obtained from the bone marrow from femurs of patients during total hip or knee arthroplastydue to osteoarthritis or hip fracture The Ethics ReviewBoard of Orthopedic Hospital of Shenyang ShenyangCity Liaoning Province approved our study All hMSCswere obtained from postmenopausal women with anaverage age of years old age range “ years oldDensitometric examinations were performed using aLunar iDXA apparatus GE Lunar Madison WI USADiagnosis of oste ia or osteoporosis were made usingthe WHO Tscore criteria ˆ’ Tscore ˆ’ or Tscore ‰ ˆ’ respectively All the subjects in the osteoporosis group had vertebral fracturesCell separationThe RosetteSep Isolation kit STEMCELL Canada wasused to isolate hMSCs Cells were cultured at °C in awet environment with CO2 The culture mediumwas refreshed every week When cells reached confluence they were trypsinized and used immediatelyCell cultureWe cultured hMSCs in αminimum essential mediumαMEM containing fetal bovine serum FBS Invitrogen antibiotics and glutamax IGIBCO USAOsteogenesis was induced by fresh osteoblast inductionmedium OIM with ˆ’ M dexamethasone SigmaAldrich D4902 mM lascorbic acid SigmaAldrichA8960 mM glycerophosphateSigmaAldrichG9422 and mM 125vitaminD3 Alkaline phosphatase ALP was used to assess osteoblast phenotypeAlizarin Red staining was used to test matrixmineralization The medium was changed every dthroughout the experiments and cells were harvested atindicated time pointsqRTPCRTotal RNAs were extracted from serum bone tissues orhMSCs by Trizol Invitrogen USA The Reverse Transcription Kit Applied Bio USA was used to synthesizecDNAs The qRTPCR reactions were prepared usingSYBR Select Master Mix Applied Bio USA and PCRwas carried out on an ABI 7900fast thermocycler Applied Bio USA The relative expression was calculatedby 2ΔΔCT method The sequences of the primers arelisted belowSNHG14F ²GGGTGTTTACGTAGACCAGAACC3²SNHG14R ²CTTCCAAAAGCCTTCTGCCTTAG3²GAPDHF ²GAAGGTGAAGGTCGGAGTC3²GAPDHR ²GAAGATGGTGATGGGA TTTC3²OCF F ²GGCGCTACCTGTATCAATGG3²OCR ²GTGGTCAGCCAACTCGTCA3²Runx2F ²CGAATAACAGCACGCTATTAA3²Runx2R ²GTCGCCAAACAGATTCATCCA3²OSXF ²GCCAGAAGCTGTGAAACCTC3²OSXR ²GCTGCAAGCTCTCCATAACC3²ALPF ²TAGTGAAGAGACCCAGGCGCT3²ALPR ²ATAGGCCTCCTGAAAGCCGA3²miR2861F ²AACGAGACGACGACAGAC3²miR2861R ²GGGGCCUGGCGGUGGGCGG3²U6 ²GCCCCCGCCTCCGCCGCCGCC3² and ²ATATGGAACGCTTCACGAATT3²Cell transfectionsVectors with shSNHG14 miR2861 mimic and miR inhibitor all from Genepharma were transfectedto hMSCs via Lipofectamine Sigma USA At dposttransfection qRTPCR was conducted to detect 0cDu BMC Musculoskeletal Disorders Page of gene expressions The miR2861 mimic sequence was²GGGGCCUGGCGGCGGGCGG3² Mimic controlsequence was ²UUCUCCGAACGUGUCACGUTT3²The antagomir sequence was ²CCGCCCGCCGCCAGGCCCC3² The antagomir control sequence was ²CAGUACUUUUGUGUAGUACAA3²ALP activityhMSCs were collected and washed The cells were lysedby Triton X100 for min and centrifuged at g for min The supernatant was used for ALP analysis by ALP Assay Kit Abcam USAAlizarin red stainingThe osteoblasts were cultured by OIM for weeks andthen fixed by ethanol Next the cells were incubated by Alizarin Red solution for an hour at CThe results were recorded for analysisLuciferase assayPrimers were designed for the potential miR2861 binding sequence of AKT2 ²UTR SNHG14 ²UTR andthen cloned into the Sac IXba I sites of pmirGLODualLuciferase reporter vector The reconstructed plasmidswere confirmed by sequencing and named pmirGLOSNHG14WT and pmirGLOAKT2wt1 We also commercially synthesized mutant reporter constructs by mutating three nucleotides of each potential miR2861binding site and designated as pmirGLOSNHG14MUT pmirGLOAKT2mut1 Cells of confluencewere seeded in triplicate in 96well plates The wildtypeWT or mutant reporter constructs Mut were cotransfected into SiHa cells in the 96well plates with nmolL miR2861 or nmolL miRNC by using lipofectamine Invitrogen CA USA respectively Reporterposttransfection using the DualLuciferase Reporter AssayKit Promega following the manufacturer™s instructionsFirefly luciferase activity values were normalized fortransfection efficiency using the corresponding Renillaluciferase activity Three independent experiments wereperformedassays weregeneperformed hWestern blot analysisCell protein lysates were separated in or SDSPAGE gel h posttransfection followed by transferring to polyvinylidene difluoride membrane PVDFWestern blot analysis was performed with monoclonalantip53 Santa Cruz antiAKT2 Abcam primary antibodies AntiGAPDH antibody Santa Cruz was used asan internal control The membrane was washed and incubated with horseradish peroxidase HRPconjugatedsecondary antibody Cell Signaling Technology USAComplexes were visualized with SuperSignal West PicoChemiluminescent Substrate Pierce and the expressionlevels of these proteins were evaluated by Quantity OnesoftwareStatistical analysisData were shown as mean ± stand deviation SD Comparisons were performed by ttest between groups oroneway ANOVA among multiple groups P wasconsidered statistical significant differencesResultsSNHG14 was upregulated but miR2861 was downregulatedin serum and hMSCs from patients with osteoporosisThe expression of SNHG14 and miR2861 in serum andhMSCs of osteoporosis patients were analyzed Compared to participants without osteoporosis n theexpression levels of SNHG14 in serum and hMSCs ofn were greatly elevatedosteoporosis patiensFig 1a and c In addition the expression of miR2861was dramatically downregulated in hMSCs of osteoporosis group Fig 1d In addition a negative relationshipbetween the expression of SNHG14 and miR2861 in theserum of the osteoporosis group was observed Fig 1bfurtherinvestigated theSNHG14 was targeted by miR2861Werelationship betweenSNHG14 and miR2861 As shown in Fig 2a the common binding site between SNHG14 and miR2861 wasobserved After successfully transfecting miR2861 intohMSCs Fig 2b the cotransfection of SNHG14 ²UTR with miR2861 led to the suppression of luciferaseactivities compared with that of SNHG14 MUT Figue2C Moreover the transfection of shSNHG14 elevatedthe expression levels of miR2861 Fig 2d The expression levels of SNHG14 were also reduced in cells transfected with miR2861 Fig 2e Thees data indicated thatSNHG14 was targeted by miR2861reduced in cellsshSNHG14 suppressed osteoblast differentiation of hMSCTo investigate the effects of SNHG14 on hMSC osteoblast differentiation we induced hMSCs differentiationto osteoblasts after transfection with shSNHG14 orshNC As shown in Fig 3a the expression levels ofSNHG14 weretransfected withshSNHG14 The suppression of SNHG14 markedly lowered osteoblastic differentiation which was indicated bylower expression levels of the osteoblastspecific genesRUNX2 Osterix OSX ALP OC and decreased ALP activity Figs 3bd We observed matrix mineralizationin vitro by Alizarin red staining in shSNHG14“transfected hMSCs compared with cells transfected withshNC It was obvious that shSNHG14 could suppresshMSCs differentiation to osteoblasts weeks posttransfection 0cDu BMC Musculoskeletal Disorders Page of Fig SNHG14 was upregulated but miR2861 was downregulated in serum and hMSCs from patients with osteoporosis a Expressions ofSNHG14 in the serum of nonosteoporosis people and osteoporosis patients n b The negative relationship between the expression ofSNHG14 and miR2861 in the serum of osteoporosis patients n c Expression of SNHG14 in hMSCs of nonosteoporosis people andosteoporosis patients n d Expression of miR2861 in hMSCs of nonosteoporosis people and osteoporosis patients n p Fig SNHG14 was targeted by miR2861 a Common binding sequences between SNHG14 and miR2861 b Expression of miR2861 mRNA inhMSCs c Dualluciferase reporter assay d Expression of miR2861 mRNA in hMSCs e Expression of SNHG14 mRNA in hMSCs N p 0cDu BMC Musculoskeletal Disorders Page of Fig shSNHG14 suppressed osteoblast differentiation of hMSC a The expression of SNHG14 mRNA in hMSCs b ALP activities in shSNHG14 orshNC transfected hMSCs on day day and day c Osteoblast differentiation assessed through osteoblast marker genes of RUNX2 OSX ALPand OC normalized to actin on day day and day d ALP and Alizarin Red staining on day N p MiR2861 suppressed osteoblast differentiation of hMSCTo further evaluate the effects of miR2861 on hMSCosteoblast differentiation we induced hMSCs to differentiate to osteoblasts after transfection with miR2861mimic or miRNC Overexpression of miR2861 significantly suppressed osteoblastic differentiation which wasindicated by decreased ALP activity Fig 4a lower expression levels of RUNX2 OSX ALP and OC Fig 4b 0cDu BMC Musculoskeletal Disorders Page of Fig MiR2861 suppressed osteoblast differentiation of hMSC a ALP activities measured at day day and day of osteoblastdifferentiation b osteoblast differentiation assessed by the mRNA expression of RUNX2 OSX ALP and OC day day and day c ALP andAlizarin Red staining results on day N p and reduced in vitro matrix mineralization Fig 4c inmiR2861mimic transfected hMSCs in contrast to cellstransfected with miRNCThe effects of SNHG14 on osteoblast differentiation ofhMSC were attenuated by miR2861Whether miR2861 could attenuate the effects ofSNHG14on osteoblast differentiation of hMSCFigure 5a illustrated that shSNHG14 decreased ALP activity but the effects were attenuated by cotransfectionwith miR2861 inhibitor Figure 5b demonstrated thatdownregulation of miR2861 greatly lowered osteoblastic differentiation induced by shSNHG14sinceshSNHG14 decreased osteogenesisAKT2 was targeted by miR2861Finally the mechanisms by which miR2861 functionedto affect the differentiation of hMSCs were exploredOur bioinformatics analysis and luciferase assay resultsshowed that AKT2 could bind with miR2861 Fig 6aand b In addition overexpression of miR2861 decreased the expression levels of AKT2 and downregulation of SNHG14 reduced the expression of AKT2Fig 6c and dDiscussionsOsteoblastic differentiation from hMSCs many originates from many cell events that are affected by variousmolecular and cellular procedures during the development of bone and skeleton It is crucial to reveal important factors that mediate this phenomenon and to studythe underlying mechanisms Owing to the successfulfindings from the previous studies different lncRNAshave been shown to participate in the osteoblast differentiation by targeting corresponding cell signaling pathways One study revealed the expression profiling of 0cDu BMC Musculoskeletal Disorders Page of Fig The effects of SNHG14 on osteoblast differentiation of hMSC were attenuated by miR2861 a ALP activities in cells transfected withcontrol shSNHG14 or shSNHG14 miR2861inhibitor at day b Expression of osteoblast marker genes of RUNX2 OSX ALP and OC at day N p lncRNAs in C3H10T12 mesenchymal stem cells undergoing early osteoblast differentiation [] LncRNA H19promoted osteoblast differentiation via the TGF1Smad3HDAC signaling pathway by deriving miR675[]Various lncRNAs and miRNAs are dysregulated during the hMSCs differentiation of osteoblast [ ] Inour study we found a similar phenomenon We firstlyanalyzed the expression of SNHG14 and miR2861 inserum and hMSCs of osteoporosis patients Comparedto nonosteoporosis participants the expression levels ofSNHG14 in serum and hMSCs of osteoporosis patientswere greatly elevated The expression of miR2861 wasdrastically downregulated in hMSCs of osteoporosisgroup A negative relationship was established betweenthe expression of SNHG14 and miR2861 in serum ofosteoporosis group Similar to previous studies we identified that lncRNA SNHG14 was upregulated but miR was downregulated in serum and hMSCs from patients with osteoporosisFig AKT2 was targeted by miR2861 a Shared binding sequences between AKT2 and miR2861 b Dualluciferase reporter assay c and dWestern blot assay of AKT2 protein expression levels N p 0cDu BMC Musculoskeletal Disorders Page of With the common shared binding sequences lncRNAscould target their specific miRNAs and exert the biological roles in the pathogenesis of many cellular procedures [] For examplelncRNA DGCR5 acts as atumor suppressor in papillary thyroid carcinoma via targeting miR2861 [] We first confirmed the commonbinding sequences between SNHG14 and miR2861 Cotransfection of SNHG14 ²UTR with miR2861 led tothe suppression of luciferase activities compared withthat of SNHG14 MUT Moreover shSNHG14 elevatedthe expression levels of miR2861 The relative expression levels of SNHG14 were also lowered in cells transfected with miR2861 As far as we know we are thefirst to reveal that SNHG14 is targeted by miR2861 during the hMSCs differentiation to osteoblastAccording to previous reports ALP is highly expressedin osteoblast which is an important indicator for maturedifferentiation of osteoblast [] Osteoblastspecific genesRUNX2 Osterix ALP and OC are also critical genes to indicate the existing of osteoblast [ ] To investigatethe effects of SNHG14 on hMSC osteoblast differentiation we induced hMSCs differentiation to osteoblastsafter transfection with shSNHG14 or shNC The expression of SNHG14 was suppressed in cells transfected withshSNHG14 Suppression of SNHG14 markedly loweredosteoblastic differentiation which was indicated by lowerexpression levels of the osteoblastspecific genes RUNX2Osterix ALP and OC decreased ALP activity and in vitromatrix mineralization by Alizarin red staining inshSNHG14 transfected hMSCs compared with cells transfected with shNC Similar to previous reports [ ] wealso observed that silencing of SNHG14 could suppresshMSCs differentiation to osteoblastsA novel regulation role of Runx2miR3960miR2861was demonstrated in mouse osteoblast differentiation []MiR2861 was found to promote osteoblast differentiationby increasing the expression of Runx2 [] To investigatethe effects of miR2861 on hMSC osteoblast differentiationwe induced hMSCs to differentiate to osteoblasts aftertransfection with miR2861mimic or miRNC Overexpression of miR2861 greatly suppressed osteoblastic differentiation which was indicated by lower expression levelsof the osteoblastspecific genes RUNX2 OSX ALP andOC and decreased ALP activity and reduced in vitromatrix mineralization in miR2861mimic transfectedhMSCs compared to cells transfected with miRNC Different from the previous study [] we noticed that miR2861suppressed osteoblast differentiation of hMSC Moreoverwe observed that the effects of SNHG14 on osteoblast differentiation of hMSC were attenuated by miR2861ConclusionsIn our data confirmed that the induction effects of SNHG14 on osteoblast differentiation of hMSCswere attenuated by miR2861 SNHG14 could induceosteogenic differentiation of hMSC in vitro by targetingmiR2861Supplementary informationSupplementary information accompanies this paper at httpsdoi101186s12891020035069Additional file AbbreviationhMSCs Human bone marrow mesenchymal stem cellAcknowledgmentsNot applicableIndividual persons dataNot applicableAuthors™ contributionsMD BW SF YL XM XF contributed to data analysis drafting or revising the gave final approval of the version to be published and agree to beaccountable for all aspects of the workFundingThere is no funding sourceAvailability of data and materialsThe analyzed data sets generated during the study are available from thecorresponding author on reasonable requestEthics approval and consent to participateThe ethics review board of the Orthopedic Hospital of Shenyang ShenyangCity Liaoning Province approved our study Written informed consent wasobtained from all individual participants included in the studyConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived January Accepted July ReferencesAggarwal S Pittenger MF Human mesenchymal stem cells modulateallogeneic immune cell responses Blood “Sonoyama W Liu Y Fang D Yamaza T Seo BM Zhang C Liu H GronthosS Wang CY Shi S Mesenchymal stem cellmediated functional toothregeneration in swine PLoS One 200611e79Nuttelman CR Tripodi MC Anseth KS Dexamethasonefunctionalized gelsinduce osteogenic differentiation of encapsulated hMSCs J Biomed MaterRes Part A “Dawson E Mapili G Erickson K Taqvi S Roy K Biomaterials for stem celldifferentiation Adv Drug Deliv Rev “Nguyen MK Jeon O Krebs MD Schapira D Alsberg E Sustained localizedpresentation of RNA interfering molecules from in situ forming hydrogels toguide stem cell osteogenic differentiation Biomaterials “Eskildsen T TaipaleenmÃki H Stenvang J Abdallah BM Ditzel N Nossent AYBak M Kauppinen S Kassem M MicroRNA138 regulates osteogenicdifferentiation of human stromal mesenchymal stem cells in vivo ProcNatl Acad Sci “Yang G Lu X Yuan L LncRNA a link between RNA and cancer BiochimBiophys Acta “Voorhoeve PM Le Sage C Schrier M Gillis AJ Stoop H Nagel R Liu YP VanDuijse J Drost J Griekspoor A A genetic screen implicates miRNA372 andmiRNA373 as oncogenes in testicular germ cell tumors Cell “ 0cDu BMC Musculoskeletal Disorders Page of Zhu L Xu PC Downregulated LncRNAANCR promotes osteoblastdifferentiation by targeting EZH2 and regulating Runx2 expression BiochemBiophys Res Commun “Liao J Yu X Hu X Fan J Wang J Zhang Z Zhao C Zeng Z Shu Y Zhang RlncRNA H19 mediates BMP9induced osteogenic differentiation ofmesenchymal stem cells MSCs through notch signaling Oncotarget Qi X Shao M Sun H Shen Y Meng D Huo W Long noncoding RNASNHG14 promotes microglia activation by regulating miR1455pPLA2G4Ain cerebral infarction Neuroscience “ Hamam D Ali D Vishnubalaji R Hamam R AlNbaheen M Chen L KassemM Aldahmash A Alajez N microRNA320RUNX2 axis regulates adipocyticdifferentiation of human mesenchymal skeletal stem cells Cell Death Dis2014510e1499 Hu R Liu W Li H Yang L Chen C Xia ZY Guo LJ Xie H Zhou HD Wu XP A Runx2miR3960miR2861 regulatory feedback loop during mouseosteoblast differentiation J Biol Chem “ Zuo C Wang Z Lu H Dai Z Liu X Cui L Expression profiling of lncRNAs inC3H10T12 mesenchymal stem cells undergoing early osteoblastdifferentiation Mol Med Rep “ Huang Y Zheng Y Jia L Li W Long noncoding RNA H promotesosteoblast differentiation via TGF1S mad3HDAC signaling pathway byderiving mi R675 Stem Cells “Tye CE Gordon JA MartinBuley LA Stein JL Lian JB Stein GS CouldlncRNAs be the missing links in control of mesenchymal stem celldifferentiation J Cell Physiol “Schoolmeesters A Eklund T Leake D Vermeulen A Smith Q Aldred SF FedorovY Functional profiling reveals critical role for miRNA in differentiation of humanmesenchymal stem cells PLoS One 200945e5605 M Kumar M Goyal R LncRNA as a therapeutic target for angiogenesis CurrTop Med Chem “ Mizuno M Kuboki Y Osteoblastrelated gene expression of bone marrowcells during the osteoblastic differentiation induced by type I collagen JBiochem “Jang WG Kim EJ Kim DK Ryoo HM Lee KB Kim SH Choi HS Koh JTBMP2 protein regulates osteocalcin expression via Runx2mediated Atf6gene transcription J Biol Chem “Salingcarnboriboon R Tsuji K Komori T Nakashima K Ezura Y Noda MRunx2 is a target of mechanical unloading to alter osteoblastic activity andbone formation in vivo Endocrinology “Lu YF Liu Y Fu WM Xu J Wang B Sun YX Wu TY Xu LL Chan KMZhang JF Long noncoding RNA H19 accelerates tenogenic differentiationand promotes tendon healing through targeting miR29b3p and activatingTGF1 signaling FASEB J “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

test the hypothesis that levobupivacaine has anti‘tumour effects on breast cancer cellsResults Colony formation and transwell assay were used to determine breast cancer cells proliferation Flow Cytom‘etry annexin V and PI staining was used to investigate breast cancer cells apoptosis The effects of levobupivacaine on cellular signalling and molecular response were studied with Quantitative Polymerase Chain Reaction and western blot Induction of apoptosis was confirmed by cell viability morphological changes showed cell shrinkage rounding and detachments from plates The results of the western blot and Quantitative Polymerase Chain Reaction indicated activation of active caspase‘ and inhibition of FOXO1 The results of the flow Cytometry confirmed that levobupiv‘acaine inhibited breast cancer cell proliferation and enhanced apoptosis of breast cancer cells Quantitative Polymer‘ase Chain Reaction and Western blot analysis showed increased p21 and decreased cyclin D Quantitative Polymerase Chain Reaction and western blot analysis showed that levobupivacaine significantly increased Bax expression accom‘panied by a significant decreased Bcl‘ expression and inhibition of PI3KAktmTOR signalling pathway These findings suggested that levobupivacaine inhibits proliferation and promotes breast cancer cells apoptosis in vitroKeywords Levobupivacaine Proliferation Invasion Apoptosis Breast cancerIntroductionBreast cancer is one of the most recorded cancer illness among women [] In the United States it is estimated that more than women die every year from breast cancerrelated illness despite the advance in chemotherapy and targeted treatments []Correspondence yanqiu63126com wqp89163com Department of Anaesthesiology Dalian Medical University Dalian China Department of Biochemistry and Molecular Biology Dalian Medical University Dalian ChinaFull list of author information is available at the end of the Molecular signalling pathways that are involved in breast cancer transformation have become targets for treatment [] The mechanisms of the PI3KAktmTOR signalling pathway have present some promising targets for cancer treatments This signalling pathway hinders the functions of several tumour suppressor genes such as Bad GSK3 FOXO transcription factors and tuberinhamartin complex which control cell survival proliferation and growth [“] Suppressing this signalling pathway may inhibit cancer cells proliferation and also stimulate them toward cell deathThe growing evidence of local anaesthetics inhibiting cancer cell growth seems promising though limited [] The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cKwakye a0et a0al BMC Res Notes Page of At the tissue level administration of a certain amount of local anaesthetics topical or local has shown to have a direct inhibitory effect on the action of epidermal growth factor receptor EGFR which is a potential target for antiproliferation in cancer cells [] Evidence also shows that ropivacaine and lidocaine hinder cancer cells growth invasion migration and enhance apoptosis of lung cancer cells [“] To the best of our knowledge the effect of levobupivacaine on breast cancer cells is yet to be determined The present study therefore aimed to investigate the antitumour effects of levobupivacaine on breast cancer cellsMain textMaterials and a0methodsEthics statementThe ethical committee of the Dalian Medical University First Affiliated Hospital approved for this study to be carried outCell cultureWe purchased MCF7 and MDAMB231 breast cancer cells from the ATCC Beijing Zhongyuan limited China We maintained the MCF7 and MDAMB231 cells with highglucose DMEM or DMEMF12 Gibco USA medium The medium was supplemented with fetal bovine serum FBS Gibco USA penicillin a0unitsml and streptomycin a0µgml TransGen Biotech China to maintain the cells The MCF7 and MDAMB231 cells were then maintained in an incubator at a0ºC humidified air with CO2 atmospheric condition The cells were routinely subcultured subsequentlyAntibodies and a0reagentsEPR17671 Akt monoclonal Antibody Abcam China Y391 mTOR Polyclonal Antibody Abcam China A2845 Bcl2 Polyclonal Antibody ABclonal Technology A11550 Bax Polyclonal Antibody ABclonal Technology A0265 PIK3CA Polyclonal Antibody ABclonal Technology A2934 FOXO1 Polyclonal Antibody ABclonal Technology EPR21032 Active caspase monoclonal Antibody Abcam China AFO931 Cyclin D1 Polyclonal Antibody Affbiotech China AF6290 p21 Polyclonal Antibody Affbiotech China AntimTOR phospho S2448 Antibody Abcam China PA517387 PhosphoPI3K p85p55 Tyr458 Tyr199 Polyclonal Antibody ThemoFisher Scientific PosphopanAKT123 Ser473 Antibody Affbiotech China Peroxidaseconjugated goat antirabbit IgG Proteintech China PRAP antibodies Proteintech China and GAPDH antibodies Proteintech ChinaCell viability assay and a0IC50We determined the MCF7 and MDAMB cells viability using CCK8 assay Levobupivacaine at a concentration of or a0mM was used to treat MCF7 and MDAMB cells plated in 96well plates — a0cellswell and then incubated for or a0h respectively in an incubator at the atmospheric condition of a0 °C with CO2 The rest of the procedures used for the CCK8 assay were the same as described elsewhere []Flow cytometryAnnexin V and propidium iodide PI staining assay were used to investigate the apoptosis of MCF7 and MDAMB cells following levobupivacaine treatment After treating the cells for a0h trypsin was used to harvest the treated cells and centrifugation at rcf for a0min The MCF7 and MDAMB treated cells were again suspended with — Binding Buffer and then a0 µl of fluorochromeconjugated annexin V SigmaAldrich Saint Louis USA was added into a0µl of the cell suspension to stain intracellular phosphatidylserine PS The cells were then incubation in a dark under room temperature The cells were again suspended and a0 µl propidium iodide staining solution SigmaAldrich Saint Louis USA added into a0µl of the cell suspension We detected the percentage of the apoptotic cells via FlowJo software Treestar Ashland USA and Flow cytometry FACS Calibur Becton Dickinson and Sunnyvale CA USAQuantitative polymerase chain reaction qPCRThe procedures used for the qPCR were the same as previously described [] The primers sequences were BAX 5TGG CAG CTG ACA TGT TTT CTG3 F 5TCC CGG AGG AAG TCC AAT G3 BCL2 5ACG GTG GTG GAG GAG CTC TT3 F 5GCC GGT TCA GGT ACT CAG TCAT3 R p21 5GCG ACT GTG ATG CGC TAA TG3 F 5GAA GGT AGA GCT TGG GCA GG3 R GAPDH ²CAT GTT CGT CAT GGG TGT GAA² F ²GGC ATG GAC TGT GGT CAT GAG3² RR Western blotAt the log phase of treated MCF7 and MDAMB cells growth we harvested the cells and then washed twice with icecold PBS The rest of the procedures used for the western blot were the same as described elsewhere []Colony formation assayThe procedures used for the colony formation assay were the same as previously described [] 0cKwakye a0et a0al BMC Res Notes Page of Transwell assayThe MCF7 and MDAMBA231 cells — that were pretreated with different dose of Levobupivacaine a0mM for a0h and resuspended in culture medium with the same concentrations of levobupivacaine were seeded onto the coated membrane in the upper chamber of the transwell 24well millicell cell culture insert a0mm diameter a0μm pores Merck KGaA P18P01250 China The procedures used for the Transwell assay were the same as previously describe []Data analysisValues were expressed as the mean ± SD Statistical analysis was performed with GraphPad Prism version 501GraphPad Software La Jolla CA US Oneway ANOVA was used to measure significance p Dunnett™s post hoc tests were used to test the difference between groupsResultsLevobupivacaine decreases breast cancer cell invasionTranswell assay analysis showed significantly decreased in the invasion ability of MCF7 and MDAMB231 cells in a dosedependent manner compared with the untreated cells Additional file a0 Fig S1a b Levobupivacaine inhibits proliferation in a0breast cancer cellsThe MCF7 and MDAMBA231 cell viability decreased as the concentrations of levobupivacaine or a0mM increased The MCF7 cells showed a cytotoxic effect while the MDAMB231 cells showed a similar cytotoxic effect of Fig a01a Under a fluorescence microscope cells treated with levobupivacaine showed morphological changes including cell rounding cell shrinkage and cells detachment from the plates Additional file a0 Fig S2a b The viability of breast cancer cells decreased in a dosedependent manner The results showed significantly decreased in the number of clones of the treated cells compared with the untreated cells Fig a01b c The data showed that the mRNA level of p21 significantly increased following levobupivacaine treatment Fig a0 1d e Western blot analysis showed a similar increased in p21 and decreased in FOXO1 and cyclin D1 expressions in a dosedependent manner compared with the untreated cells Fig a01f g Additional file a03f gLevobupivacaine promote apoptosis in a0breast cancer cellsLevobupivacaine significantly reduced the number of cells showing nuclear staining when compared with the untreated cells Fig a0 2a b The qPCR data showed a decreased in Bcl2 and increased in Bax expressions in MCF7 and MDAMB231 cells compared with the untreated cells Fig a0 2c d Western blot analysis also showed a similar decreased in Bcl2 and increased expressions of active caspase and Bax compared with the untreated cells Fig a02e f Additional file a03e fLevobupivacaine inhibits proliferation and a0promotes apoptosis in a0breast cancer through a0PI3KAktmTOR signalling pathwayWestern blot analysis showed a significant decreased in the expression of the nuclear localization of pPI3K pAkt and pmTOR compared with the untreated cells Fig a03a b Additional file a03a bDiscussionBreast cancer remains a common cause of mortality among women worldwide Though current orthodox drugs have demonstrated promise in breast cancer therapy its treatment options remain limited These therefore supports the concept that effective therapeutic approaches for breast cancer are critically needed Several retrospective studies have demonstrated that regional anaesthesia is associated with a decreased risk of recurrence or metastasis of multiple carcinomas including breast prostate and cervical cancers [“] Recent growing evidence demonstrates that local anaesthetics have an antitumour effect and may suppress the motility of cellular function and invasiveness more likely via voltagegated sodium channel inhibition [] A study report indicates that lidocaine inhibits the growth of human hepatocellular carcinoma cells HCC by increasing the Caspase activity whereas ropivacaine inhibits the growth of HCC cells by stopping the cell cycle in G2 phase [] Lee et a0al demonstrated that local anaesthetics potentiate TNFα mediated apoptosis in HK2 cells [] The cellular modification of treated cells is likely dependent on the duration of exposure and the dose of the local See figure on next pageFig Levobupivacaine inhibits proliferation in breast cancer cells MCF‘ and MDA‘MB‘ cells were treated with different concentrations of levobupivacaine a Cell viability was measured by CCK‘ assay IC50 results of levobupivacaine on MCF‘ and MDA‘MB cells b c Colony formation of MCF‘ and MDA‘MB cells treated with various concentrations of Levobupivacaine and stained with crystal violet d e The mRNA expression levels of p21 and GAPDH were analysed by qPCR f g Protein expression assessment of MCF‘ and MDA‘MB‘ cells by western blot against antibodies FOXO1 p21 Cyclin D1 and GAPDH used as control The data was statistically significant at indicates P indicates P indicates P compared with untreated cells This data corresponds to the mean ± SEM of three independent experiments 0cKwakye a0et a0al BMC Res Notes Page of 0cKwakye a0et a0al BMC Res Notes Page of Fig Effects of levobupivacaine on apoptosis of breast cancer cells a b MCF‘ and MDA‘MB cells were treated with different concentrations of levobupivacaine for h The cells were then stained with fluorescein‘conjugated annexin V and PI and analysed by flow cytometry Error bars represent standard error of the mean P versus the control c d Relative gene expression of Bax and Bcl‘ following the treatment of breast cancer cells with different concentrations of levobupivacaine for h and analysed by qPCR e f MCF‘ and MDA‘MB cells were treated with different concentrations of levobupivacaine for h and the activities of Bax Bcl‘ and Active caspase were examined by Western blot analysis using specific antibodies GAPDH was used as internal controls The data was statistically significant at indicates P indicates P compared with control The data correspond to the mean ± SEM of three independent experiments 0cKwakye a0et a0al BMC Res Notes Page of Fig MCF‘ and MDA‘MB cells were treated with different concentrations of levobupivacaine for h a b The cells were lysed and subjected to SDS‘PAGE and analysed by western blotting and probed with specific antibodies p‘PI3K p‘Akt and p‘mTOR The results showed a decrease in the expressions of p‘PI3K p‘Akt and p‘mTOR proteins GAPDH was used as internal controls The data represent the mean ± SD of three independent experimentsanaesthetic [“] In this study we employed MCF and MDAMB231 cells as models and found that different concentrations of levobupivacaine could effectively inhibit breast cancer cell proliferation and promote apoptosis in a0vitro The antiproliferation and apoptosis effects observed in this study suggest that levobupivacaine may have therapeutic effects on breast cancerPI3KAktmTOR signalling pathway plays a vital role in cell proliferation survival development metabolism motility and regulation of the immune response Breast cancer cell resistance to therapies can result from the activation of PI3KAktmTOR signalling pathway [“] This has made the PI3KAktmTOR signalling pathway an important object of study for understanding the development and progression of breast cancer In patients with breast cancer PI3KAktmTOR signalling pathway can be a target for diagnostic prognostic and treatment purposes [“] Akt plays a role in the activation and inactivation of many transcription factors Activation of Akt correlated with the activation of mTOR Phosphorylation of the FOXO proteins by Akt may results in cytoplasmic retention by interacting with other proteins thereby isolating them from their targeted genes Cyclin D1 classified as a pro oncogene is often overexpressed in several human malignancies including breast colon lung and prostate cancers [] Reports show that overexpression of cyclin D1 and underexpression of tumour suppressor p21 is required for cancer initiation as it is confirmed that downregulation of cyclin D1 and overexpression of p21 in xenograft model discontinues the formation of cancer in the early stages [] Datta et a0al reported that Akt can phosphorylate the proapoptotic Bcl2 family member Bad causing its isolation from the mitochondrial membrane by other proteins [] Local anaesthetics modify the protein levels of key members of the Bcl2 family in a manner that presents an increase in the ratio of BaxBcl2 which may contribute to the response of cancer cells to apoptosis In the present study the role of levobupivacaine on the expression of PI3K Akt and mTOR was investigated to illustrate the potential molecular mechanism We observed a significantly decreased expression of pAkt pPI3K pmTOR and subsequent decreased expression of FOXO Cyclin D1 and Bcl2 following levobupivacaine treatment which correlated with decreased breast cancer cells proliferation and increased apoptosis These emerging pieces of evidence suggest that levobupivacaine may inhibit proliferation and promote apoptosis by suppressing PI3KAktmTOR signalling pathway which demonstrated an antitumour effect on breast cancer cells in this studyConclusionlevobupivacaine has the potency of reducing breast cancer cell viability proliferation and also causes cell death by suppressing the PI3KAktmTOR signalling pathway These findings could lead to clinical studies which will seek to examine the anticancer effects of levobupivacaine and may also increase the benefits in cancer patient as well as improve patient care 0cKwakye a0et a0al BMC Res Notes Page of LimitationsNumerous studies have reported on the antitumour effects of local anaesthetics on various cancer cells [“] However our work is not without limitations In a0vivo and clinical studies on the antitumour effects of levobupivacaine are neededBiology Dalian Medical University Dalian China Department of Anaesthesia and Critical Care School of Medicine University of Health and Allied Sciences Ho Ghana Department of Biochemistry and Molecular Medicine School of Medicine and Health Sciences University for Development Studies Tamale Ghana Departments of Anaesthesia and Critical Care Ridge Hospital Accra Ghana Department of Medicine Princefied University Ho Ghana Received June Accepted July Supplementary informationSupplementary information accompanies this paper at https doi101186s1310 ‘‘ ‘Additional file a0 Figure S1 Levobupivacaine decreases breast cancer cell invasionAdditional file a0 Figure S2 Effect of levobupivacaine on the morphology of MCF‘ and MDA‘MB cellsAdditional file a0 Original gelsblots scan used in Fig 1f g Fig 2e f and Fig 3a b for MCF‘ and MDA‘MB‘ cellsAbbreviationsEGFR Epidermal growth factor receptor HCC Hepatocellular carcinoma cells NC Nitrocellulose PI Propidium iodide PS Phosphatidylserine qPCR quanti‘tativepolymerase chain reactionAcknowledgementsWe thank the First Affiliated Hospital and The Department of Biochemistry of Dalian Medical University for making available all the necessary materials needed for this work We also thank the Key Laboratory of Liaoning Provincial Education Department Grant NO LZ2016002 and Liaoning Natural Science Foundation Grant NO of China for supporting this work Our thanks also go to the China Scholarship Council and the Government of the Republic of Ghana for giving financial aid to some of the authors to study at Dalian Medical UniversityAuthors™ contributionsAKK SK QY and QPW conceived and designed this study QPW and QY were responsible for the supervision and coordination of this study AKK SK JL MNR QY and QPW conducted the data collections SK led the data analysis with inputs from AKK QY and QPW AKK and SK wrote the first draft of the manuscript and JL MNR SAR AAF JA and EAN contributed to revising and reviewing the manuscript All authors read and approved the final manuscriptFundingThis study was supported by the Key Laboratory of Liaoning Provincial Education Department Grant NO LZ2016002 and Liaoning Natural Science Foundation Grant NO Availability of data and materialsThe data used andor analysed in this study are available from the correspond‘ing author upon reasonable requestEthics approval and consent to participateThe ethical committee of the First Affiliated Hospital of Dalian Medical Univer‘sity approved the study protocol and because this study used breast cancer cells consent to participate was not applicable for the studyConsent for publicationsNot applicableCompeting interestsAuthors declare that they have no competing interestsAuthor details Department of Anaesthesiology Dalian Medical University Dalian China Department of Anaesthesiology First Affiliated Hospital of Dalian Medi‘cal University Dalian China Department of Biochemistry and Molecular References American Cancer Society Breast Cancer Facts and Figures “ Atlanta American Cancer Society American Cancer Society Cancer Facts and Figures Atlanta Ameri‘ can Cancer Society Siegel R Naishadham D Jemal A Cancer statistics CA Cancer J Clin “ Chang YC Hsu YC Liu CL Huang SY Hu MC Cheng SP Local anaesthetics induce apoptosis in human thyroid cancer cells through the mitogen‘activated protein kinase pathway PLoS ONE 20149e89563 Gomez‘Gutierrez JG Souza V Hao HY de Montes Oca‘Luna R Dong YB Zhou HS McMasters KM Adenovirus‘mediated gene transfer of FKHRL1 triple mutant efficiently induces apoptosis in melanoma cells Cancer Biol Ther “Sunters A de Fern¡ndez Mattos S Stahl M Brosens JJ Zoumpoulidou G Saunders CA Coffer PJ Medema RH Coombes RC Lam EW FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel‘treated breast cancer cell lines J Biol Chem “Fu Z Tindall DJ FOXOs cancer and regulation of apoptosis Oncogene “ Barnes DM Gillett CE Cyclin D1 in breast cancer Breast Cancer Res Treat “Sherr CJ Roberts JM CDK inhibitors positive and negative regulators of G1‘phase progression Genes Dev “ Pelengaris S Khan M Evan G c‘MYC more than just a matter of life and death Nat Rev Cancer “ Di Padova M Barbieri R Fanciulli M Arcuri E Florida A Effect of local anaesthetic ropivacaine on the energy metabolism of Ehrlich ascites tumour cells Oncol Res 199810491e8 Xing W Chen DT Pan JH Chen YH Yan Y Li Q Xue RF Yuan YF Zeng WA lidocaine induces apoptosis and suppresses tumour growth in human hepatocellular carcinoma cells in vitro and in a xenograft model in vivo Anesthesiology “ Drasner K Lidocaine spinal anaesthesia a vanishing therapeutic index Anesthesiology “ Wang HW Wang LY Jiang L Tian SM Zhong TD Fang XM Amide‘linked local anaesthetics induce apoptosis in human non‘small‘cell lung cancer J Thorac Dis “ https doi1021037 jtd20160966 Piegeler T Votta‘Velis EG Liu G Place AT Schwartz DE Beck‘Schimmer B Minshall RD Beat A Anti‘metastatic potential of amide‘linked local anaesthetics inhibition of lung adenocarcinoma cell migration and inflammatory Src signalling independent of sodium channel blockade Anesthesiology “ Lirk P Berger R Hollmann MW Feigl H Lidocaine time and dose‘depend‘ently demethylates deoxyribonucleic acid in breast cancer cell lines in vitro Br J Anaesth “ Villar‘Garea A Fraga MF Espada J Esteller M Procaine is a DNA‘demethyl‘ating agent with growth‘inhibitory effects in human cancer cells Cancer Res 4984e634984e9 Kampo S Ahmmed B Zhou T Owusu L Anabah TW Doudou NR Kuug‘bee ED Cui Y Lu Z Yan Q Wen Q‘P Scorpion venom analgesic peptide BmK AGAP inhibits stemness and epithelial‘mesenchymal transition by down‘regulating PTX3 in breast cancer Front Oncol Hirata M Sakaguchi M Mochida C Sotozono C Kageyama K Yoshihiro K Munetaka H Lidocaine inhibits the tyrosine kinase activity of the epidermal growth factor receptor and suppresses proliferation of corneal epithelial cells Anesthesiology “ 0cKwakye a0et a0al BMC Res Notes Page of Grouselle M Tueux O Dabadie P Geescaud D Mazat JP Effect of local anaesthetics on mitochondrial membrane potential in living cells Biochem J “ Fraser SP Diss JKJ Chioni AM Mycielska ME Pan H Yamaci RF Pani F Siwy Z Krasowska M Grzywna Z Brackenbury WJ Theodorou D Koyuturk M Kaya H Battaloglu E De Tamburo Bella M Slade MJ Tolhurst R Palmieri C Jiang J Latchman DS Coombes RC Djamgoz MBA Voltage‘gated sodium channel expression and potentiation of human breast cancer metastasis Clin Cancer Res “ Le Gac G Angenard G Clement B Laviolle B Coulouarn C Beloeil H Local anaesthetics inhibit the growth of human hepatocellular carcinoma cells Anesth Analg “ Lee HT Xu H Siegel CD Krichevsky IE Local anaesthetics induce human renal cell apoptosis Am J Nephrol “ Unami A Shinohara Y Ichikawa T Baba Y Biochemical and microarray analyses of bupivacaine‘induced apoptosis J Toxicol Sci “ Villar‘Garea A Fraga MF Espada J Esteller M Procaine is a DNA‘demethyl‘ating agent with growth‘inhibitory effects in human cancer cells Cancer Res “ Sakaguchi M Kuroda Y Hirose M The antiproliferative effect of lidocaine on human tongue cancer cells with inhibition of the activity of epidermal growth factor receptor Anesth Analg “ Chang YC Liu CL Chen MJ Hsu YW Chen SN Lin CH Chen CM Yang FM Hu MC Local anaesthetics induced apoptosis in human breast tumour cells Anesth Analg “ Kawasaki C Kawasaki T Ogata M Sata T Chaudry IH Lidocaine enhances apoptosis and suppresses mitochondrial functions of human neutrophil in vitro J Trauma “ Hodgkin AL Huxley AFA quantitative description of membrane current and its application to conduction and excitation in nerve J Physiol “ Besson P Driffort V Bon ‰ Gradek F Chevalier S Roger S How do voltage‘gated sodium channels enhance migration and invasiveness in cancer cells Biochim Biophys Acta “ Roger S Gillet L Le Guennec JY Besson P Voltage‘gated sodium channels and cancer is excitability their primary role Front Pharmacol Roger S Potier M Vandier C Besson P Le Guennec JY Voltage‘gated sodium channels new targets in cancer therapy Curr Pharm Des “ Driffort V Gillet L Bon E Marionneau‘Lambot S Oullier T Joulin V Collin C Pag¨s JC Jourdan ML Chevalier S Bougnoux P Le Guennec JY Besson P Roger S Ranolazine inhibits NaV15‘mediated breast cancer cell invasive‘ness and lung colonization Mol Cancer Nelson M Yang M Dowle AA Thomas JR Brackenbury WJ The sodium channel‘blocking antiepileptic drug phenytoin inhibits breast tumour growth and metastasis Mol Cancer Yuan T Li Z Li X Yu G Wang N Yang X Lidocaine attenuates lipopoly‘saccharide‘induced inflammatory responses in microglia J Surg Res “ Brocco MC Paulo DNS Almeida CED Carraretto AR Cabral SA Silveira AC Gomez RS Baptista JF A study of interleukin IL‘ and tumour necrosis factor‘alpha TNF‘α serum levels in rats subjected to faecal peritonitis and treated with intraperitoneal ropivacaine Acta Cirurgica Brasileira “ Piegeler T Schl¤pfer M Dull RO Schwartz DE Beat A Minshall RD Beck‘Schimmer B Clinically relevant concentrations of lidocaine and ropivacaine inhibit TNFα‘induced invasion of lung adenocarcinoma cells in vitro by blocking the activation of Akt and focal adhesion kinase Br J Anaesth “ Shankar S Chen Q Srivastava RK Inhibition of PI3KAKT and MEKERK pathways act synergistically to enhance antiangiogenic effects of EGCG through activation of FOXO transcription factor J Mol Signal Qian J Zou Y Rahman JSM Lu B Massion PP Synergy between phosphatidylinositol kinaseAkt pathway and Bcl‘xL in the control of apoptosis in adenocarcinoma cells of the lung Mol “ Ortega MA Fraile‘Mart™Ä±nez O As™Unsolo A Buj™an J Garc™Ä±a‘Honduvilla N Coca S Signal transduction pathways in breast cancer the important role of PI3KAktmTOR J Oncol Royds J Khan AH Buggy DJ Update on existing ongoing prospective trials evaluating the effect of anaesthetic and analgesic techniques during primary Cancer surgery on Cancer recurrence or metastasis Int Anesthesiol Clin 2016544e76“ Burgering BMT Medema RH Decision on life and death FOXO forkhead transcription factors are in command when PKBAkt is off duty J Leukoc Biol “ Chen Q Ganapathy S Singh KP Shankar S Srivastava RK Resveratrol Induces Growth Arrest and Apoptosis through Activation of FOXO Tran‘scription Factors in Prostate Cancer Cells PLoS ONE 20105e15288 Arnold A Papanikolaou A Cyclin D1 in breast cancer pathogen‘esis J Clin Oncol “ Santarius T Shipley J Brewer D Stratton MR Cooper CS A census of amplified and overexpressed human cancer genes Nat Rev Cancer “ Datta SR Brunet A Greenberg ME Cellular survival a play in three Akts Genes DAev “ Lirk P Hollmann MW Fleischer M Weber NC Feigl H Lidocaine and ropivacaine but not bupivacaine demethylate deoxyribonucleic acid in breast cancer cells in vitro Br J Anaesth 2014113Suppl 1i32“i3838 Li K Yang J Han X Lidocaine sensitizes the cytotoxicity of cisplatin in breast cancer cells via the up‘regulation of RARβ2 and RASSF1A demeth‘ylation Int J Mol Sci “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in pub‘lished maps and institutional affiliations¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your field¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research over 100M website views per year ¢ At BMC research is always in progressLearn more biomedcentralcomsubmissionsReady to submit your research Choose BMC and benefit from 0c'

Thyroid_Cancer

RUFYs a Family of EffectorProteins Involved in IntracellularTrafficking and CytoskeletonDynamicsRmy Char1 and Philippe Pierre123 Aix Marseille Universit Centre National de la Recherche Scientifique Institut National de la Sant et de la RechercheMdicale Centre d™Immunologie de MarseilleLuminy Marseille France Institute for Research in Biomedicine and IlidioPinho Foundation Department of Medical Sciences University of Aveiro Aveiro Portugal Shanghai Instituteof Immunology School of Medicine Shanghai Jiao Tong University Shanghai ChinaIntracellular trafficking is essential for cell structure and function In order to performkey tasks such as phagocytosis secretion or migration cells must coordinate theirintracellular trafficking and cytoskeleton dynamics This relies on certain classes ofproteins endowed with specialized and conserved domains that bridge membraneswith effector proteins Of particular interest are proteins capable of interacting withmembrane subdomains enriched in specific phosphatidylinositol lipids tightly regulatedby various kinases and phosphatases Here we focus on the poorly studied RUFY familyof adaptor proteins characterized by a RUN domain which interacts with small GTPbinding proteins and a FYVE domain involved in the recognition of phosphatidylinositol3phosphate We report recent findings on this protein family that regulates endosomaltrafficking cell migration and upon dysfunction can lead to severe pathology at theanismal levelKeywords RUFY cancer neurodegenerative diseases immunity RUN FYVE phosphatidylinositol 3phosphatecytoskeletonINTRODUCTIONThe anization of cells into multiple membranous compartments with specific biochemicalfunctions requires complex intracellular traffic and sorting of lipids and proteins to transport themfrom their sites of synthesis to their functional destination Intracellular transport involves lipidvesicles or tubules with the capacity to fuse with one another or to be secreted They collectivelyparticipate in the dynamic exchanges necessary for cell homeostasis Rothman S¸reng Membrane traffic is tightly coordinated with protein synthesis signal transduction ofenvironmental stimuli and cytoskeleton anization allowing the implementation of key cellularfunctions such as endocytosis exocytosis or migration McMahon and Gallop Habtezion VegaCabrera and PardoLpez MacGillavry and Hoogenraad Margaria Tapia Buratta Stalder and Gershlick Several families of molecular components required for orchestrating membrane vesicle exchangeand transport during this process are conserved They include adaptor and coat proteins smallGTPbinding proteins GTPases as well as Synaptosome Associated Protein SNAP ReceptorEdited byRoberto BotelhoRyerson University CanadaReviewed byDaniel G S CapellutoVirginia Tech United StatesBrian Paul CeresaUniversity of Louisville United StatesCorrespondenceRmy CharcharcimlunivmrsfrPhilippe PierrepierrecimlunivmrsfrSpecialty sectionThis was submitted toMembrane Traffica section of the journalFrontiers in Cell and DevelopmentalBiologyReceived June Accepted July Published August CitationChar R and Pierre P TheRUFYs a Family of Effector ProteinsInvolved in Intracellular Traffickingand Cytoskeleton DynamicsFront Cell Dev Biol 103389fcell202000779Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingSNARE proteins and SNARE binding proteins Juliano The vast superfamily of GTPases is involved in the establishmentor regulation of virtually every step of intracellular membranetrafficking They behave as molecular switches that can alternatebetween active and inactive states through GTP binding andhydrolysis into GDP Takai Stenmark Thelargest group of GTPases involved in intracellular membranetraffic is the Rab proteins family Lamb Rab GTPasesspecifically localize to diï¬erentintracellular compartmentsregulating vesicle formation and sorting as well as transportalong the cytoskeletal network Each Rab protein can be recruitedto specific membrane subdomains of a defined anelle andis associated to multiple eï¬ectors controlling membrane fusionand trafficking Rab interaction with the membrane fusioncomplexes and cytoskeleton regulators is therefore crucial forcellular functions including endocytosis and autophagy Chenand WandingerNess Bruce Geng Thomas and Fromme Yuan and Song Here we review the literature concerning a lesswell knownfamily of proteins involved in the complex biochemical crosstalkestablished between the cytoskeleton and intracellular vesiclesThis small group of proteins was named RUFY for œRUN andFYVE domaincontaining RUFYs share a common structuraldomain anization including an Nterminal RUN domainone or several coiledcoil CC repeats and a Cterminal FYVEdomain A The molecular structures of the diï¬erentRUFY proteins has been described Dunkelberg and GutierrezHartmann Mari KukimotoNiino Kitagishi and Matsuda but their function in endocyticregulation and their physiological relevance at the anismallevel are still poorly characterized Kitagishi and Matsuda Terawaki We revisit here how the rufy genefamily was annotated and propose the addition of a novelmember the fyco1 FYVE and Coiledcoil containing domain gene given its sequence and functional similarities withthe other rufy genes Pankiv Terawaki We also highlight recent findings on the implication ofRUFY proteins in the regulation of cytoskeleton and endosomedynamics and their contribution to immunity cancer andneurodegenerative diseasesEndocytosis and AutophagyEndocytosis and autophagy are membrane traffic pathwaysrequired for degradation and recycling ofextracellularand intracellular components respectively Birgisdottir andJohansen These pathways have a common endpoint at thelysosome where their cargo is degraded These both pathwaysintersect at several stages throughout vesicle formation transportand fusion and share some of the components of their molecularmachineries BThere are numerous coexisting endocytic pathways whichinitiate by the formation of nascent endocytic vesicles formedfrom plasma membrane invaginations and scissions Theseendocytic vesicles undergo homotypic fusion and are rapidlytargeted to sorting endosomes SE Sorting events initiated inSE determine the fate of internalized cargo molecules such asrecycling to plasma membrane degradation in lysosomes orother trafficking events Naslavsky and Caplan BOn their way to degradation sorted cargo accumulate inearly endosomes EE that further mature into late endosomesLEthrough multiple events of cargo and lipid sortingLate endosomes adopt a membrane anization termedmultivesicular bodies that are enriched in lysobisphosphatidicacid and contain intraluminal vesicles Gruenberg NextLE potentiate their hydrolytic competence by fusing withlysosomes Pillay resulting in the degradationoftheir contents providing nutrients and key factors tothe cell Doherty and McMahon Kaksonen and Roux Notably endosomes play a role in signal transductionby serving as signaling platforms either for surface activatedreceptors like Tolllike receptors and epidermal growth factorreceptor or metabolic sensors such as mechanistic targetof rapamycin complex mTORC1 Arg¼ello Often they promote the degradation of their targets leadingto signaltermination Chung The endocyticpathway has also specialized functions in diï¬erentiated cellssuch as neurotransmitter release and recycling in neurons orantigen processing and presentation in professional antigenpresenting cellsArg¼ello SolDom¨nech Hinze and Boucrot Endocytosis events and endosomes positioning ishighly dependent on the dynamic and spatial reanizationofinclude actinintermediate filaments or microtubules Fletcher and Mullins Pegoraro the diï¬erent cytoskeleton networks thatlike B cells or dendritic cellsComplementary to endocytosis autophagy is an intracellularprocess by which cells degrade and recycle their own cytoplasmicmaterials Mizushima and Komatsu Autophagy plays acentral role in many physiological processes including stressmanagement development immunity and aging Puleston andSimon Zhong F®lfan Moretti Doherty and Baehrecke Autophagy ispartially controlled though mTORC1 activity and is responsiblefor degradation and recycling of misfolded proteins as wellas obsolete anelles Galluzzi The endpoint ofautophagy is to deliver cytoplasmic material to lysosomes wherelike for endocytosed cargo it is degraded Several autophagyprocesses can be distinguished based on the entry mode ofthe cytosolic components destined for degradation BMacroautophagy involves engulfment of cytoplasmic contentsinto a double membrane vesicle termed the autophagosomeThe autophagosome fuses then with lysosomes becomingin which its cargo is degraded Galluzzian autolysosome The presence ofspecific phosphoinositideslipidstogether with Rab GTPases at a given membranecompartment is often directly correlated with compartmentfunction One of the common mechanism regulating endocytosisand autophagy is an accumulation of phosphatidylinositol phosphate PtdIns3P at surface of EE and on intraluminalvesicles of multivesicular endosomes and on autophagosomesNascimbeni B PtdIns3P is also observedat sites of LC3ˆ’associated phagocytosis another pathway ofinternalization used by the cells to ingest large particulatematerial or microbes PtdIns3P is therefore a beacon used by theFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingFIGURE RUN and FYVE domain containingproteins in the endolysosomal pathway A Schematic representation of the RUFY proteins family B Description ofthe endolysosomal and autophagy pathways and presumed functional locations of RUFY proteins Extracellular material is ingested by endocytosis orphagocytosis The action of different endosomes allows cargo to be sorted recycled or degraded in a complex and regulated process involving fusion maturationand transport along the cytoskeleton Alternatively during autophagy obsolete components present in cytosol are captured in autophagosomes prior fusion withlysosomes and degradation macroautophagy or directly internalized through endosomal invagination microautophagy SE sorting endosome EE earlyendosome TGN trans golgi network LE late endosome MVBs multi vesicular bodies RE recycling endosome MT microtubule CT centrioles ER endoplasmicreticulum The location of PI3P and RUFY proteins known activity is shown Created with BIoRendercomFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and Traffickingcellular machinery to regulate endosomal sorting and autophagyBirgisdottir and Johansen RUN DomainsThe presence of a single copy of a RUN and a FYVE domainat their extremities is the key characteristic defining the RUFYfamily members RUN domains were named after three proteinsbearing similar peptide motifs RPIP8 UNC14 and NESCAnew molecule containing SH3 at the carboxyˆ’terminus Ogura Matsuda RUN domains are present inmultiple proteins RUN proteins in a large panel of anismsFigure and principally allow direct interactions with smallGTPases of the Rap and Rab families Callebaut Yoshida RUN domains adopt a hydrophobicglobular structure bearing six conserved blocks named A to FFigure 3A These blocks correspond to eight αhelices andsome 310helices The first helix is crucial to limit hydrophobicexposure and maintain protein solubility of RUNcontainingproteins Callebaut KukimotoNiino In spite of strong conservation among the domains presentin RUNcontaining proteins the proteins they interact withtheir eï¬ectors are highly variable Mari and thestructural features of the RUN domain alone are not sufficientto define binding specificity for one or several members of theGTPase superfamily Fukuda Most RUN domainbearing proteins bind small GTPases but interactions with othermolecules like kinesin have also been described Boucrot A direct physical link between RUN proteins withactin filaments and microtubules has been also demonstratedTorti reinforcing the idea that these molecules arealso critical for cellular functions requiring actin remodelingsuch as migration or phagocytosis Price and Bos Bos Miertzschke Xu Figure 4AAdditional functions for RUN domains have been describedfor example for the RUN domain present in NESCA whichblocks TRAF6mediated polyubiquitination of the NFkappaBessential modulator and consequently induces NFkB activationThis is just one of the ways in which RUN proteins canact in signal transduction and the coordination of membranetraffic with actin dynamics upon external stimulation Yoshida As well as promoting endosomal fusion throughtheir binding to Rab or Rap GTPases Callebaut FIGURE Evolution of RUN and FYVE domain or rufy genes among living anisms Diagram illustrating the evolution of the rufy genes Species representative ofvarious taxonomic groups are listed data were extracted from the Differential Expression Atlas Genes database EMBLEBI Next to each species studied thenumber corresponds to the number of genes having in its sequence a FYVE green RUN blue or both red domain The œX corresponds to the appearance of acommon rufy ancestor geneFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingFIGURE Molecular anization of RUN and FYVE domains from the RUFY proteins family Alignment of the protein sequences of the RUN A and FYVE Bdomains of the RUFY proteins family in human and mouse A RUN consensus blocks are represented by segments A“F Rpip8 sequence is used as RUN domainreference B FYVE conserved motives and zinc fingers are represented by segments In the alignment œx is any amino acid and œ represents positively chargedamino acid Eea1 sequence is used as FYVE domain reference For all alignment amino acids are colored according to their properties Cyan for hydrophobicpositions A V I L M turquoise for aromatic positions F Y W H red for basic residues K R purple for acidic residues D E green for polar uncharged N Q ST salmon for cysteine C orange for glycine G and yellow for proline P Gray numbers below alignment means the amino acids position after alignment Blacknumbers surrounding the alignments represent the start left and end right positions of the domains in the peptide sequence of each protein Alignment wererealized with Seaviewer analyzer software Gouy Accession numbers for protein are following human Rpip8 NP_0011382971 mouse Rpip8NP_0580391 human Eea1 NP_0035573 mouse Eea1 NP_0010019321 human RUFY1 NP_0794343 mouse RUFY1 NP_7661451 human RUFY2NP_0604574 mouse RUFY2 NP_0817012 human RUFY3 NP_0557761 mouse RUFY3 NP_0818061 human RUFY3XL NP_0010325191 mouseRUFY3XL NP_0012767031 human RUFY4 NP_9408852 mouse RUFY4 NP_0011641121 human FYCO1 NP_0787892 mouse FYCO1NP_0011037232Yoshida their interaction with motor proteins likekinesin or myosin suggests a role for RUN domains in regulatingvesicular and anelle transport Callebaut Yoshida Via these diï¬erent mechanisms RUN proteins havebeen implicated in neuronal development Honda 2017bsignaling Sun migration Yoshida and regulation of various cellular function like endocytosis orexocytosis Kitagishi and Matsuda FYVE DomainsFYVEdomainbearing proteins for Fab1 YOTBZK63212Vac1 and EEA1 are specifically found in association withmembranous anelles enriched in PtdIns3P and highlyconserved among eukaryotes including yeast Hayakawa Figure FYVE domains adopt a zinc finger conformationMisra and Hurley Kutateladze and Overduin Inaddition to FYVE ten types of zinc finger folds have beenFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingFIGURE RUFY proteins are important for intracellular trafficking signaling and cytoskeleton dynamics A Schematic representation of the RUN and FYVEdomains activity of RUFY proteins RUN domains act on signaling endosomal protein trafficking and cytoskeletal network dynamics via small GTPase proteins FYVEdomains bind PtdIns3P and regulates autophagy and endosome trafficking B Function of RUFY proteins in homeostatic conditions C Consequences ofalterations in RUFY proteins functions at the cellular and anismal levelcharacterizedincluding conventional Gal4 GATA1 TFIISMetRS LIM RING domain PKC CRD and PHD domainsZinc fingers are structural conformations adopted by peptidechains upon coordination of two Zn2 cations within a cysteinerich region Schwabe and Klug Stenmark Unlike most molecules bearing zinc fingers FYVE proteinsdisplay only one copy of the domain located at any positionalong the peptide chain highlighting its autonomy as a structuralunit FYVE zinc fingers can stabilize proteinprotein or proteinDNARNA interactions Dunkelberg and GutierrezHartmann A œclassical FYVE domain has eight potential zinccoordinating tandem cysteine positions and is characterized byhaving basic amino acids around the cysteines Many membersof this family also include two histidine residues in a sequencemotif including WxxD CxxC RHHCxCG and RVC whereœx means any amino acid and œ a positively charged aminoFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and Traffickingacid Figure 3B Most deviations from this sequence canreduce the domain affinity for zinc and destabilize it Stenmark Misra and Hurley Stenmark and Aasland Kutateladze and Overduin Within this structuralframework specific modifications in the nonconserved residuesof the domain can radically aï¬ect FYVE protein subcellularlocalization and function by forming a œturret loop and adimerization interface Hayakawa With regard to their affinity for PtdIns3P FYVE domaincontaining proteins are mostly found associated to EE orphagosomes Stenmark Gaullier Stenmark and Aasland Figure 4A The presence ofFYVE domains is therefore correlated to the regulation ofmembrane traffic through specific recognition of PtdIns3Pdomains by œRHHCxCG motifs Gaullier and modulation by associated phosphatidylinositol kinasesPtdIns3P is generated from phosphatidylinositol by Class IIIPtdIns 3kinases PI3Klike Vps34 on target membranessuch as nascent autophagosome omegasomes Melia or EE Di Paolo and De Camilli Raib Scott B In turn accumulation ofPtdIns3P recruits and activates eï¬ector proteins containingFYVE domains favoring transport or fusion of target anellesStenmark and Gillooly Axe Burman andKtistakis Schink Affinity for PtdIns3P isdetermined by the pair of histidine residues present in theœRHHCXCG motif of the FYVE domain Stahelin Diraviyam Lee He This affinity can also be harnessed by FYVE proteins to linkendosomes with mRNA ribonucleoprotein ps mRNPand associated ribosomes playing a role in their longdistancetransport in the cell Pohlmann Importantlymany FYVE proteins homodimerize Dimerization multiplies theconserved residues displayed by the diï¬erent signature motifspresent in the FYVE domain and contributes to a network ofhydrogen bonding and electrostatic interactions that providespositive selection for binding several PtdIns3P head groupsPHdependent insertion of FYVE domain into cell membranesHe Pankiv is reinforced by additionalhydrophobic membrane interactions with the turret loop andortandem lysine residues These nonspecific interactions promoteFYVE domain access to phosphate head groupsthat arehindered by the close packing of lipid molecules This bivalentmechanism increases therefore greatly FYVE domains specificityfor PtdIns3Penriched domains and discrimination againstother mono or polyphosphorylated PtdIns species Misra andHurley Stenmark and Aasland Dumas Kutateladze and Overduin FYVE proteins are therefore key players in endocytosis andautophagy and mutations in FYVE domains can alter profoundlythese functions as well as cellular homeostasis Kamentseva For example EEA1 protein early endosome antigen isknown to be crucial for endosome dynamics and any mutation inits conserved residues or the oligomerization site can drasticallyreduce the affinity between its FYVE domain and PtdIns3PStenmark Gaullier In this contextRUFYs proteins by bearing a Nterminal RUN domain oneor several copies of a coiledcoil domain next to a CterminalFYVE domain A have all the features required tocarryout specific adaptor functions to regulate endocytosis orautophagy by impacting on anelle fusion and mobility alongthe cytoskeletonThe RUFY Proteins FamilyThe RUFY family encompass four genes named rufy1 to sharing homologies and displaying specific tissue expressionand alternative splicing Rufy genes are relatively conservedgenes absent from prokaryotes and fungi Upon evolution theemergence of the common ancestor appeared in vertebrates andarthropods which possess one ortholog CG31064 Figure No RUFY protein could be detected In Caenorhabditis elegansand only a FYVEbearing protein T10G35 considered as anortholog of human EEA1 shows some sequence similaritieswith the RUFY family T10G35 exhibits PtdIns3P bindingactivity and is involved in endocytosis being mostly expressedin epidermis and intestine of C elegans Hayakawa In chordates Rubicon RUN domain and cysteinerichdomain containing Beclin 1interacting protein and FYVE AndCoiledCoil Domain Autophagy Adaptor FYCO1 displaystructural and functional features potentially categorizing themas RUFY proteins Rubicon was identified as a componentthe Class III PI3K complex and a negative regulatorofof autophagy and endosomaltrafficking Matsunaga Zhong Like RUFYs Rubicon containsmultiple functional domains that interact with other proteinsincluding a RUN a CC and a FYVElike domains Wong However despite these similaritiesthe poordegree of sequence homology and the lack of conservationof its FYVElike domain which was found not to bind toPI3P Burman and Ktistakis prevented Rubicon™sintegration within the RUFY proteins family conversely toFYCO1 which we propose here to name RUFY5 and detail thecharacteristics belowRUFY1RUFY1 previously named Rabip4 is an kDa proteinmainly expressed in the brain kidneylung placenta andtestis There are two RUFY1 isoforms Rabip4 and Rabip4™that has an additional amino acid upstream oftheNterminal RUN domain A They were both shownto interact with the small endosomal GTPases Rab4 Rab5and Rab14 Fouraux Vukmirica Table RUFY1 inactivation inhibits efficient recycling ofendocytosed transferrin implicating RUFY1 in the regulationof EE functions through cooperative interactions with Rab4and Rab14 Cormont Yamamoto Nag This was further demonstrated by the alteration ofepidermal growth factor receptor endocytic trafficking kineticsin cells depleted of RUFY1 Gosney and thehijacking of RUFY1 by the bacteria P gingivalis to escapelysosomal degradation Takeuchi In melanocytesRUFY1 was found to form a complex with rabenosyn5Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingTABLE Summary of RUFY proteins functional interactionsProtein AliasesBinding partnerFunctionsRUFY1Rabip4 Rabip4™ZFYVE12RUFY2LZFYVE Rabip4rKIAA1537 FYVE13RUFY3Singar1 RIPXZFYVE30 KIAA087RUFY4ZFYVE31FYCO1RUFY5ZFYVE7 RUFY3CTRCT18 CATC2Rab4EtkRab14AP3Rabenosyn5KIF3ABRab4AAP3 complexPODXL1Rab33ARab4ARab6ARETRap2FascinRab5Rab33AGPM6aRap2STEFYial2complexPAK1FOXK1HOXD9Rab7Recycling endosomal traffickingRegulation of endocytosis through its interaction with RUFY1RUFY1™s recruitment endosome tethering and fusionRegulates spatial distribution of lysosomeSorting endosome pathway in endosomal membrane inmelanocytes and segregates tyrosinaserelated protein1Increases cell proliferation migration and invasionEndosome dynamic Golgi complexassociated Rab33 andautophagosome formation on omegasomesLead to a fusion of the RET tyrosine kinase domain to a RUNdomain and a coiledcoil domain appear to be critical fortumorigenesisControl neuronal polarityControl the growth of axons and neuronal growth coneActs on endosomal traffickingFacilitates cell polarityInduce cell migration and invasion in gastric cancerIncreases cells migration RUFY3mediated with metastasis invasionin colorectal cancerHOXD9 transactivate RUFY3 and it overexpression induce gastriccancer progression proliferation and lung metastasisAutophagosome formation and lysosome clusteringMAP1LC3ABAutophagosome formation and elongationRab7Kinesin1Endosomal transport by acting with microtubule plus enddirectiontransportAllows translocation from the late endosome lysosome andautophagosome to the plasma membrane through plusendmicrotubule transportStudyCormont Yang Yamamoto Ivan Nag Zhi Fukuda Kitagishiand Matsuda Staubitz JanoueixLerosey Wei Yoshida Fukuda Honda 2017aWang Xie 2017aZhu Terawaki Cheng Olsvik Wang KrauŸ and Haucke Raib KIF3AB Rab4A and adaptor protein3 AP3 to diï¬erentiallyregulate tyrosinaserelated protein1 and tyrosinase sorting inendosomes contributing to melanosome maturation Nag Table Moreover silencing the Rabip4™isoform ofRUFY1 was shown to promote outgrowth of plasma membraneprotrusions and to regulate the spatial distribution of lysosomesat their tips through an interaction with AP3 Ivan Figures 1B 4B RUFY1 is also capable of controllingcell migration by regulating integrin trafficking Vukmirica presumably via endocytosis In full agreementwith a role of RUFY1 in regulating endosomal dynamics asingle nucleotide polymorphism S705A in the rufy1 gene wasassociated with high blood glucose levels and type diabetesmellitus susceptibility in an exomewide association studyEWAS Yamada This result is consistent with theearly finding that Rabip4 expression leads to Glucose transporter Glut1intracellular retention Cormont Interestingly RUFY1 display a SH3binding motif œPxxPxPembedded in the FYVE domain and is able to interactingwith the epithelial and endothelialtyrosine kinase ETKand possibly regulates endocytosis through this interactionYang Another EWAS aiming to find earlyonsetAlzheimer™s Disease AD susceptibility genes identified RUFY1among genes involved in endolysosomal transport and knownto be important for the development of AD Kunkle Figure 4CRUFY2lungRUFY2 or Leucine zipper FYVEfinger protein LZFYVE is a kDa protein originally identified as an activating transcriptionfactor2 interactor embryogenesis Dunkelberg and GutierrezHartmann preferentially located in the nucleus andexpressed during After development RUFY2 expression remainshigh in the brainliver and the gastrointestinal tractYang RUFY2 displays two Nterminalleucinezipper domains as well as a Cterminal FYVEfinger domainAlthough it is likely to have a nuclear function at early stagesof embryonic development the presence of a FYVE domainsuggests a cytoplasmic role for RUFY2 in regulating membranetraffic in fully diï¬erentiated cells Importantly the RUN domainof RUFY2 was shown to associate specifically with the Golgicomplexassociated Rab33A Fukuda Table GivenFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and Traffickingthe reported interaction of Rab33A and Rab33B with Atg16Land its putative role in regulating autophagy Fukuda and Itoh RUFY2 could contribute to autophagosome formationthrough a dual interaction with Rab33A and PtdIns3P onomegasomes Figures 1B 4B Irrespective of its function rufy2expression is subject to modulation by the micro RNA miR155BofillDe Ros which is an important regulator ofimmune cells development and inflammatory responses Ceppi The rufy2 gene is also frequently found mutatedin cancer cells with the most frequent mutations convertingit into a strong target for nonsense mediated mRNA decaythereby decreasing considerably its expression Shin Figure 4CRUFY3RUFY3 also known as Rap2interacting protein X RIPXKukimotoNiino or Single AxonRelated Singar1Mori is the best characterized member of theRUFY family RUFY3 the smallest of the RUFY proteins witha molecular weight of kDa A is mostly expressedin neurons Kitagishi and Matsuda Neuronal RUFY3 isatypical since it lacks a FYVE domain and is considered as partof the RUFY family based on strong sequence similarities withthe other members notably in the RUN and coiledcoil domainsFigure 2A RUFY3 is distributed between the cytosol and at theplasma membrane but not in intracellular vesicles presumablybecause it lacks a FYVE domain In artificial conditions likefollowing expression of the dominant gain of function mutantform of Rab5 Q79L in U937 cells RUFY3 was found associatedin large vesicle structures and to coimmunoprecipitate withRab5 via an interaction with its carboxyl terminal domain andsurprisingly not its RUN domain Yoshida LikeRUFY2 RUFY3 was also shown in a 2hybrid screen and bycoimmunoprecipitation to bind Rab33 through its coiledcoildomain CC1 Fukuda In 293T and 3Y1 celllines however RUFY3 was shown not to interact with severalsmall GTPasesincluding Rab2 Rab5 Rab7 Rho and RasThis suggests that either RUFY3 requires cell specific partnerproteins or posttranslation modifications to be able to bind tosmall GTPases RUFY3 was first described as interacting withRap2 a small Raslike GTPase via a residue fragment “ located in the RUN domain JanoueixLerosey KukimotoNiino Table Together with Rap1 Rap2interacts with Ras eï¬ectors such as Raf PI3K and Ral guaninenucleotide dissociation stimulator inhibiting activation of theirdownstream targets and thus suppressing Ras oncogenic activityKukimotoNiino Nussinov In the adultnervous system Rap1 and Rap2 also regulate the maturationand plasticity of dendritic spine and synapses By forminga complex together with Rap2 and Fascin RUFY3 interactswith the filamentous actin network and controls the growth ofaxons and neuronal growth cone Wei Table Recent mechanistic studies indicate that RUFY3 accumulates inlipid rafts by forming a Glycoprotein M6A GPM6aRUFY3Rap2STEFYial2 complex Honda 2017a Table This complex activates the Rac guanine nucleotide exchangefactor Honda 2017b impacting actin anization andpromoting neuronal polarity and growth Figure 4B RUFY3seems therefore to have diï¬erent axogenic functions in brainMori Honda 2017b and not surprisingly rolesfor RUFY3 in amyotrophic lateral sclerosis Arosio major depressive disorder Aberg and AD Zelaya have been reported Olfactory dysfunction occurs in of AD cases and is correlated with elevated rufy3 expressionin glomerular and mitral layers of the olfactory bulb Zelaya RUFY3 is cleaved by caspase and critically required forcaspasemediated degeneration of tropomyosin receptor kinaseA positive sensory axons in vitro and in vivo Hertz Figure 4C Removal of neuronally enriched RUFY3 is able toblock caspase 3dependent apoptosis while dephosphorylation ofRUFY3 at residue S34 appears required for its degradation Hertz Analysis of rufy3deficient mice supports a seconddistinct function for RUFY3 in neuronal growth and polaritysince mutant embryos show defects in axonal projection patternsThese occur in addition to the prevention of CASP3dependentapoptosis in dorsal root ganglions RUFY3 appears therefore to bekey for nervous system development remodeling and functionexplaining the embryonic lethality displayed upon rufy3 geneticinactivation in mouse Hertz With the current advance in genomics and single cell RNAsequencing specific gene expression patterns can be revised andmore accurately defined Analysis of several genomic databasesBioGPS NCBI Human Atlas Protein ImmGen Ensembl revealthat in addition to neurons RUFY3 expression can be detectedin other tissues and cell types Moreover the rufy3 gene appearsto have many transcriptional variants leading to the expressionof diï¬erent protein isoforms Two of these isoforms display aCterminal region extended by amino

Thyroid_Cancer

"Ovine pulmonary adenocarcinoma OPA is a neoplastic disease caused by exogenous Jaagsiekte SheepRetrovirus exJSRV The prevalence of JSRVrelated OPA in Eastern European countries including Romania is unknownWe aimed to investigate the prevalence and morphological features of OPA classical and atypical forms in theTransylvania region Romania the immunophenotype of the pulmonary tumors and their relationships with exJSRVinfection A total of adult ewes slaughtered between and in two private slaughterhouses fromTransylvania region Romania was evaluated Lung tumors were subsequently assessed by cytology histologyimmunocytochemistry immunohistochemistry electron microscopy and DNA testingContinued on next page Correspondence mariantaulescuusamvclujro1Department of Veterinary Pathology University of Agricultural Sciences andVeterinary Medicine Calea Manastur ClujNapoca Romania2Laboratory of Genomics Biodiversity Animal Breeding and MolecularPathology Institute of Life Sciences University of Agricultural Sciences andVeterinary Medicine ClujNapoca RomaniaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cToma BMC Veterinary Research Page of Continued from previous pageResults Out of examined sheep had OPA prevalence The diaphragmatic lobes were the most affectedGrossly the classical OPA was identified in of investigated cases and the atypical OPA in that includedsolitary myxomatous nodules Histopathology results confirmed the presence of OPA in all suspected cases which wereclassified into acinar and papillary types Myxoid growths MGs were diagnosed in classical OPA cases and in cases ofatypical form Lung adenocarcinoma was positive for MCK and TTF1 and MGs showed immunoreaction for VimentinDesmin and SMA Ki67 expression of classical OPA was higher than atypical OPA and MGs JSRVMA was identified by IHC in both epithelial and mesenchymal cells of OPA Immunocytochemistry and electron microscopy alsoconfirmed the JSRV within the neoplastic cells ExJSRV was identified by PCR in of analyzed samples Phylogeneticanalysis revealed the presence of the exJSRV type MT8096781 in Romanian sheep affected by lung cancer andshowed a high similarity with the UK strain AF1052201Conclusions In this study we confirmed for the first time in Romania the presence of exJSRV in naturally occurring OPAin sheep Additionally we described the first report of atypical OPA in Romania and to the best of our knowledge inEastern Europe Finally we showed that MGs have a myofibroblastic originKeywords Atypical OPA Epidemiology Jaagsiekte sheep retrovirus type Myxoid growths Ovine pulmonaryadenocarcinomaincluding JSRV []BackgroundOvine pulmonary adenocarcinoma OPA represents avirusrelated neoplastic disease caused by an exogenousbetaretrovirus [] The disease was first reported inSouth Africa in the late 1800s as an important cause ofchronic respiratory distress in sheep [] The etiologicalagent of OPA is exogenous Jaagsiekte Sheep RetrovirusexJSRV A specific U3 long terminal repeatLTRsequence of exJSRV was detected in lungs from affectedanimals [ ] The LTR region varies among differentretrovirusessheep endogenousretrovirus [] and ovine enzootic nasal tumour virus []Based on the U3 sequence and restriction profiles of thevirus some authors suggest that there are two types ofexogenous retroviral sequencesKenyan andSouth African and type II Wyoming USA and UK isolates [ ] ExJSRV has a specific tropism for the differentiated epithelial cellstype II pneumocytes andnonciliated bronchiolar Clara cells of the lung and it isthe only virus known to cause pulmonary adenocarcinoma in naturally infected animals [] ExJSRV is mainlytransmitted by infected aerosols [ ] and in naturalcases the incubation period is very long ranging from to years Therefore it is most often encountered inadult sheep [] but the lambs can also manifest clinicalsigns [] There is no evidence for breed or sex relatedOPA susceptibility [] Other animal species includinggoats and moufflons were occasionally diagnosed withOPA [ ]type IOPA shares many histological similarities with the human pulmonary adenocarcinoma representing an important animal model for understanding the mechanisms ofviral oncogenesis [] Although JSRV was found in humanpulmonary neoplastic cells its role in the development oflung cancer in humans is not fully elucidated []There are two recognized forms of OPA which showgross histological and immunohistochemical differences[ ] The lesions in classical OPA predominantlyaffect all pulmonary lobes and are located in the cranioventral area They can be either nodular or exhibit a diffuse growth type showing a grey moist appearance on thecross section Atypical OPA consists of hard nodulespearlywhite that have a dry cut surface the tumors arewelldelimited by the surrounding pulmonary tissue []such asThe classical form is more common than the atypicaltype In Europe the classical OPA has been reported inseveral countriesIreland [] UK []Scotland [] Italy [] Germany [] Spain [] Theincidence of the disease is usually low but in some geographical areas it reaches up to [] More than of the affected animals usually die because of progressive respiratory failure [] therefore causing importanteconomic losses The atypical OPA is less contagious thanclassical form [] The atypical OPA has been reported inSpain Peru Iran and India [ ] but there is noevidence of its occurrence in other countries where OPAis commonly foundClinically the affected sheep develop chronic and progressive respiratory distress especially when exercised Acommon sign of classical OPA is mucous nasal discharge because of production of large fluids amounts inthe lung [] This fluid might be absent in some casesparticularly in the atypical form of OPA where the tumors remain incidentally found in abattoir studies orwhen the animals are necropsied for other reasons []Currently there are no efficient methods to clinicaldiagnose the disease and a full diagnosis can be obtainedonly postmortem by histological examination [] Polymerase chain reaction PCR analysis of bronchoalveolarlavage BAL samples collected from living animals may 0cToma BMC Veterinary Research Page of be useful for preclinical identification of infected individuals with exJSRV However this technique is not ableto identify all the early stages of classical OPA and atypicalform where the mucus production is absent orfewer infected cells are present in the BAL sample [] Currently this method is not extensively used because the sample collection requires sedation Furthermore intravitam diagnosis of exJSRV infection by PCRfrom blood offers many false negative results probablybecause the numbers of infected white blood cells is verylow [ ] Additionally the viral genetic material orproteins can also be detected by PCR and immunohistochemistry IHC respectively in pulmonary and lymphoid tissues [ ]Romania is an important European country in sheepfarming with over million heads reported in according to Romanian Minister of Agriculture There areonly two studies regarding OPA presence in Romaniawith reported incidences of and [ ] However no evidence of exJSRV infection in relation withOPA nor atypical OPA had been previously describedCurrently no information about the prevalence of classical OPA in Romania is available Furthermore to theauthor’s knowledge exJSRVrelated OPA has not beenreported so far in other countries from Eastern Europein the last decadeIn this study we aimed to investigate the prevalenceand morphological features of OPA classical and atypicalforms in Turcana sheep breed in the Transylvania regionRomania immunohistochemical features of the neoplastic epithelial components and myxoid growths MGsand identification of exJSRV by electron microscopyimmunocytochemistry IHC and PCR methods A comparison between nucleotide sequences of exJSRV identified in sheep lung tumors and other reported exJSRVstrains from different geographical regions was also previewed in order to identify the strain present in RomaniaResultsPrevalence distribution patterns and gross features ofOPAOut of examined slaughtered ewes cases weresuspected of OPA after gross examination Howeverhistological examination confirmed the presence of OPAin cases therefore a prevalence of of the disease in Transylvania Romania Data of sheep includedin the study and confirmation of JSRV infection aresummarized in Table In the remaining cases n the diagnoses ofchronic suppurative bacterial bronchopneumonia andverminous bronchopneumonia associated with extensivefibrosis and atelectasis were histologically differentiatedfrom OPA All diagnosed sheep belonged to Turcanabreed and were adult females the age ranging from to yearsOn postmortem examination the distribution patterns of pulmonary lesions were divided into three majroups Fig 1a Group I in cases the lesions were focal or solitary multifocal to coalescing anddiffuse large masses affecting a single pulmonary lobewith higher prevalence in the left diaphragmatic lobe cases Fig 1b Group II in cases the lesionswere located unilateral but affecting or more pulmonary lobes Fig 1c Group IIIin the other cases neoplastic processes were bilaterally locatedmainly affecting the diaphragmatic lobes and involvingup to of the pulmonary parenchyma Fig 1d Inthese severe cases the lungs failed to collapse and theywere heavy and denseGrossly the morphological features of pulmonary lesions were classified according to GarciaGoti []into two main pathologic forms classical OPA and atypical OPA The classical OPA was identified in cases and consisted of pink to light grey or whitesolitary nodules of varying size cm or large denseconfluent masses reaching up to cm in diameter Insome cases the neoplastic masses were delimited fromthe normal parenchyma by a fine line of atelectasia or bya zone of emphysema On the cut surface the tumorsincluded in this form were moist had a homogenous appearance and the airways usually contained a viscousfrothy fluid Fig 1d In two cases large areas of lytic necrosis and cavitation were randomly distributed withinthe neoplastic tissue In most cases multifocal to diffusepleural fibrosis over the neoplastic masses was a common gross findingThe atypical OPA was encountered in four cases and characterized by white dried nodular orconfluent lesions of approximately – cm in diametermainly located in the subpleural area of the left diaphragmatic lobe Fig 1e and e1 No or small amount of lungfluid was detected in these cases In two of the cases thepulmonary lesions consisted of whitegrey solitary welldelimited and unencapsulated nodules Fig 1f on the cutsurface these nodules showed a multilobular soft to denseand gelatinous appearance and consisted with myxomalike masses Fig 1gCytological and histopathological featuresCytological examination of both classical and atypicalforms of OPA revealed numerous well differentiated cuboidal or polygonal neoplastic epithelial cells arranged insmall acini clusters or individually Fig 2a The neoplastic cells had a moderate amount of pale blue finelygranular cytoplasm moderate nuclear cytoplasmic NC ratio and round to oval centrally located nuclei withfinely stippled chromatin and – distinct blue nucleoli 0cToma BMC Veterinary Research Page of Table Data of sheep included in the study and confirmation of JSRV infectionCase samples115MAY17Pathologic formClassicalGenderageFHistological typePapillaryBreedTurcanaIHCJRSVPCRJSRVICCNPTEMNP225MAY17356JUN17433DEC17534DEC17636DEC17702JAN18803JAN189012JAN18101JAN18112JAN18125JAN181310JAN18143JUL18155JUL18166JUL18177JUL181802AUG181903AUG18201AUG18212AUG18223AUG18235AUG18246AUG18251OCT18262OCT18273OCT18284OCT18295OCT18306OCT18317OCT18328OCT18333MAY19TurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFClassicalAtypicalClassicalClassicalClassicalAtypicalClassicalClassicalAtypicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalAtypicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassical344MAY19Age adult sheep – years F female MGs myxoid growths NP not performedClassicalTurcanaFPapillaryMGsAcinarAcinarAcinarAcinarAcinarAcinarAcinarPapillaryPapillaryAcinarPapillaryAcinar MGsPapillaryAcinarPapillaryPapillary MGsAcinar MGsPapillaryAcinarAcinarMGsAcinarPapillary MGsAcinarAcinarAcinarAcinar MGsAcinarAcinarPapillary MGsAcinar–––NPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNP––NPNP–NPAnisocytosis and anisokaryosis were mild to moderatewith rare mitotic figures Large numbers of macrophagesand mature lymphocytes were admixed with the neoplastic cells OPA containing MGs were poorly cellular and composed of spindle stellate and elongatedcells with round to oval nuclei and inserted in apale blue slightly vacuolar extracellular myxoid material Alltumors diagnosed by cytological examwere subsequently confirmed by histopathology andimmunohistochemistryHistologically all pulmonary tumors classified as classical form of OPA n were composed of a singlelayer of neoplastic cuboidal columnar or polyhedral epithelial cells lining the alveolar lumina or bronchiolessurrounded by a fine to moderate fibrovascular stromaThe predominated histologicaltypes of pulmonaryadenocarcinoma were represented by acinar n Fig 2b and papillary n Fig 2c The diameter ofindividual neoplastic epithelial cells ranged from to μm showed moderate amount of pale acidophilic 0cToma BMC Veterinary Research Page of Fig Gross features of spontaneous OPA in sheep a Distribution of pulmonary lesions b Classical form of OPA involving the diaphragmaticlobe arrow c Classical form of OPA affecting multiple pulmonary lobes arrows d Classical form of OPA on cut surface showing the junctioninterrupted line between the tumor and normal tissue e and e1 Atypical form of OPA showing a subpleural whitegrey nodule arrow On cutsurface the tumor is pearly white and dried arrow f Gross aspects of pulmonary myxomalike nodule arrow g the myxomatous mass shows amultilobular whitegray gelatinous feature on cut surfacefinely granular cytoplasm and intermediate NC ratioAnisokaryosis and anisocytosis were low to moderatethe nuclei were round to oval centrally located with afine granular to lacy chromatin with and – distinctbasophilic nucleoli The average of mitotic rate was per HPF without atypical features Multifocal thestroma was heavily infiltrated with macrophages lymphocytes and few plasma cells The adjacent parenchymaof the neoplastic mass showed atelectasis and at the periphery of the tumors the neoplastic cells were occasionally arranged in a lepidic growth pattern The myxoidgrowths consisting of sparsely cellular structures of spindles cells embedded in an abundant extracellular matrixwere observed in cases of classical OPA The MGswere admixed with the neoplastic epithelial componentor in some areas were disposed as multiple poorly demarcated masses Fig 2d eThe histological features of the atypical form n were similar to those described in classical form andtwo cases were classified as acinar type The distinguishof atypical OPA features were represented by moreprominent limits between normal tissue and neoplasticmasses due to higher fibroblast proliferation and inflammatory infiltrates predominated by mature lymphocytesand macrophages Fig 2fIn the other two cases of pulmonary solitary nodulesthe neoplastic epithelial component was absent and theneoplasm was composed by short bundles and streamsof spindle stellate or individual cells Fig 2g The mesenchymal cells were embedded into an abundant myxoidmatrix that contains moderate amounts offoamyamphophilic ABPAS positive material mucin Fig 2hand i The individual cells had variable distinct bordersintermediate NC ratio and moderate amounts of paleacidophilic homogenous cytoplasm The nuclei wereoval to elongated – μm in diameter in transversesection centrally located with clumped chromatin andindistinct nucleoli No mitotic figures were present anisocytosis and anisokaryosis were low Based on thehistological features a presumptive diagnosis of pulmonary myxomas was madeOverall mesenchymal proliferations and myxomatouschanges named as myxoid growths MGs were identified in out of tumors cases of classical 0cToma BMC Veterinary Research Page of Fig Microscopical findings of OPA a Cytological exam of pulmonary tumors showing multiple nest of cubical to polygonal epithelial cellsinterpreted as type II alveolar cells DQ stain b Histological appearance of OPAclassical form tubular type and c papillary type HE stain d and eThe microphotographs of classical OPA showing myxoid growths white arrow interspersed with neoplastic epithelial component blue arrowHE stain f Histological exam of atypical OPA showing a welldelimited neoplastic nodule surrounded by fibrous tissue and infilammatoryinfiltrates arrow HE stain g Myxomalike tumor without neoplastic epithelial component The mass is multinodular white arrow andcomposed of spindle to stellates cells and abundant extracellular matrix the inset HE stain h and i ABPAS stained sections from myxomalikenodules showing abundant pale blue extracellular myxomatous matrix the Goblet cells of bronchial lining epithelium are used as positivecontrol for ABPAS stainform and cases of atypical form consistent with pulmonary nodules without epithelial cell neoplasia myxomalike nodulesImmunohistochemical features of pulmonary massesImmunohistochemically in both forms of OPA the neoplastic epithelial component showed a strong and diffusereaction for MCK Fig 3a and TTF1 Fig 3b the epithelial cells were negative for vimentin Fig 3c Thecells of MGs were diffusely and intense positive forvimentin Fig 3d desmin Fig 3e selective positive foralphaSMA Fig 3f and negative for MCK Fig 3g suggesting their true mesenchymal origin probably frompulmonary interstitial myofibroblasts All mesenchymalcomponents were also negative for S100 protein Fig3h and TTF1 The proliferative index ki67 of theepithelial component of classical OPA was higher meanvalue of Fig 3i than atypical OPA mean valueof whereas in the MGs this labelling was low inall forms mean value of classical form and in atypical OPA consisting of myxomalike noduleswithout neoplastic epithelial component Fig 3jstainingJSRV identification methodsImmunocytochemistry and immunohistochemistryImmunocytochemicalsmearsimprintsshowed a positive reaction consistent with the presenceof intracytoplasmic JSRV antigen within the neoplasticcells Fig 4a and a1 No immunocytochemical expression of JSRVMA was detected in the imprints smearsfrom normal lungsof 0cToma BMC Veterinary Research Page of Fig Immunophenotypical characterization of OPA a The neoplastic epithelial cells of classical form are diffusely and intensely immunopositivefor MCK b TTF1positive nuclei of the epithelial cells are present within the neoplastic masses Inset the bronchial epithelium was used aspositive control for TTF1 c All epithelial cells lining neoplastic acini are negative for vimentin blue arrow in contrast to immunopositive stromalcells white arrow d Myxoid growths showing an intense immunopositive reaction for Vimentin Inset detail of diffuse cytoplasmic labeling forVimentin e Mesenchymal cells of myxoid growths have diffuse and strong cytoplasmic labeling for Desmin Inset The smooth muscle cells ofpulmonary arteries and bronchioles are strongly positive for desmin and served as internal positive control f The cells of myxoid growths haveselective and moderate cytoplasmic labeling for alphaSMA Inset The bronchiolar smooth muscle cells are positive for SMA internal positivecontrol g The myxoid component of OPA showing a negative immunoreaction of MCK blue arrow compared to the neoplastic epithelial cellswhich are strongly immunopositive h Neoplastic cells of myxomalike nodules showing a negative reaction for S100 protein Inset the bronchialcartilage is diffusely positive for S100 protein internal positive control i The proliferative index characterized by ki67 immunopositive nuclei washigher in the epithelial neoplasia compared to the MGs and myxomalike nodules blue arrows j Inset Bronchialassociated lymphoid tissueBALT hyperplasia was used as internal positive control for ki67 DAB and hematoxylin counterstainIn both classical and atypical OPAimmunohistochemical evaluation of pulmonary tumors showed a diffuse positive JSRVMA reaction of neoplastic cells in cases The positive reaction was characterizedby a finely granular brown staining of the cellular cytoplasm of both neoplastic epithelial component Fig 4band MGs Fig 4c Additionally a positive reaction wasalso observed for the inflammatory cells mainly macrophages and lymphocytes Moreover the intensity andnumber of JSRVpositive cells in atypical tumours werereduced compared to the classicalform of OPA Anegative JSRVMA reaction was found in all normal pulmonary tissues n selected as negative controlTransmission electron microscopy TEMUltrastucturally the neoplastic type II pneumocytes havebeen recognized by the presence of microvilli basal orcentrally located nuclei numerousintracytoplasmicmoderately electronodense round and lamellar structures of approximately – nm in diameter interpreted as lamellar bodies Fig 4d Additionally roundoval structures of – μm in diameter delimited by a 0cToma BMC Veterinary Research Page of Fig JSRV detection from neoplastic tissues a and a1 Immunocytochemistry showing intracytoplasmic JSRVMA expression within the neoplastic epithelialcells DAB and hematoxylin counterstain B Diffuse expression of the JSRVMA marker by neoplastic epithelial cells forming tubular structures c Expression ofthe JSRVMA marker by MGs and epithelial tumor Left inset Cell cytoplasm of MGs white arrow is strongly labelled with JSRVMA antibody Right insetNeoplastic epithelial cells have diffuse cytoplasmic labelling for JSRVMA bue arrow DAB and hematoxylin counterstain d JSRVinfected type pneumocytes Note the presence of intracytoplasmic lamellar body white arrow and aggregates and solitary intracytoplasmic viral ps grey arrowTEM Bar μm Inset Detail of a viral replication site showing specific virions compatible with JSRV white arrow TEM Bar nm e Electrophoresis profile ofthe amplified DNA fragments bp evidencing the presence of proviral Jaagsiekte DNA in ovine pulmonary adenocarcinoma OPA L DNA ladder PCRpositive samples PCR negative − samples M1 and M2 control samples amplified from healthy ovine lung tissue NTC no template control negativecontrol f Phylogenetic analysis of different JSRV strains and other retroviruses based on the LTR region The Romanian strains of exJSRV JSRVRO GenBankMT8096781 showed homology at nucleotide level of with UK strain GenBank AF1052201 and with the South African strain GenBank M80216 0cToma BMC Veterinary Research Page of moderately electrondense membrane and containing anabundant electron lucent material intracytoplasmic vacuoles were also identified within the cytoplasm of typeII alveolar cells JSRV ps were found within theaforementioned intracytoplasmic vacuoles or in the cytosol of the neoplastic cells in out of examined samples The viral ps were arranged into small groups– virions or individually measured about –nm in diameter and showed a moderately electrondense central area Fig 4d Extracellular JSRV pswere not observed in none of the examined examplesPCR DNA sequencing and phylogenyAll pulmonary lesions histologically diagnosed as OPAwere submitted for PCR analysis The expected pbamplicons obtained from exJSRV proviral DNA werevisualized in out of analysed samples while no bands were visible in the negative controlsFig 4e The unaffected tissues adjacent to the neoplastic masses were also negative for the virus presenceBased on the obtained LTR nucleotide sequences ofJSRV deposited in GenBank under accession numberMT8096781 from lung tumours and their comparisonwith other retroviral strains available in GenBank weevidenced a homology at nucleotide level of withexJSRV UK strain GenBank AF1052201 and only with the South African strain GenBank M80216Fig 4f Therefore we concluded that affected animalsare infected with Jaagsiekte Sheep Retrovirus type which is the most probable responsible for the adenocarcinoma found in Romanian Turcana sheepDiscussionsPrimary respiratory tract and lung tumors in animalshave a lower incidence compared with other systems aswell as compared to human patients In contrast thelung is the preferential site of metastases in all animals[] In sheep the most common tumor of the respiratory system is ovine pulmonary adenocarcinoma OPAalso known as jaagsiekte or ovine pulmonary adenomatosis []This work confirms the presence ofJSRVinfectedsheep in Romania To the author’s knowledge JSRVrelated OPA has not been reported in other countries ofEastern Europe in the last decadeFor this study the samples were collected from twoslaughterhouses which are representative for Transylvania not for the whole country Unfortunately due tospecific slaughtering procedures we collected the pulmonary tissue samples only at the end of slaughteringAs a consequence an exact match between examinedlungs and ear tag for each slaughtered sheep was notpossible to be determined Therefore neither the exactorigin and age of each sheep were not possible to beestablished However according to the records of theslaughterhouses all slaughtered individuals were adultfemales and originated from different counties of Transylvaniaincluding Cluj BistritaNasaud Mures Sibiuand Alba Thus the authors can relate that all the positive OPA cases originated from Transylvania a historicalregion that is located in center of Romania The presentstudy shows that the prevalence of OPA is about and is higher compared to the previous reports regarding OPA situation in Romania and respectively[ ] ExJSRV presence was not determined in any ofthese two previous studies Furthermore the prevalenceof OPA in Romania is higher than in other Europeancountries including UK [] and Ireland [] We can think about two possible explanations ofthis prevalence of OPA in Romanian Turcana sheepFirst it can be related with a high number of sheep thatare raised in Romania According to the Ministry ofAgriculture data the number of sheep in was over million heads This places the Romanian sheep industry in the third position in Europe after Spain and UKAnother important causative factor could be related withsheep management in Romania The majority of animalsare raised in free pastures and there is a lot of sheepmovements between regions to find fresh grasslandsThis is a traditional sheep breeding management and itis a way of life for the sheep breeders that is still verypresent in Romania These two factors might explain ahigher OPA prevalence in Romanian sheep comparedwith other European countries In contrast to the prevalence of OPA in Romania the disease is quite commonin South America South Africa and Scotland where – of infected animals develop pulmonary tumors []OPA represents a continuous issue regarding propermethods of diagnosis and prevention [ ] Despitesome specific clinical signs of classical OPA includingdyspnea and tachypnea in combination with progressive weight loss and nasal mucous discharge [] andsuggested imaging modalities radiography computedtomography and ultrasonographic examination [ ]currently the gold standard diagnostic method for bothclassical and atypical OPA relies in gross and histologyexams performed during post mortem evaluation [] Inour study data about clinical signs of slaughtered sheepwith OPA were not available Postmortem evaluation including gross and histological exams of the affectedsheep is essential for confirmation of OPA Althoughgross changes are relatively specific for OPA in somecases chronic bronchopneumonia should be included inthe differential list A recent study showed that numerous cases of suspected OPA based on grosschanges were false positive lesions due to its similaritieswith chronic bronchopneumonia moreover wereexJSRVnegative by RTPCR and IHC In some cases 0cToma BMC Veterinary Research Page of in sheepare usuallyOPA and bronchopneumonia can occur concurrentlyand the pneumonic lesions may obscure the characteristic histological features of OPA [] Lesions of chronicbronchopneumonialocallyextensive dense greydark red in colour affecting thecranioventral region ofthe lung [] In our studychronic suppurative bacterial bronchopneumonia without neoplastic lesions was histologically diagnosed onlyin four cases of suspected OPA characterized by cranioventral consolidation of the pulmonary lobes Our findings showed that the distribution of both classical andatypical forms of OPA was different than those causedby chronic bacterial bronchopneumonia and the diaphragmatic lobe was the most affected part of the pulmonary parenchyma In the other two cases OPA hadconcurrent pulmonary necrosis fibrosis atelectasis andabscesses caused by Corynebacterium pseudotuberculosis and these findings are in agreement with a previousstudy []The histological features of OPA consist of proliferation of epithelial cells into acini papillary and solidstructures supported by a fibrovascular stroma In atypical form in addition to the epithelial neoplasia markedfibroblast proliferation collagen deposition and mononuclear cellinfiltration are characteristic microscopicchanges [] These inflammatory changes may be causedby intercurrent infections or a specific immune responseof the host [] Occasionally small nodules of neoplasticloose mesenchymal tissue appear admixed with the neoplastic component [] Both epithelial and mesenchymalcomponents showed rare mitoses and a ki67 index meanvalue of [] and this is in good agreement withour findings where ki67 index of the epithelial componentof classical OPA was higher mean value of thanatypical OPA mean value of Another subtype of solitary tumor associated withexJSRV in sheep is grossly characterized by round orelongated white–grey nodules of bright gelatinous appearance and sharply demarcated from the surroundingparenchyma [] These nodules are histologically classified as myxoid nodules or myxoid growths or mesenchymal growths MGs [ ] MGs resemble characteristicfeatures of benign mesenchymal tumors and have beendescribed in a variable proportion of tumoursintermingled with the neoplastic epithelial component [] insome cases they were identified in metastases [] TheMGs are not an uncommon feature encountered in OPAbut interestingly they were not reported in experimentalstudies []In the current study out of ovine pulmonarytumors presented MGs accounting of the samples although there are reports of up to of theOPAs that contain also the neoplastic mesenchymalcomponent

Thyroid_Cancer

lack of COVID19 diagnostic tests for the whole Spanish population thecurrent strategy is to identify the disease early to limit contagion in the communityAimTo determine clinical factors of a poor prognosis in patients with COVID19 infectionDesign and settingDescriptive observational retrospective study in three primary healthcare centres with anassigned population of MethodExamination of the medical records of patients with COVID19 infections confirmed by polymerase chain reaction Logistic multivariate regression models adjusted for age and sexwere constructed to analyse independent predictive factors associated with death ICUadmission and hospitalizationResultsWe included patients mean age years female aged � years were health workers doctors nurses auxiliaries Predictors of ICUadmission or death were greater age OR 95CI to male sex OR 95CI to autoimmune disease OR 95CI to bilateral pulmonary infiltrates OR 95CI to elevated lactatedehydrogenase OR 95CI to elevated Ddimer OR 95CI to and elevated Creactive protein OR 95CI to Myalgia or arthralgia OR 95CI to was protective factor against ICU admission andPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsdeath Predictors of hospitalization were chills OR 95CI to feverOR 95CI to dyspnoea OR 95CI to depressionOR 95CI to lymph ia OR 95CI to andelevated Creactive protein OR 95CI to Anosmia OR 95CI to was the only significant protective factor for hospitalization after adjusting forage and sexConclusionDetermining the clinical biological and radiological characteristics of patients with suspected COVID19 infection will be key to early treatment and isolation and the tracing ofcontactsIntroductionOn December the health authorities of Wuhan city Hubei Province China reporteda cluster of cases of pneumonia of unknown aetiology with onset of symptoms on December including severe cases with a common exposure identified in a city market [] whichwas closed on January On January the Chinese authorities identified a newCoronaviridae family virus initially named coronavirus 2019nCoV and later coronavirusSARSCoV2 as the causal agent [] The genetic sequence was shared by the Chinese authorities on January On January the first case was detected in the USA in Washingtonstate [] On January the World Health anization declared the SARSCoV2 outbreak in China a public health emergency of international concern [] Subsequently the outbreak has spread outside China with Europe especially affected []The first positive case diagnosed in Spain was confirmed on January on the islandof La Gomera while the first death occurred on February in Valencia city the date was confirmed twenty days later The first confirmed case in Barcelona was on February and fromthen until June there have been confirmed cases in Spain []The most common signs of infection are respiratory symptoms fever cough and shortnessof breath In more severe cases the infection may cause pneumonia severe acute respiratorysyndrome renal failure and death [] Transmission appears to be mainly persontopersonvia the airway through respiratory droplets measuring microns when the patient has respiratory symptoms cough and sneezing and contact with fomites [] Most estimates of theincubation period of COVID19 range from to days with most around five days Evidenceon the transmission of the virus before symptom onset is unclear There is currently no specifictreatment for COVID19 infections To date the most important scientific efforts have focusedon three areas strategies to contain the spread of the disease the initiation of clinical trialswith antivirals and multiple therapies and the design of a new vaccine which is still unclearThese strategies include some of a community nature where primary healthcare plays a centralrole in disease prevention and control [] Few studies have described the clinical characteristics of the disease fewer the predictive factors and virtually none have described the Mediterranean population compared with the rest of the world Therefore this study aimed todescribe the clinical biological and radiological manifestations the evolution treatments andmortality rate of patients with COVID19 infection in the population of Barcelona city anddetermine the most important predictors of a poor prognosisPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsMaterials and methodsA multicentre observational descriptive study was carried out in three urban primary healthcare centres serving an assigned population of with one reference hospital The studyincluded all consecutive adult patients with COVID19 confirmed by polymerase chain reaction PCR from nasal and pharyngeal samples during the study period of February to April Diagnostic confirmation was made in the hospital laboratories as PCR is not available in primary healthcare centres Signs and symptoms the main available haematologicaland biochemical data and the results of imaging tests were recorded as were comorbiditiesthe evolution the hospitalization rate intensive care unit ICU admission and the treatmentsreceived The study population was divided into four age groups “ years “ years“ years and � years Other variables recorded were the type of followup the need fortemporary work disability and the source of possible contacts The time to first medical visitwas defined as the difference in days between symptom onset and medical visit by a familyphysician The factors that determined a poor prognosis hospitalization ICU admissiondeath were collected The data were obtained from the electronic medical record Missingdata were collected by telephone interviews with patients when possible Patients from nursinghomes were excluded as the rate of infections and mortality has been shown to be muchhigher than in the noninstitutionalized population The study was approved by the EthicsCommittee of the Hospital Clinic of Barcelona registration number HCB20200525 Thestudy was conducted according to the Helsinki Declaration and Spanish legislation on biomedical studies data protection and respect for human rightsStatistical analysisCategorical variables are presented as absolute frequencies and percentages and continuous variables as means and standard deviations SD Predictors of death ICU admission andhospitalization were determined using the student™s t test for continuous variables and the chisquare test for categorical variables Logistic multivariate regression models adjusted for ageand sex were constructed to analyse independent predictive factors associated with death ICUadmission and hospitalization Odds ratios OR and their confidence intervals 95CIobtained in the adjusted regression analysis were calculated Forest plots were used to represent OR and 95CI Values of p005 were considered statistically significant The statisticalanalysis was performed using the R version for WindowsResultsClinical characteristics and comorbiditiesWe included patients mean age years female aged � yearsThe mean time from symptom onset to the medical visit was SD days Clinical characteristics are shown in Table Notably were health workers doctors nurses auxiliaries The most frequentclinical symptoms were cough fever general malaise fatigue myalgia or arthralgia dyspnoea diarrhoea headache anosmia and dysgeusia Physical examination in patients showed had auscultatory alterations tachypnoea and an oxygen saturation of � ICU admission and death were associated with a greater mean age years vs yearsp male sex vs p dyspnoea vs p fever vs p auscultatory alterations vs p and low oxygen saturation vs p Table Myalgia or arthralgia vs PLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Clinical and exploratory factors predicting hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admissionHospitalizationAge”yearsDistribution”no “ years“ years“ years� yearsMale”no Occupation”no n No n YesPAdjusted ORNoYesPAdjusted OR ± ± n ± [“][ CI]n ± n ± [“][ CI] [“] [“]Other type of exposure Health professionalOther health workersSmoking exsmokersmoker”nototal no Temperature at admission” ˚C Patients with fever �˚C”nototal no Time from symptom onset tomedical visit”days¡Symptoms”no ¶ ± ± ± ± ± ± NANANA [“] [“] [“] [“] ± ± ± [“] [“] ± [“] NANANA [“]CoughGeneral malaiseFatigue [“] [“] [“] [“] [“] [“]Myalgia or arthralgia [“] [“]DyspnoeaDiarrhoeaHeadacheAnosmiaDysgeusiaSore throatBlocked noseNausea or vomitingSputum productionChillsAstheniaChest painAlterations in physical examination”nototal no §Auscultatory alterationsTachypnoeaTachycardiaPharyngitis [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] NA [“]Continued PLOS ONE 101371journalpone0237960 August PLOS ONE 0cTable ContinuedVariablesTotalDeath or ICU admissionHospitalizationPrognostic factors in Spanish COVID19 patientsOxygen saturation �”nototalno n n No n YesPAdjusted ORNoYes[ CI]n n [“] PAdjusted OR[ CI] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Temperature distribution was ˚C “ËšC “ËšC and ˚C ¡ In patients™ data on period between symptom onset and medical visit were lacking¶ Symptoms with a frequency of patients were disorientation n conjunctivitis n haemoptysis n and cutaneous lesions n § patients had a physical examination The alterations with a frequency of patients were cutaneous lesions n and tonsillopharyngitis n Ref reference NA not applicable101371journalpone0237960t001p headache vs p dysgeusia vs p andanosmia vs p were less frequent in patients admitted to the ICU or whodied than the remaining patients Age OR 95CI to and male sexOR 95CI to were independent predictors of ICU admission and deathMyalgia or arthralgia OR 95CI to was the only significant protective factor against ICU admission and death after adjusting for age and sex Fig The best clinicalpredictors of hospitalization were chills OR 95CI to fever OR 95CI to and dyspnoea OR 95CI to Anosmia OR 95CI to was the only significant protective factor for hospitalization after adjusting for age and sex Table and Fig Comorbidities were presented by patients the most common were hypertension in diabetes mellitus in and obesity in Table Heartdisease vs p autoimmune disease vs p diabetes vs p hypertension vs p and chronic kidney disease vs p were the comorbidities significantly associated with ICUadmission and death Table Autoimmune disease was the only significant predictivecomorbidity for ICU admission and death after adjusting for age and sex OR CI to Fig Depression was the best predictor of hospitalization among allcomorbidities OR 95CI to Fig Having � comorbidity was associated with ICU admission and death OR 95CI to and hospitalizationOR 95CI to independently of age and sexImaging and laboratory testsChest Xray was necessary in patients and showed lobar pulmonary infiltrates in bilateral pulmonary infiltrates in and an interstitial pattern in Table Chest CT was required in patients and pulmonary ultrasound in Biologically of patients had lymph ia mm3 Likewise had a lactate dehydrogenase LDH Uml and liver test alterations were commonelevated ASTGOT in and ALTGPT in In of cases Ddimer waselevated 500mgL The most important factors for ICU admission and death were bilateralpulmonary infiltrates OR 95CI to elevated lactatedehydrogenaseOR 95CI to elevated Ddimer OR 95CI to andelevated Creactive protein OR 95CI to Fig Significant predictivePLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsFig Prognostic factors for death and ICU admission �The upper limits of the confidence intervals were restricted to in order not to mask the significant effects of other variables with smaller ranges101371journalpone0237960g001factors associated with hospitalization after adjusting for age and sex were lymph iaOR 95CI to and elevated Creactive protein OR 95CI to Fig Treatment complications and evolutionTreatment included hydroxychloroquine in patients azithromycin in lopinavirritonavir in glucocorticoids in and tocilizumab in among others Table and of patients required hospitalization Phone follow up was registered in patients patients were monitored at homePLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsFig Prognostic factors for hospitalization �The upper limits of the confidence intervals were restricted to inorder not to mask the significant effects of other variables with smaller ranges101371journalpone0237960g002 of the patients of working age sought work disability due to COVID19 TheICU admission rate was The evolution included pneumonia in patientsadult respiratory distress syndrome in severe renal failure in pulmonarythromboembolism in and sepsis in patients Occupational contact with persons with confirmed or suspected COVID19 infection was reported by patientswhile reported that contact occurred in the family setting Occupational contactwas a protective factor against hospitalization OR 95CI to ICU admission and death OR 95CI to after adjusting for age and sex The mortalityrate to date was PLOS ONE 101371journalpone0237960 August PLOS ONE 0cTable Comorbidities associated with hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admissionHospitalizationn NoYesPAdjusted OR [NoYesPAdjusted OR [n n CI]n n CI]Prognostic factors in Spanish COVID19 patientsComorbidities”no  Any comorbidityHypertensionDiabetesObesityDyslipidaemiaCancer [“] [“] [“] [“] [“] [“] [“] [“] Chronic kidney disease Heart disease Autoimmune disease Chronic obstructive pulmonary diseaseDepressionCardiac arrhythmiaThyroid alterationsAsthmaLiver diseaseCerebrovascular diseaseAlzheimer disease [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Comorbidities with a frequency of patients were bronchiectasis n fibromyalgia n anaemia n arthritis n HIV n syphilis n andtuberculosis n 101371journalpone0237960t002DiscussionThis study summarizes the clinical biological and radiological characteristics evolution andprognostic factors of patients with COVID19 disease in primary and community healthcareTo date we are aware of three published Spanish studies [“] The first reported data from patients on ICU admissions in a region where the pandemic was reported early [] Thestudy by Borobia [] describes the first adult patients with COVID19 consecutivelyadmitted to a University Hospital in Madrid The third focuses on the differences by agedependent categories in the clinical profile presentation management and shortterm outcomes [] Although there have been two systematic reviews and metaanalysis that analysethe clinical characteristics of COVID19 they are limited to Chinese cohorts or case series [] and a large USA cohort [] that did not analyse clinical predictors of a poor prognosisClinically the same main symptoms of cough and fever are reported in all series Howeverin Barcelona city we have observed diarrhoea anosmia and dysgeusia which is hardlyreported in the Chinese series [] which unlike ours comes principally from hospitals diarrhoea occurred in of cases very similar to the in New York [] and clearly higherthan the reported in China Nearly of patients had anosmia and dysgeusia similar tothe results obtained in French patients [] In contrast expectoration was found in only compared with in the Chinese seriesPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Analytical and radiological predictors of hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admission n Hospitalization n n NoYesPAdjusted ORNoYesn n [ CI]n n PAdjusted OR[ CI]Alterations in chest Xray”nototalno  Bilateral pulmonary infiltratesInterstitialground glass patternLobar pulmonary infiltrateAlterations in chest CAT scan”nototal no ¡ [“] [“] [“] [“] [“] [“]Bilateral pulmonary infiltrates Interstitialground glass pattern Laboratory parameters”nototalno [“] [“] [“] [“]Leukocytes mm3 [“] [“]Lymphocytes mm3Platelets mm3 [“] [“] [“] [“]Haemoglobin gdl [“] [“]Creactive protein mglitreProcalcitonin ngmlLactate dehydrogenase UlitreAminotransferase aspartate UlitreAlanine aminotransferase UlitreTotal bilirubin mgdL [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]Creatine kinase Ulitre [“] [“]Creatinine 15mgdL Ddimer mglitreSodium mEqlitrePotassium mEqlitre [“] [“] [“] [“] [“] [“] [“] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  patients had a chest Xray The alterations with a frequency patients were pneumothorax n and pleural effusion n Chest Xray resultswere not available in patients¡ patients had a chest CAT scan Alterations with a frequency of patients were pulmonary thromboembolism n emphysema n lobarpulmonary infiltrates n pneumonia n atelectasis n and pleural effusion n CAT scan results were not available in five patients101371journalpone0237960t003PLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Predictors of the evolution complications and treatment in patients hospitalized or with ICU admissiondeathVariablesTotalDeath or ICU admission n Hospitalization n n NoYesPAdjusted OR [NoYesPAdjusted OR [n n CI]n n CI]Complications”no  Any complicationPneumonia NA [“] [“] [“]Adult respiratory distress [“ [“]syndromeRenal failurePulmonary thromboembolismTreatments”no ¡HydroxychloroquineAzithromycinLopinavirRitonavir [“] [“] [“]NA] [“] [“] [“] [“] [“] [“]Oxygen therapy [“] [“]Intravenous antibiotics [“] [“]GlucocorticoidsTocilizumabCephalosporinsLow molecular weight heparin Remdesivir Covid19 infection”no [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]Any cohabitant [“] [“]Any work colleague [“] [“]Any contact person in other [“] [“]settingsIn bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Complications in patients were sepsis n multian failure n electrolyte alterations n hematologic alterations n and lung cancer n ¡ Treatments with a frequency of patients except remdesivir were amoxicillin n interferon n rituximab n darunavir n and entecavirn NA not applicable101371journalpone0237960t004Chinese patients had a mean age of years ten years lower than our series and ofour patients were aged � years compared with and in China Germany andthe USA respectively but in Italy [ “] Older age and male sex predisposed to ahigher mortality rate in our and all large series [ ] In our patients comorbidities werethree times higher than in the Chinese cohort [] and were similar to the findings of the NewYork study [] Any comorbidity was a risk factor for hospitalization ICU admission anddeath Depression was an independent risk factor for hospitalization which has not beenobserved in other cohorts studied Depression was often accompanied by a vulnerable socialsituation which may have justified hospitalization Likewise autoimmune diseases were independent risk factor for ICU admission and death Various hypotheses have been postulated onpossible autoimmune alterations in the pathogenic evolution of the disease With respect toPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientstreatment no drug has proved effective against Covid19 until now Moreover many treatments were unavailable in the outpatient setting Currently we are only certain that treatmentwith tocilizumab showed better survival rates in retrospective cohorts [] although its efficacy has not been tested in randomized clinical trials Therefore the results on the outcomesassociated with treatment should be interpreted with cautionThe same comorbidities were identified with hypertension and diabetes being the twomost common while in the USA and Italy obesity seems to be higher Our results show thatobesity was close to being an independent risk factor for hospitalization OR CI to Strikingly of our patients were healthcare workers compared with in Wuhanand in Germany [ ] Although these studies recognized an important degree ofunderreporting of cases in health workers the difference remains important There are at leasttwo possible explanations first the lack of personal protective equipment in the initial phaseof the epidemic a constant revindication of health professionals who felt undersupplied Secondly many cases were health professionals from primary healthcare or the reference hospitalwho reside in the same area where they workIn all reported series bilateral pneumonia was the most common radiological finding waspresent in more than half the cases [] and was a factor of a poor prognosis and mortality Incontrast an interstitial radiological pattern did not confer an increased risk of mortality TheWuhan study reported a CAT scan use of compared with in Barcelona In contrast chest Xrays were carried out in and respectively the availability of diagnostic means was higher in China A recent international consensus states that radiologicalassessment is not necessary in asymptomatic patients or those with mild disease but is requiredin patients with moderate or severe disease regardless of whether a definite diagnosis ofCOVID19 has been made [] In addition simple chest Xrays are preferable in a resourceconstrained environment with difficulties in accessing CAT scans [] The possible use of pulmonary ultrasound for the pointofcare diagnosis of COVID19 pneumonia has not been sufficiently analysed but might be an efficient alternative due to its portability and reliability []In fact the regional Catalan government has recently acquired ultrasound machines toenable family physicians to make doctors can make pointofcare home or nursing homediagnoses of pneumonia [] Biologically lymph ia and increased CRP LDH and Ddimer were usually constant and similar in all series and were associated with an increased riskof mortality A differential variable in our series is a greater number of alterations in liver testswhich was present in “ of patients data similar to the USA and Italian cohorts but different from the Chinese cohort where it was [] We also found hypokalaemia in of patients a factor not reported in other studiesWe found a hospitalization rate of compared with “ in the USA and inChina and an ICU admission rate of which was similar to the Chinese USA “ and German results While the protocols of action and admission are similarand depend on the level of clinical involvement the therapeutic protocols differ between hospitals cities and countries There remain many unknowns in the treatment of COVID19The only truth is that we do not have a vaccine an etiological treatment or a treatment withsufficient scientific evidence to generalize its use Currently the systematic review of antiretroviral treatments has not offered conclusive results [] and despite in vitro results for hydroxychloroquine COVID19 infections are currently intractable [ ]The mortality rate in our study was compared with in New York in hospitalized patients in China in Germany and in Italy Different informationand recording systems the availability of diagnostic tests and above all the anization ofnational health systems may have contributed to the differences observedPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsThe study had some limitations due to the observational retrospective design However itis sufficiently representative of the population with confirmed COVID19 to permit betteridentification of the factors of a poor prognosis of the disease from a clinical perspective Wecannot rule out some heterogeneity in data codification due to observers™ interpretations of themedical records However this bias is minimal as most clinical factors included are clearlydefined in the electronic medical record Another limitation of this study is the percentage ofpatients without laboratory parameters more than Even though in real clinical practicethese percentages may be expected the results corresponding to laboratory parameters shouldbe interpreted with cautionFour months after the declaration of the pandemic there is not a sufficiently reliable available and generalizable diagnostic test that can analyse the seroprevalence of COVID19 evenin the most industrialized countries Given this lack determining the clinical biological andradiological characteristics of probable cases of COVID19 infection will be key to the initiation of early treatment and isolation and for contact tracing especially in primary healthcareSupporting informationS1 DatasetXLSXAcknowledgmentsThe authors thank David Buss for editorial assistanceAuthor ContributionsConceptualization Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuData curation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuFormal analysis Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuInvestigation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuMethodology Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuProject administration Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuResources Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuSupervision Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuValidation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsVisualization Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuWriting “ original draft Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuWriting “ review editing Antoni Siso´Almirall Belchin Kostov Minerva MasHerediaSergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana AugustAnguitaGuimet Jaume BenaventÃreuReferences World Health anization Pneumonia of unknown cause”China wwwwhointcsrdon05january2020pneumoniaofunkowncausechinaen accessed April Centers for Disease Control and Prevention Novel Coronavirus 2019nCoV Situation Summarystackscdcgovviewcdc84806cdc_84806_DS1pdf accessed April Holshue ML DeBolt C Lindquist S Lofy KH Wiesman J Bruce H First Case of Novel Coronavirus in the United States N Engl J Med “ 101056NEJMoa2001191 PMID World Health anization Novel Coronavirus2019nCoV wwwwhointdocsdefaultsourcecoronavirusesituationreports20200130sitrep10ncovpdfsfvrsnd0b2e480_2 accessed AprilJohns Hopkins University CSSE COVID19 Dashboard by the Center for Systems Science and Engineering CSSE at Johns Hopkins University JHU gisanddatamapsarcgiscomappsopsdashboardindexhtmlbda7594740fd40299423467b48e9ecf6 accessed April Ministerio de Sanidad Gobierno de España Situacio´n del COVID19 en España covid19isciiies accessed April Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Clinical Characteristics of Coronavirus Disease in China N Engl J Med 101056NEJMoa2002032 PMID Yeo C Kaushal S Yeo D Enteric involvement of coronaviruses is faecaloral transmission of SARSCoV2 possible Lancet Gastroenterol Hepatol “ 101016S2468 PMID Chang BB Chiu TY Ready for a long fight against the COVID19 outbreak an innovative model oftiered primary health care in Taiwan BJGP 103399bjgp 20X101068PMID Barrasa H Rello J Tejada S Martı´n A Balziskueta G Vinuesa C SARSCov2 in Spanish Intensive Care Early Experience with 15day Survival In Vitoria Anaesth Crit Care Pain Med 101016jaccpm202004001 PMID Borobia AM Carcas AJ Arnalich F A´ lvarezSala R MonserratVillatoro J Quintana M A Cohortof Patients with COVID19 in a Major Teaching Hospital in Europe J Clin Med Martı´nSa´nchez FJ Del Toro E Cardassay E Valls Carbo´ A Cuesta F Vigara M Clinical presentation and outcome across age categories among patients with COVID19 admitted to a Spanish Emergency Department Eur Geriatr Med 101007s41999020003592 PMIDFu L Wang B Yuan T Chen X Ao Y Fitzpatrick T Clinical characteristics of coronavirus disease COVID19 in China A systematic review and metaanalysis J Infect 101016jjinf202003041 PMID RodriguezMorales AJ CardonaOspina JA Gutie´rrezOcampo E VillamizarPeña R HolguinRiveraY EscaleraAntezana JP Clinical laboratory and imaging features of COVID19 A systematicreview and metaanalysis Travel Med Infect Dis 101016jtmaid2020101623PMID Richardson S Hirsch JS Narasimhan M Crawford JM McGinn T Davidson KW PresentingCharacteristics Comorbidities and Outcomes Among Patients Hospitalized With COVID19 inthe New York City Area JAMA 101001jama20206775 PMID Goyal P Choi JJ Pinh

Thyroid_Cancer

"At the time of surgery approximately “ of the patients with pancreatic cancer are consideredunresectable because of unexpected liver metastasis peritoneal carcinomatosis or locally advanced disease This leads to futilesurgical treatment with all the associated morbidity mortality and costs More than of all liver metastases develop in thefirst six months postoperatively These subcentimeter liver metastases are most likely already present at the time of diagnosisand have not been identified preoperatively due to the poor sensitivity of routine preoperative contrastenhanced CT CECTMethods The DIAPANC study is a prospective international multicenter diagnostic cohort study investigating diffusionweighted contrastenhanced MRI for the detection of liver metastases in patients with all stages of pancreatic cancerIndeterminate or malignant liver lesions on MRI will be further investigated histopathologically For patients with suspected liverlesions without histopathological proof follow up imaging with paired CT and MRI at and 12months will serve as analternative reference standardDiscussion The DIAPANC trial is expected to report highlevel evidence of the diagnostic accuracy of MRI for the detection ofliver metastases resulting in significant value for clinical decision making guideline development and improved stratification fortreatment strategies and future trials Furthermore DIAPANC will contribute to our knowledge of liver metastases regardingincidence imaging characteristics their number and extent and their change in time with or without treatment It will enhancethe worldwide implementation of MRI and consequently improve personalized treatment of patients with suspectedpancreatic ductal adenocarcinomaTrial registration ClinicalTrialsgov Identifier NCT03469726 Registered on March 19th Retrospectively registeredKeywords Pancreatic cancer Liver metastases MRI Staging Correspondence JohnHermansradboudumcnl G Litjens and D M Rivi¨re contributed equally to this work1Department of Radiology and Nuclear Medicine Radboudumc NijmegenThe NetherlandsFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLitjens BMC Cancer Page of BackgroundPancreatic ductal adenocarcinoma PDAC is one of themost lethal forms of cancer and expected to become thesecond leading cause of cancerrelated deaths before Developments in pancreatic cancer diagnosticssurgical techniques and treatment have hardly improvedthe survival rate in the past years The 5year relativesurvival rate as reported by the American Cancer Societyremains only [ ]Only “ of all patients are eligible for surgery todate the only potential cure [] Approximately “ ofall patients with pancreatic cancer have metastatic diseaseat diagnosis and of all patients have locally advanceddisease with tumor involvement of surrounding vessels orans At the time of surgery approximately “ ofthe patients are considered unresectable because of unexpected liver metastasis peritoneal carcinomatosis or locally advanced disease [“]More than of all liver metastases develop in thefirst six months postoperatively [] These liver metastases are most likely already present at the time of diagnosis and have not been identified preoperatively as theyare too small to be detected by routine preoperativeultrasound and contrastenhanced CT CECT [ ]CECT is highly accurate in assessing the relationship ofthe tumor to critical arterial and venous structures sincetheir involvement can preclude surgical resection HoweverCECT has a poor sensitivity “ for the detection andcharacterization of liver metastases [ “] especiallyfor subcentimeter metastases which are often present inpancreatic cancer [] This leads to futile surgical treatment with all the associated morbidity mortality and costsMoreover patients who were explored with curative intentand were found unresectable due to peritoneal or liver metastases had a worse overall survival compared to patientswith unexpected locally advanced disease []Nowadays diffusionweighted MR imaging DWI appears to be valuable in both detection and characterizationof focal liver lesions with a high sensitivity “ evenfor subcentimeter lesions “ [“] This technique can be used to detect and characterize liver lesionsbased on decreased diffusion of water molecules caused bytumoral hypercellularity and reduced extracellular spaceDWI is especially useful for detecting subcentimeter livermetastasesit is more accurate than conventional T2weighted imaging techniques because signal suppression ofintravascular flow is obtained black blood effect whilemaintaining good residual signal of the liver lesions [] Itis easy to implement and adds very little time to a standardMRI examination However without highquality evidenceof the benefit of MRI the use of MRI as part of the routineworkup is questioned and therefore not implemented Currently most guidelines advise to use MRI as a problemsolving tool in addition to CECT eg when the primarytumor cannot be visualized or in case of undefined liver lesions [“] The American Society of Clinical OncologyASCO leaves the choice of imaging modality in the handsof the physician [] MRI is advised for all patients according to the Japanese guideline however the level of evidenceis low grade C []Most studies that have been performed for liver metasincluding our singletases of PDAC are retrospectivecenter study in patients with potentially resectable pancreatic cancer without liver metastases on CECT [] Inthis study Gadolinium Gd enhanced MRI with DWIdetected synchronous liver metastases in of patientswith potentially resectable pancreatic cancer on CECTwith a sensitivity of DWI showed more lesions thanGdenhanced MRI most of which were particularlysmall mm Correspondingly the only prospectivestudy to our knowledge showed that Gdenhanced MRIespecially DWI depicted small liver metastases in approximately of patients with a potentially resectablepancreatic cancer without liver metastases on CECT[] The reported sensitivity was “ and the specificity “ However due to the relatively low prevalence of patients with liver metastases in their studypopulation in total only patients with liver metastaseswere included in this studyIn the DIAPANC study we will determine the diagnosticaccuracy of Gdenhanced MRI with DWI in the detectionof liver metastases in patients with all stages of PDACMethodsDesigninternationalThe DIAPANC study is a prospectiveinvestigatingmulticenterstudydiffusionweighted Gdenhanced MRI for the detectionof liver metastases in patients with pancreatic cancerdiagnosticcohortThis protocol was written and reported according to theStandard Protocol Items Recommendations for Interventional Trials SPIRIT Guidance and Checklist []Study populationAll patients with suspected pancreatic ductal adenocarcinoma are eligible to be included in this study and will beactively recruited at the outpatient clinic by the treatingphysician Written informed consent will be obtained byone of the members of the research team We will includepatients until patients with liver metastasis are included with a maximum total of patients Exclusioncriteria are age below years previous treatment forpancreatic cancer concomitant malignancies except foradequately treated basocellular carcinoma of the skin subjects with prior malignancies must be diseasefree for atleast years contraindications for MRI or CECT ie untreatable contrast allergy severe renal function impairment not MRI compatible medical implants insufficient 0cLitjens BMC Cancer Page of command of the local language and pregnancy This studyhas been approved by the ethical board of our universitymedical center Approval of the local medical ethicalboard is obliged before the start of inclusion in the participating hospitalsSpecific withdrawal of patientsPatients with adenocarcinoma of the distal common bileduct papilla of Vater or duodenum patients with aneuroendocrine tumor or patients with benign tumorswill be excluded from analysis and followupPrimary outcomeThe sensitivity and specificity of Gdenhanced MRI withDWI for the detection of liver metastases in patientswith pancreatic cancerSecondary outcomesThe secondary outcomes of this study are sensitivityand specificity of CECT for the detection of liver metastases sensitivity and specificity of MRI and CECT forthe prediction of resectability and the effect of the MRIon patient managementData collectionAll patients will be assigned a unique participant codeThe key will be stored separately from the data We planto collect the following baseline data age sex performance status WHO performance score American Societyof Anesthesiologists physical status body mass indexweight loss decreased appetite diabetes mellitus previousliver or pancreatic diseases smoking and alcohol statusand tumor markers CEA and CA19“ using the datamanagement system Castor EDC Castor Electronic DataCapture Ciwit BV Amsterdam The Netherlands Dataon diagnostic procedures like endoscopic imaging and biopsies treatment and clinical followup will be collectedduring the entire study period by the local treating physicians or the trial coordinators using Castor EDC Patientswill be asked to fill in validated quality of life questionnaires EORTC QLQC30 and QLQPAN26 at baselineand after and 12months followupMRI and CTMRI scans will be made on a T scanner with T2 weightedimaging using an intravenous gadoliniumbased contrastagent with a T1 weighted precontrast arterial and portalvenous phase DWI with bvalues of and smm2and with a Magnetic Resonance CholangioPancreatographyMRCP CECT scans are performed with intravenous iodinecontrast agent with a pancreatic phase of the upper abdomen a portal venous phase of the entire abdomen Additionally the chest will be staged using chest CT MRI and CECTwill be performed at baseline and after and 12monthsfollowup the schedule is displayed in a flowchart in Fig Interpretation of MRI and CTAll MRI and CECT scans will initially be evaluated by thelocal radiologist and the findings will be included in theclinical decision making The MRI and CECT scans willalso be independently evaluated by a second radiologistblinded for findings of the first evaluation and the clinicaloutcome If the MRI and CECT of one patient is evaluatedby the same radiologist a minimum interval of weeks willbe used to minimize the risk of recall biasThe MRI and CECT scans will be analyzed for localresectability and suspicious liver lesions Number of liverlesions lesion size liver segment presumed diagnosis ofsuspicious liver lesions indeterminate or malignant andimaging characteristics on MRI will be notedReference standardIndeterminate or malignant liver lesions will be furtherinvestigated histopathologically The first step in obtaining histological proof of suspected liver lesions on CECTandor MRI is transabdominal ultrasound of the liverBiopsy will be performed of visible liver lesions and analyzed with routine histological examination When lesions are not visible or there is no histological proof ofthe visible lesions the next step is surgical explorationlaparoscopic or in borderline resectable pancreatic cancer In case liver lesions are identified a frozensection is performed Hereafter patients are treated according to standard care protocolFor patients with suspected liver lesions without histopathological proof followup imaging with paired CECTand MRI at and months will serve as an alternativereference standard Lesions that are growing or increasingin number over time will be considered metastasesDefinitionsOn MRI liver lesions are defined as malignant on DWIwhen they are moderately hyperintense at b smm2and remains hyperintense at b smm2 A lesion isconsidered benign when it is hyperintense at b smm2and shows a substantial decrease in signal intensity athigher b values b and b smm2 If none ofthe criteria is met a lesion is classified as indeterminateOn CECT liver lesions are defined as malignant if theyare hypodense not showing typical features of a simplecyst fluid attenuation measurements roundoval welldefined borders no contrast enhancement hemangiomalocalization next to vessels peripheral nodular enhancement centripetal fillin or focal fatty infiltration geographic hypodense area angular margins typical locationIf a lesion is showing signs of simple cyst hemangioma or 0cLitjens BMC Cancer Page of Fig Flowchart of study schedule and proceduresfocal fatty infiltration it is defined as benign If a lesion istoo small to characterize it is classified as indeterminateTNM status is classified according to the AmericanJoint Committee on Cancer AJCC 8th edition []Lymph nodes are defined as suspicious ifthey arerounded and ‰¥ mm or if they are notrounded with theshortest axis ‰¥ mmSafety and ethicsThere is a low risk and low burden for patients participating in this study Patients might benefit fromstudy participation due to possible improvement ofdetection of liver metastases The contrast agent usedfor MRI has few known side effects and rarely leadsto a severe allergic reaction [] Extra CECT scansmight be performed in some study patients with theassociated radiation and contrast exposure Patientsdiagnosed with pancreatic cancer have a 5year overall survival of Radiationinduced cancer has a latency yearsTherefore the health risk for this specific oncologicpatient group is almost negligiblesubstantiallyexceedsperiodthat 0cLitjens BMC Cancer Page of MRI can lead to earlier detection of liver metastaseshowever in some patients these lesions might be toosmall to biopsy Consequently we cannot always providethe patient certainty about the nature of the liver lesionsdetected with MRI Furthermore in followup local recurrence or metastases might be detected before a patient has symptoms This may be seen as a disadvantageby some individualsStatisticsSample sizeThe sample size for the study was calculated for the primary endpoint sensitivity and specificity of MRI for thedetection of liver metastasesThe sample size is calculated based on a method forpower calculations for diagnostic studies described by Jones [] Based on literature and our previously performedretrospective study [ “] we estimate the sensitivity ofMRI will be approximately In literature the specificityfor MRI is usually higher than the sensitivity therefore webased our sample size calculation on the sensitivity onlyWith an expected sensitivity of confidence interval of Z and α patients with metastasisare required for analysis Based on literature the expectedpercentage of patients with liver metastases is approximately [ ] With an expected inclusion rate of assuming cannot be analyzed optimally eg becauseno representative liver biopsies could be acquired mortalitybefore first followup or withdrawal we need approximately patients In case the proportion of patients withmetastases is not equal to in our cohort we will include until we reach patients with liver metastasis orup to a maximum total of patientsAnalysisAnalysis will be done using SPSS IBM Corp ArmonkNew York USA Continuous variables will be summarized with standard descriptive statistics including meanstandard deviation median and range Categorical variables will be summarized with frequencies A pvalueless than is considered statistically significantFor the analysis of the diagnostic accuracy sensitivityand specificity a — cross tabulation will be madecomparing MRI and CECT to histopathology and followup Performance of CECT and Gdenhanced MRI withDWI will be compared using McNemar™s test We willreport the changes made in patient management in a descriptive manner Median and 1year survival will be reported Survival endpoints disease free survival andoverall survival will be analyzed using KaplanMeierplots Survival curves are compared using the log ranktest We will compare the results of both readers to determine the interobserver variability A Cohen™s Kappak value of “ is interpreted as excellent “ substantial agreement “ moderate agreement “fair agreement and “ pooragreementWe partly anticipated missing data by introducing thecomposite reference standard of follow up Unfortunatelymissing data still can occur when for instance a patientsuspected of having metastatic disease does not have histopathological confirmation and dies before the compositereference standard follow up could take place If necessaryadditional analysis will be performed to determine the robustness of the results and to deal with missing dataTrial statusThe first patient was included on December 21st Atthe time of protocol submission July 23th active inclusion of patients has started in six centers RadboudUniversity Medical Center Nijmegen the NetherlandsKonstantopouleio General HospitalAthens GreeceMedisch Spectrum Twente Enschede The Netherlandsand Jeroen Bosch Hospital Den Bosch The NetherlandsUniversity Medical Center Groningen Groningen TheNetherlands and University Hospital Ram³n y CajalMadrid Spain and a total of patients have been included Four centers are preparing to start with inclusionInselspital Universit¤tsspital Bern Bern SwitzerlandUCHealth University of Colorado HospitalDenverUnited States of America Azienda Ospedaliera Universitaria Integrata Verona Verona Italy and Policlinico AGemelli Rome Italy Inclusion of patients is expected tobe finished December accuracy ofDiscussionThe purpose of the DIAPANC trial is to investigate thediagnosticcontrastenhanced diffusionweighted MRI in patients with suspected PDAC for thedetection of liver metastases Additionally we will evaluatewhether performing contrastenhanced diffusionweightedMRI will improve the detection of liver metastases compared to CECT by determining the sensitivity and specificity of CECT for the detection of liver metastasesDespite the good diagnostic performance of MRI forliver metastases the benefits of MRI remain unclearmostly because of low level of evidence heterogeneityand bias in the performed studies Two recently published metaanalyses have suggested the results shouldbe confirmed by performing a welldesigned and sufficiently powered study directly comparing liver CT andMRI in the same cohort [ ]A major difficulty in the interpretation of the currentliterature is that most studies are retrospective oftenonly reporting on a subset of patients actually undergoing a resection patients with borderline resectable tumors or patients with indeterminate liver lesions onCECT These patients have a higher probability of 0cLitjens BMC Cancer Page of having liver metastases However in an era of neoadjuvant therapy local ablative therapy for advanced tumorsexpensive targeted therapies and resection of oligometastases MRI may be beneficial to patients with allstages of PDAC Therefore all patients with suspectedPDAC are eligible for inclusion in the DIAPANCMRI field strength T versus T was a significant factor in the heterogeneity between studies that was found ina metaanalysis T MRI had a higher sensitivity anda lower specificity for diagnosing liver metastasiscompared to T MRI sensitivity and specificity[] Because the signaltonoise ratio and thelesiontoliver contrast are higher on T MRI than on T MRI it is reasonable that a T MRI permits a higher lesion detection rate [ ] In the DIAPANC study weplan to perform all MRIs on a T scanner A potentialdownside of a multicenter design is the intervendor variability that could occur when comparing the quantitativeApparent Diffusion Coefficient ADC value this variabilityseems to be more pronounced at T than at T []Availability of MRI is not expected to be an issue asMRI is available in every expert center for pancreaticdiseases However problems with MRI capacity couldarise due to the need for MRI within a short intervalafter CT A time interval of two weeks was chosen toprovide a feasible time frame for MRI to be performedand no intervallesions are expected within this timeinterval []The DIAPANC trial is the first international prospective multicenter cohort study about the diagnostic accuracyof contrastenhanced diffusionweighted MRI On theWorld Health anization trial registry website ICTRPincorporating all inter national trial registries there areonly four other prospective trials registered in this fieldThe first trialis a completed French prospectivemulticenter trial presumably the only one prospective study that has been published [] The studyhas been performed in patients with potentiallyresectable pancreatic cancer on a T scanner usinggadobenate dimeglumine MultiHance as contrastagent The study has been performed to assess thediagnostic performance of diffusionweighted MRIfor the preoperative diagnosis of liver metastasis andthe modification of therapeutic strategy as a consequence ofliver metastasis ondiffusionweighted MRI []the diagnosis ofThe second trial is a British single center observational study with a target sample size of patientswith confirmed or suspected pancreatic cancer referred for pancreaticoduodenectomy and is completed recently The primary outcome of this studyis the proportion of patients correctly identified byMRI to have lymph node peritoneal or liver metastases To our knowledge the results have not beenpublished and there is no information on scan parameters and contrast agent available []The third trial from Australia is the only randomizedcontrolled trial The study has a target sample size of patients and is not yet recruiting The aim of the studyis to compare the 12month recurrence rate in patientswith locally operable pancreatic adenocarcinoma managed with standard preoperative assessment of liver metastases with CECT versus preoperative assessment withliver specific contrast MRI []The fourth trial is a Chinese comparative study and isnot yet recruiting The study aims to compare liver specific contrast MRI and CECT in liver metastasis of pancreatic cancer with a target sample size of patients []The DIAPANC trial hypothesizes a superior value ofMRI for the detection of liver metastases compared toCECT To reliably determine the diagnostic accuracy thegold standard is histopathology of the liver lesions Considering it is not always possible and sometimes even unethical to obtain histopathological proof of every lesionfollowup is used as a reference standard Hence we areable to simultaneously gather information on early localrecurrence or metastases after resection disease progression and therapy response evaluation on MRI and CECTIn conclusion the DIAPANC trial is expected to report highlevel evidence of the diagnostic accuracy ofMRI for the detection of liver metastases compared toCECT resulting in significant value for clinical decisionmaking guideline development and improved stratification for treatment strategies and future trials Furthermore DIAPANC will contribute to our knowledge ofliver metastases regarding incidence imaging characteristics their number and extent and their change in timewith or without treatment When our hypothesis is confirmed it will enhance the worldwide implementation ofMRI and consequently improve personalized treatmentof patients suspected of PDACAbbreviationsADC Apparent Diffusion Coefficient AJCC American Joint Committee onCancer Castor EDC Castor Electronic Data Capture CEA CarcinoembryonicAntigen CECT Contrast Enhanced Computed Tomography CA19“ Carbohydrate Antigen “ DIAPANC Diagnostic accuracy of contrastenhanced diffusionweighted MRI for liver metastases of pancreatic cancerDWI DiffusionWeighted Imaging EORTC European anization forResearch and Treatment EUS Endoscopic Ultrasound FNA Fine NeedleAspiration FNB Fine Needle Biopsy Gd Gadolinium ICTRP InternationalClinical Trials Registry Platform MRCP Magnetic Resonance CholangioPancreatography MRI Magnetic Resonance Imaging PDAC Pancreatic ductaladenocarcinoma SPSS Statistical Package for the Social SciencesTNM Tumor Node Metastasis WHO World Health anization QLQC30 Quality of life questionnaire including questions QLQPAN26 Pancreatic cancer module of quality of life questionnaire including questionsAcknowledgementsWe acknowledge all patients who participated and will participate in thestudy Secondly we acknowledge all participating institutions conduct ofthe study would be impossible without contribution of these institutions 0cLitjens BMC Cancer Page of Authors™ contributionsGL and DR drafted the manuscript of the protocol JH is principalinvestigator of the study and participated in the design of the study MP isthe study sponsor and participated in the design of the study GL DR EGSR LB and CL participated in the design of the study GL primarilycoordinates the study All authors critically reviewed the manuscript andapproved the final manuscript Publications of the study results will be inaccordance with international recognized scientific and ethical standardsconcerning publications and authorship including the UniformRequirements for Manuscripts Submitted to Biomedical Journals establishedby the International Committee of Medical Journal EditorsFundingThe Dutch Cancer Society KWF reviewed and financially funded the DIAPANC study Research Project grant reference number They do notinfluence the data collection interpretation of data the manuscript or thedecision to publishAvailability of data and materialsThe complete dataset will be property of the Sponsor all participatinginstitutions will own the dataset of the included patients from their centerPublic access to the full trial protocol trialrelated documents participantlevel dataset and statistical code may be made available on requestEthics approval and consent to participateThe DIAPANC study will be conducted according to the principles of theDeclaration of Helsinki 64th version October and in accordance withthe Medical Research Involving Human Subjects Act WMO The independent ethics review board region ArnhemNijmegen Nijmegen TheNetherlands has approved the trial protocol NL6047309117 Furthermoresecondary approval for all participating centers from The Netherlands was orwill be individually obtained from all local ethics committees According toDutch law ethical approval by the ethics review board of the study sponsorie initiating center Radboudumc Nijmegen The Netherlands is appropriatefor all Dutch centers For all participating centers outside of The Netherlandsapproval from a local independent ethics review board was or will be obtained The trial is registered in the registry provided by the US National Library of Medicine clinicaltrialsgov with identification number NCT03469726Patients can only participate if written informed consent has been providedProtocol modifications will be communicated to all relevant parties egparticipating centers funder after approval of the ethical committee and willbe updated in the trial registry Possible substudies like Biobank sampleswill be stored at the Radboud Biobank or artificial intelligence analysis areincluded on the informed consent form Patients must give separate consentto participate in these substudies The study will be monitored according tothe guidelines of The Netherlands Federation of University Medical CentresNFU and adverse events related to study procedures will be recordedThere is a study subject insurance for patients that suffer harm from trialparticipationConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Radiology and Nuclear Medicine Radboudumc NijmegenThe Netherlands 2Department of Gastroenterology and HepatologyRadboudumc Nijmegen The Netherlands 3Department of MedicalOncology Radboudumc Nijmegen The Netherlands 4Department ofPathology Radboudumc Nijmegen The Netherlands 5Department ofPathology University Medical Center Utrecht The Netherlands 6Departmentof Surgery Radboudumc Nijmegen The NetherlandsReceived June Accepted July ReferencesSiegel R Ma J Zou Z Jemal A Cancer statistics CA Cancer J Clin“Rahib L Smith BD Aizenberg R Rosenzweig AB Fleshman JM Matrisian LMProjecting Cancer incidence and deaths to the unexpected burden ofthyroid liver and pancreas cancers in the United States Cancer Res “ Willett CG Czito BG Bendell JC Ryan DP Locally advanced pancreaticcancer J Clin Oncol “Raman SP Reddy S Weiss MJ Manos LL Cameron JL Zheng L Impactof the time interval between MDCT imaging and surgery on the accuracyof identifying metastatic disease in patients with pancreatic cancer AJR AmJ Roentgenol 20152041W37“Glant JA Waters JA House MG Zyromski NJ Nakeeb A Pitt HA Doesthe interval from imaging to operation affect the rate of unanticipatedmetastasis encountered during operation for pancreatic adenocarcinomaSurgery “Allen VB Gurusamy KS Takwoingi Y Kalia A Davidson BR Diagnosticaccuracy of laparoscopy following computed tomography CT scanning forassessing the resectability with curative intent in pancreatic andperiampullary cancer Cochrane Database Syst Rev 20167CD009323Van den Broeck A Sergeant G Ectors N Van Steenbergen W Aerts R TopalB Patterns of recurrence after curative resection of pancreatic ductaladenocarcinoma European J Surg Oncol “Haeno H Gonen M Davis MB Herman JM IacobuzioDonahue CA Michor FComputational modeling of pancreatic cancer reveals kinetics of metastasissuggesting optimum treatment strategies Cell ““Holzapfel K ReiserErkan C Fingerle AA Erkan M Eiber MJ Rummeny EJ Comparison of diffusionweighted MR imaging and multidetectorrowCT in the detection of liver metastases in patients operated for pancreaticcancer Abdom Imaging “ Balci NC Semelka RC Radiologic diagnosis and staging of pancreatic ductaladenocarcinoma Eur J Radiol “ Paik KY Choi SH Heo JS Choi DW Analysis of liver metastasis afterresection for pancreatic ductal adenocarcinoma World J GastrointestinalOncol “ Motosugi U Ichikawa T Morisaka H Sou H Muhi A Kimura K et alDetection of pancreatic carcinoma and liver metastases with gadoxeticacidenhanced MR imaging comparison with contrastenhanced multidetector row CT Radiology “Schima W BaSsalamah A Kolblinger C KulinnaCosentini C Puespoek AGotzinger P Pancreatic adenocarcinoma Eur Radiol “ Danet IM Semelka RC Nagase LL Woosely JT Leonardou P Armao D Livermetastases from pancreatic adenocarcinoma MR imaging characteristics JMagnetic Resonance Imaging “Kneuertz PJ Cunningham SC Cameron JL Torrez S Tapazoglou N HermanJM Palliative surgical Management of Patients with Unresectablepancreatic adenocarcinoma trends and lessons learned from a large SingleInstitution Experience J Gastrointest Surg “Eiber M Fingerle AA Brugel M Gaa J Rummeny EJ Holzapfel K Detectionand classification of focal liver lesions in patients with colorectal cancerretrospective comparison of diffusionweighted MR imaging and multisliceCT Eur J Radiol “Lowenthal D Zeile M Lim WY Wybranski C Fischbach F Wieners G et alDetection and characterisation of focal liver lesions in colorectal carcinomapatients comparison of diffusionweighted and GdEOBDTPA enhancedMR imaging Eur Radiol “ Holzapfel K Bruegel M Eiber M Ganter C Schuster T Heinrich P et alCharacterization of small mm focal liver lesions value of respira

Thyroid_Cancer

Inflammation plays a leading role in the pathogenesis of nephrolithiasis The association of the dietary inflammatory index DII with urinary lithogenic factors is unclear This study aimed to evaluate the relation of DII to urinary risk factors of kidney stones formationResults Of participants n n n n and n had hyperoxaluria hypercreatininuria hypercalciuria hyperuricosuria hypocitraturia respectively There was a signifi‘cant increasing trajectory in urinary calcium uric acid and creatinine as well as a decreasing trend in urinary citrate across tertiles of DII score all P ‰ After multivariate adjustment for energy intake age physical activity and body mass index high DII scores were associated with elevated odds of having hypercreatininuria OR 95CI “ Ptrend hypercalciuria OR 95CI “ Ptrend ‰ hyperuricosuria OR 95CI “ Ptrend and hypocitraturia OR 95CI “ Ptrend ‰ No association was identi‘fied between DII and hyperoxaluriaKeywords Dietary inflammatory index Kidney stones Hypercalciuria Hypocitraturia Hyperoxaluria Hyperuricosuria HypercreatinuriaIntroductionNephrolithiasis is one of the most prevalent urologic disorders and impose a substantial burden on human health globally [] The high recurrence rate of kidney stones is yet unsolved [ ]Thus there is an urgent need to target modifiable risk factors to prevent the development and recurrence of renal stonesHigher urinary excretions of oxalate calcium creatinine and uric acid as well as lower excretions of citrate are potential modifiable a0 urinary lithogenic risk factors Correspondence mina_mirzaei101yahoocom Department of Community Nutrition School of Nutritional Sciences and Dietetics Tehran University of Medical Sciences TUMS Tehran PO Box ‘ IranFull list of author information is available at the end of the involved in the formation of kidney stones [“] Inflammation is also another mechanism which plays a leading role in the pathogenesis of nephrolithiasis [] Dietary modifications toward decreasing inflammation may have a potential to prevent kidney stones or their recurrence Several micronutrients or foods such as magnesium vitamin E vitamin C carotenoids fruits and fish had an antiinflammatory impact [“] In contrast simple sugars red meats highfat dairy products and refined grains are associated with elevated inflammatory markers [] Nevertheless nutrients or foods are not consumed separately but as part of the whole diet [“] The Dietary Inflammatory Index DII is developed to measures the overall inflammatory potential of diets [] which has been recognized to be related to the biomarkers of inflammation [] A proinflammatory diet has The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cMaddahi a0et a0al BMC Res Notes Page of been found to be related to the reduced kidney function [] However there is no study investigating the relation of DII to urinary lithogenic factors Therefore this study aimed to assess the association of DII with hypercalciuria hypocitraturia hyperoxaluria hyperuricosuria and hypercreatinuria in patients with nephrolithiasisMain textMethodsSubjectsThis crosssectional study was performed on a total of stone former men aged “ a0years in Tehran Iran in Participants were recruited from the Urology Research Center of Sina Hospital Tehran Iran Inclusion criteria for this study were having a history of kind stone formation and age ‰¥ a0years People with a history of thyroid disease fatty liver disease malignancy stroke diabetes cardiovascular disease and hypertension were excluded Participants who were on medications such as corticosteroids diuretics anticancer drugs multivitamins potassium citrate calcium and vitamin D or C supplements were not eligible for this study Furthermore all alcohol drinkers and drugs abusers were excluded Patients were included in the study after signing written informed consentsDietary assessmentUsual food intake of patients during the previous year was measured by a validated semiquantitative 168item food frequency questionnaire FFQ[] DII was calculated using the method reported by Shivappa et a0al [] The DII is based on scientific papers scoring food parameters based on whether they elevated reduced ˆ’ or had no impact on six inflammatory biomarkers [Creactive protein interleukin IL1 beta IL10 IL4 IL6 and tumor necrosis factoralpha As mentioned Shivappa et a0 al calculated DII according to the food parameters As dietary patterns of different populations are different with each other some food parameters used in the study by Shivappa may not be available in different FFQs Hence researchers calculate DII according to available foods in the FFQ by modification of the method used by Shivappa et a0al [] In the current study the DII score was calculated using the corresponding food parameters available from the FFQ used in our study This approach has been used broadly in the previous studies [] The DII score was calculated with the use of the corresponding nutrients or food parameters available from the FFQ including energy protein total fat carbohydrate dietary fiber monounsaturated fatty acids n3 fatty acids n6 fatty acids polyunsaturated fatty acids saturated fatty acids cholesterol trans fatty acids vitamin A thiamin niacin riboflavin Vitamin B6 folate vitamin B12 vitamin E vitamin C Vitamin D bcarotene iron magnesium zinc selenium as well as caffeine onion greenblack tea paper and garlic The inflammatory effect scores for dietary components used for calculation of DII in this study are reported in Additional file a0 Table a0S1 To calculate the DII score for each participant the mean intake of each nutrient or food parameter was standardized by subtracting mean global intake of food items from the actual individual™s intake and dividing it by the global SD to create a zscore Zscore is used to express an individual™s exposure relative to the standard global exposure This approach both anchors the individual™s exposure to a robust range of dietary patterns in a variety of cultural traditions and obviates completely the problem of noncomparability of units because the a0Zscores is independent of the units of measurement These zscores then were converted to proportions and centered by multiplying values by and subtracting to normalize the scoring system and to avoid skewness The centered percentile values for food items were then multiplied by the corresponding food itemspecific inflammatory effect scores to obtain the food itemspecific DII scores Calculation of DII for carbohydrate intake in a participant in our study as an example for DII calculation is presented in Additional file a0 Table a0S2 similar approach was followed for the calculation of DII for other nutrients Information about global daily mean intake standard deviation for global intake and overall inflammatory effect score of all nutrientsfood items used for DII calculation is reported in the study by Shivappa et a0al [] The overall DII score for each individual was calculated by summing food itemspecific DII scores [] Higher DII scores indicate a more proinflammatory diet while lower DII scores indicate a more antiinflammatory dietMeasurements of a0study outcomesThe 24h urine samples were collected from all participants and urine was analyzed using an AutoAnalyzer as described previously [] Hyperoxaluria a0 was a0 defined a0 as a0 the urinary oxalate ˃ a0 mgday hypocitraturia as a0urinary citrate of a0mgday hyperuricosuria as urinary uric acid over a0 gday hypercreatininuria as urinary creatinine of ˃ a0 mgday and hypercalciuria as a0a urinary calcium ‰¥ a0mgday []Measurement of a0other variablesGeneral Information was obtained using interview Physical activity was measured using of a0 the a0 International a0Physical a0Activity a0Questionnaires a0IPAQ [] Body weight was measured in minimal clothing after removal of shoes by a digital scale Seca Germany with a precision about a0kg Height of individuals was assessed in 0cMaddahi a0et a0al BMC Res Notes Page of standing position without shoes using a calibrated stadiometer Seca Germany to the nearest a0cm BMI was calculated as weight divided by the square of height kgm2Statistical analysesDII was categorized into tertiles T1 ˆ’ to ˆ’ T2 ˆ’ to T3 to Analysis of variance ANOVA and Chi square tests were used to compare continuous and nominalordinal variables across tertiles of DII respectively Continuous variables are reported as mean ± SE and nominalordinal variables as frequency Odds ratio OR and confidence interval CI for the relation of DII to study outcomes was calculated using the logistic regression analysis Statistical significance was set at p ‰ for all tests All analyses were undertaken using the statistical Package for Social Science Version SPSS Inc Chicago IL USAResultsParticipants in the highest tertile of the DII had significantly higher total daily energy intake P ‰ and lower physical activity P than those in the other tertiles There was a significant increasing trajectory in urinary calcium P ‰ uric acid P ‰ and creatinine P ‰ and a decreasing trend in urinary citrate P ‰ across tertiles of DII score Table a0DII score and a0urinary lithogenic factorsIn the crude model it was found that higher adherence to the DII was significantly related to the increased odds of hypercreatininuria OR 95CI ˆ’ Ptrend ‰ hypercalciuria OR 95CI Table Characteristics of a0the a0study participants across a0tertiles of a0the a0DII scoreAge yearHeight cmWeight kgBMI kgm2Physical activity scoreEnergy intake kcaldayUrinary creatininekg weight mgdaykgTotalN ± ± ± ± ± ± ± ± ± ± ± Dietary inflammatory index scoreTertile n ± ± ± ± ± ± ± ± ± ± ± Tertile n ± ± ± ± ± ± ± ± ± ± ± Tertile n ± ± ± ± ± ± ± ± ± ± ± P value‰ ‰ ‰ ‰ ‰ Urinary citrate mgdayUrinary oxalate mgdayUrinary uric acid gdayUrinary calcium mgdayJob status Engineerphysician Clerk Student Teacher Self‘employed Retired Worker UnemployedMarital status Married SingleEducation level Illiterate ‰ Diploma University degreeCategorical variables are presented as frequency n and continuous variables as mean ± SE Oneway ANOVA was used for continuous variables and person™s Chi square test for categorical variables 0cMaddahi a0et a0al BMC Res Notes Page of ˆ’ Ptrend ‰ hyperuricosuria OR 95CI Ptrend ‰ and hypocitraturia OR 95CI ˆ’ Ptrend ‰ After multivariate adjustment for energy intake age physical activity and BMI high DII scores were associated with elevated odds of having hypercreatininuria OR 95CI hypercalciuria OR 95CI ˆ’ Ptrend ‰ hyperuricosuria OR 95CI ˆ’ Ptrend and hypocitraturia OR 95CI ˆ’ Ptrend ‰ The relation of DII to hypercreatininuria hyperoxaluria and hyperuricosuria was not significant after adjustment for carbohydrate fiber and protein intake Table a0 Ptrend ˆ’ adjustment for covariates dietary intakes of protein and fiber were slightly related to the decreased odds of hypocitraturia Protein OR 95CI ˆ’ fiber OR 95CI ˆ’ and hypercalciuria Protein OR 95CI ˆ’ fiber OR 95CI ˆ’ while the intake of protein and fiber was not to associated with hypercreatininuria hyperoxaluria and hyperuricosuriaDiscussionWe revealed that in stone former men a diet with a high DII is significantly related to the increased odds of having hypercreatininuria hypercalciuria hyperuricosuria and hypocitraturia but not to hyperoxaluriaIt has been confirmed that kidney stone formers could be susceptible to recurrence in stones formation We also evaluated the association of dietary protein and fiber intake on urinary risk factors Table a0 After Table Univariate and a0 multivariate logistic regression models for a0 the a0 relation of a0 DII score to a0 urinary risk factors of a0kidney stone formationDietary inflammatory index scoreModel Crude modelModel Model Model Odds ratio CIOdds ratio CIOdds ratio CIOdds ratio CIHypercreatininuria T1 T2 T3 P value for trendHypocitraturia T1 T2 T3 P value for trendHyperoxaluria T1 T2 T3 P value for trendHyperuricosuria T1 T2 T3 P value for trendHypercalciuria T1 T2 T3 P value for trend “ “‰ “ “‰ “ “ “ “‰ “ “‰ Model adjusted for energy intakeModel additionally adjusted for age BMI and physical activityModel adjusted for carbohydrate fiber and protein intake “ “ “ “ “ “ “ “ “ “‰ “ “ “ “‰ “ “ “ “ “ “‰ “ “ “ “ “ “ “ “ “ “ 0cMaddahi a0et a0al BMC Res Notes Page of Table Multivariate logistic regression models for a0the a0relation of a0dietary fiber and a0protein intake as a0continues variable to a0urinary risk factors of a0kidney stone formationFiberOdds ratio CIProteinOdds ratio CIModel Model Model “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Model “ “ “ “ “HypercreatininuriaHypocitraturiaHyperoxaluriaHyperuricosuriaHypercalciuriaModel adjusted for energy intakeModel additionally adjusted for age BMI and physical activitybecause of unhealthy dietary patterns [] Inconsistent with our finding a study did not report any significant difference in creatinine across tertiles of DII in subjects with chronic kidney disease [] A randomized controlled trial a0 study by Noori et a0 al [] on recurrent stone formers showed that a a0DASH diet which in contrast to a diet with a high DII is featured by a high intake of whole grains fruits lowfat a0 dairy products and vegetables and a low intake of total fat cholesterol saturated fat meat and refined grains is significantly associated with a decrease in calcium oxalate supersaturation and an increase in citrate excretion Moreover another study reported that greater adherence to the Mediterranean a0dietary pattern a0is related to the reduced risk for incident kidney stones [] The relationship between systemic inflammation and nephrolithiasis has been identified previously [] Since both DASH and Mediterranean diets attenuate a0 inflammation [ ] the protective effects of these dietary patterns on kidney stones formation may be mediated at least partly by reducing systemic inflammation A crosssectional study conducted on diabetic patients also reported that higher intake of œvegetable and fish dietary pattern is related to a lower creatinine rates [] Vegetables and fish as components of DII are identified to have antiinflammatory effects [ ] The DII is a tool to assess the overall impact of a diet on inflammatory potential [] and is associated with markers of systemic inflammation including such as IL6 [] and CRP [] [] IL6 and CRP are two of the inflammatory biomarkers considered in the calculation of DII [] It has been revealed that the DII score is inversely related to the Dietary Approaches to Stop Hypertension Score DASH Mediterranean Diet Score and Healthy Eating Index2010 [ ] Taken together these findings support that a likely mechanism for the relation of DII scores to hypercreatininuria hypercalciuria hyperuricosuria and hypocitraturia could be explained by the higher systemic inflammation level among people following a proinflammatory dietSince DII positively depends on protein intake that is also a metabolic risk factor for hypercalciuria hyperuricosuria and hypocitraturia and on contrary dietary fiber has a negative impact on DII and is a factor that can reduce calcium absorption we also adjusted the analysis for protein carbohydrate and fiber intake to differentiate the metabolic impact of DII from its proinflammatory impact It was found that DII is related to hypercalciuria and hypocitraturia independent of dietary intake of protein carbohydrate and fiber however the relationship between DII and hyperuricosuria disappeared showing that this association may be resulted from metabolic effects of DII Nevertheless protein and fiber intake was inversely associated with hypercalciuria and hypercalciuriaConclusionIn conclusion this study found that a diet with high inflammatory property might be unfavorably associated with urinary risk factors of kidney stone formation in men with a history of nephrolithiasisLimitationFirst since the participants of the current study were limited to men our findings may not be generalizable to women therefore it is essential to conduct such a study on women too Third causation cannot be inferred the crosssectional design of the present investigation Finally the calculation of DII by FFQ has a potential recall bias for the evaluation of dietary intake 0cMaddahi a0et a0al BMC Res Notes Page of Supplementary informationSupplementary information accompanies this paper at https doi101186s1310 ‘‘ ‘yAdditional file a0 Table a0S1 Inflammatory effect scores for dietary components used for calculation of DII Table a0S2 calculation of DII for carbohydrate intake in a participant in our study as an example for total DII calculationAbbreviationsIPAQ International physical activity questionnaires DII Dietary inflammatory index PUFAs Polyunsaturated fatty acids CRP C‘ reactive protein FFQ Food frequency questionnaire ANOVA Analysis of variance OR Odds ratio CI Confidence interval BMI Body mass indexAcknowledgementsWe would like to thank the Tehran University of Medical Sciences This work was supported by Tehran University of Medical Sciences Grant ID13951046Authors™ contributionsSMKA designed the research and collected the samples NSM and SHA wrote the paper HY and MSY analyzed data KhM conducted research and had primary responsibility for final content All authors read and approved the final manuscriptFundingNoneAvailability of data and materialsThe data are not publicly available due to containing information that could compromise the privacy of research participantsEthics approval and consent to participateEthics approval for the study protocol was granted by The Human Ethics Committee of Tehran University of Medical Sciences Grant ID IRTUMSVCRREC13951046 All participants signed written informed consent formsConsent for publicationNot ApplicableCompeting interestsAll authors declared that they have no competing interestsAuthor details Department of Community Nutrition School of Nutritional Sciences and Dietetics Tehran University of Medical Sciences TUMS Tehran PO Box ‘ Iran Department of Urology Sina Hospital Tehran University of Medical Sciences Tehran Iran Department of Epidemiology and Biosta‘tistics School of Public Health Tehran University of Medical Sciences Tehran Iran Received March Accepted July References Li Y Zhang J Liu H et al Curcumin ameliorates glyoxylate‘induced calcium oxalate deposition and renal injuries in mice Phytomedicine Fink HA Akornor JW Garimella PS et al Diet fluid or supplements for secondary prevention of nephrolithiasis a systematic review and meta‘analysis of randomized trials Eur Urol “Littlejohns TJ Neal NL Bradbury KE Heers H Allen NE Turney BW Fluid intake and dietary factors and the risk of incident kidney stones in UK Biobank a population‘based prospective cohort study European urology focus Ong CN Minerals from drinking water Bioavailability for various world populations and health implications Nutrients in Drinking Water Yagisawa T Chandhoke PS Fan J Metabolic risk factors in patients with first‘time and recurrent stone formations as determined by comprehen‘sive metabolic evaluation Urology “ Grases F Melero G Costa‘Bauza A Prieto R March J Urolithiasis and phytotherapy Int Urol Nephrol “ Khan SR Reactive oxygen species inflammation and calcium oxalate nephrolithiasis Translational Androl Urol Bo S Durazzo M Guidi S et al Dietary magnesium and fiber intakes and inflammatory and metabolic indicators in middle‘aged subjects from a population‘based cohort Am J Clin Nutri “ Chrysohoou C Panagiotakos DB Pitsavos C Das UN Stefanadis C Adher‘ence to the Mediterranean diet attenuates inflammation and coagula‘tion process in healthy adults the ATTICA Study J Am Coll Cardiol “ Upritchard JE Sutherland W Mann JI Effect of supplementation with tomato juice vitamin E and vitamin C on LDL oxidation and products of inflammatory activity in type diabetes Diab Care “ Neale E Batterham M Tapsell LC Consumption of a healthy dietary pat‘tern results in significant reductions in C‘reactive protein levels in adults a meta‘analysis Nutri Res “ Esmaillzadeh A Kimiagar M Mehrabi Y Azadbakht L Hu FB Willett WC Dietary patterns and markers of systemic inflammation among Iranian women J Nutri “ Mohseni R Mohseni F Alizadeh S Abbasi S The Association of Dietary Approaches to Stop Hypertension DASH Diet with the risk of colorectal cancer a meta‘analysis of observational studies Nutrition and cancer ‘ Alizadeh S Djafarian K Alizadeh M Shab‘Bidar S The relation of healthy and Western dietary patterns to the risk of endometrial and ovarian cancers a systematic review and meta‘analysis Int J Vitamin Nutrition Res Alizadeh S Shab‘Bidar S Mohtavinejad N Djafarian K A posteriori dietary patterns and risk of pancreatic and renal cancers Nutrition Food Sci‘ence Mohseni R Abbasi S Mohseni F Rahimi F Alizadeh S Association between dietary inflammatory index and the risk of prostate cancer a meta‘analysis Nutr Cancer “ Shivappa N Steck SE Hurley TG et al A population‘based dietary inflammatory index predicts levels of C‘reactive protein in the seasonal variation of blood cholesterol study SEASONS Public Health Nutrition “ Xu H Sjögren P Ärnlöv J et al A proinflammatory diet is associated with systemic inflammation and reduced kidney function in elderly adults J Nutri “ Esfahani FH Asghari G Mirmiran P Azizi F Reproducibility and relative validity of food group intake in a food frequency question‘naire developed for the Tehran lipid and glucose study J Epidemiol “ Shivappa N Steck SE Hurley TG Hussey JR Hébert JR Designing and developing a literature‘derived population‘based dietary inflammatory index Public Health Nutri “ Bondonno NP Blekkenhorst LC Bird AL et al Dietary inflammatory index and the aging kidney in older women a ‘year prospective cohort study Eur J Nutri ‘ Maddahi NS Mirzaei K Aghamir SMK Modaresi SS Yekaninejad MS Major Dietary Patterns and kidney stone formation among Iranian men J Nutri Sci Dietetics ‘ Moghaddam MB Aghdam FB Jafarabadi MA Allahverdipour H Nikookheslat SD Safarpour S The Iranian version of international physi‘cal activity questionnaire IPAQ in Iran content and construct validity factor structure internal consistency and stability World Appl Sci J “ Trinchieri A Mandressi A Luongo P Longo G Pisani E The influence of diet on urinary risk factors for stones in healthy subjects and idiopathic renal calcium stone formers Br J Urol “ Rouhani MH Najafabadi MM Surkan PJ Esmaillzadeh A Feizi A Azadbakht L Dietary inflammatory index and its association with renal function and progression of chronic kidney disease Clin Nutri ESPEN “ 0cMaddahi a0et a0al BMC Res Notes Page of Noori N Honarkar E Goldfarb DS et al Urinary lithogenic risk profile in sgÃ¥rd R Rytter E Basu S Abramsson‘Zetterberg L Möller L Vessby B recurrent stone formers with hyperoxaluria a randomized controlled trial comparing DASH Dietary Approaches to Stop Hypertension‘style and low‘oxalate diets Am J Kidney Dis “ Leone A Fernández‘Montero A de la Fuente‘Arrillaga C et al Adherence to the Mediterranean dietary pattern and incidence of nephrolithiasis in the Seguimiento Universidad de Navarra Follow‘up SUN cohort Am J Kidney Dis “ Saneei P Hashemipour M Kelishadi R Esmaillzadeh A The Dietary Approaches to Stop Hypertension DASH diet affects inflammation in childhood metabolic syndrome a randomized cross‘over clinical trial Ann Nutr Metab “ Hsu C‘C Jhang H‘R Chang W‘T et al Associations between dietary patterns and kidney function indicators in type diabetes Clin Nutr “ Duda MK O™Shea KM Tintinu A et al Fish oil but not flaxseed oil decreases inflammation and prevents pressure overload‘induced cardiac dysfunction Cardiovasc Res “High intake of fruit and vegetables is related to low oxidative stress and inflammation in a group of patients with type diabetes Scand J Food Nutri “ Wood LG Shivappa N Berthon BS Gibson PG Hebert JR Dietary inflam‘matory index is related to asthma risk lung function and systemic inflam‘mation in asthma Clin Exp Allergy “ Hodge A Bassett J Shivappa N et al Dietary inflammatory index Medi‘terranean diet score and lung cancer a prospective study Cancer Causes Control “ Wirth MD Hébert JR Shivappa N et al Anti‘inflammatory dietary inflammatory INDEX scores are associated with healthier scores on other dietary indices Nutri Res “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in pub‘lished maps and institutional affiliations ¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your field¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research over 100M website views per year ¢ At BMC research is always in progressLearn more biomedcentralcomsubmissionsReady to submit your research Choose BMC and benefit from 0c'

Thyroid_Cancer

"production process errors may bediscovered which could affect the content and all legal disclaimers that apply to the journalpertain Published by Elsevier 0cSARSCoV2 another kind of liver aggressor how does it do that Sonia A LozanoSepulveda1 Kame GalanHuerta Natalia Mart­nezAcu±a Daniel ArellanosSoto and Ana Mar­a RivasEstilla1 1Department of Biochemistry and Molecular Medicine School of Medicine and Hospital Universitario œDr Jose E Gonzalez Autonomous University of Nuevo Len Monterrey Ana Mar­a RivasEstilla Department of Biochemistry and Molecular Medicine School of Medicine and Hospital Universitario œDr Jose E Gonzalez Autonomous University of NL Mxico Corresponding author Nuevo Leon Ave Francisco I Madero y Ave Gonzalitos sn Col Mitras Centro Journal Preproofbut complications such as pneumonia respiratory distress syndrome and multian Monterrey Nuevo Len Mxico Telfax Email amrivas1yahooca Abstract Clinical manifestations of SARSCoV2 infection include more frequently fever and cough failure can occur in persons with additional comorbidities Liver dysfunction is one of the most striking affections among patients suggesting that SARSCoV2 may represent a new king of liver aggressor However the molecular process underlying this phenomenon is 0cstill unclear In this work we overview the most recent findings between the molecular biology of the virus pathogenic mechanisms and its relationship to liver disease observed in patients Abbreviations AaDO2 ACE2 AIH ALT AST COVID19 GGT GI GTEx Alveolararterial Oxygen Gradient Angiotensinconverting enzyme Autoimmune Hepatitis Alanine transaminase Aspartate aminotransferase Coronavirus infectious disease Gamma glutamyl transpeptidase Gastrointestinal GenotypeTissue Expression Metabolicassociated fatty liver disease Nonstructural proteins Reading Frame Journal Preproofcomplex disease in many severely ill patients In other infected subjects an infection is Keywords SARSCoV2 liver liver impairment COVID19 ACE2 MAFLD nsp ORF Introduction SARSCoV2 is the etiological agent of the disease known as COVID19 which causes a disease characterized by pneumonia cough fever occasional diarrhea headache cardiac injury and in some patients™ liver alterations COVID19 has been found to be an extremely reported to be so severe that it can lead to a disproportionate and mortal reaction of the immune system known as a cytokine storm All of these factors make COVID19 highly unpredictable it is what specialists call a multisystem disease 0cAround the world cases of liver dysfunction denoted by elevated hepatic enzymes in serum such as AST aspartate aminotransferase and ALT alanine transaminase have been documented among patients infected with SARSCoV2 There is still no certainty whether the COVID19related liver damagedysfunction is due mainly to the viral replication per se drugs toxicity or other coexisting comorbidities whether sexrelated difference could help to explain why infected men are more healthy or harmful relationship between the liver and its viral aggressor In this paper we describe a brief overview of the implications for researchers in the field of It is important to understand how liver function can be altered by direct infection with this predisposed to develop COVID19associated liver dysfunction than infected women to analyze if there is any genetic predisposition related to impaired liver function during the œrespiratory virus which mechanisms of viral pathogenesis are involved to evaluate disease and of course the crosstalk between viral and cellular proteins that mediate this Journal Preproofliver disease of the most recent findings between the molecular biology of the virus This emerging viral illness is typically characterized by fever dry cough myalgia headache sore throat diarrhea and may be aggravated with shortness of breath and respiratory failure [] The angiotensinconverting enzyme ACE2 the functional receptor of the spike glycoprotein of SARSCoV2 is widely distributed in the anism Historically Hamming and colleagues reported ACE2 expression in the surface of lung alveolar epithelial cells enterocytes of the small intestine arterial and venous endothelial pathogenic mechanisms and its relationship to liver disease observed in patients Clinical characteristics and liver injury in patients with COVID19 0ccells and arterial smooth muscle cells [] Posterior transcriptomic and proteomic analyses confirmed their findings and added high ACE2 expression in adipose tissue bone marrow duodenum endometrium heart kidney small intestine smooth muscle testis and thyroid [] ACE2 is also expressed in liver but in lesser extent One of the most worrisome severe cases of COVID19 [] Regarding the gastrointestinal GI tract and liver over COVID19associated liver injury is defined as any liver damage that occurs during disease progression andor COVID19 treatment in patients with or without a history of previous complications is the unusual formation of blood clots in many patients with COVID19 even those who were receiving anticoagulants Researchers at Mount Sinai in New York published studies suggesting that clots in the lungs play an important role in the most of COVID19 patients develop GI symptoms such as anorexia diarrhea nausea and vomiting and a significant proportion present with altered liver function tests [] Journal PreproofDecreased albumin levels are associated with severe infection and poor prognosis Still AST elevation is the most common abnormality in patients presenting with COVID19 observed more frequently in men and is mainly documented in more severe cases [] liver disease In general the incidence of increased liver biochemical markers in hospitalized patients with COVID19 mainly AST and ALT and slightly elevated bilirubin varies between to of cases [] The increase in liver enzymes is there are no reports of acute or subacute liver failure in patients with COVID19 The largest cohort study that included cases of COVID19 from China showed that had preexisting chronic liver disease Lei and colleagues reported that impaired liver function was related to mortality in COVID19 patients [] Elevated AST was more frequent and significant than the increase of ALT in severe 0chospitalized patients Moreover elevated AST was shown to be associated with highest mortality risk [] In the study reported by Yijin Wang [] they found that of COVID patients had elevated AST activity The median levels of ALT were UL vs UL respectively AST were UL vs UL respectively in abnormal and normal aminotransferase groups Liver enzymes abnormality were associated with disease severity protein levels In addition they found by ultrastructural examination of coronavirus ps in hepatocytes in COVID19 cases SARSCoV2 infected hepatocytes displayed as well as a series of laboratory tests including higher Alveolararterial Oxygen Gradient AaDO2 higher gamma glutamyl transpeptidase GGT lower albumin and lower total conspicuous mitochondrial swelling endoplasmic reticulum dilatation and glycogen granule decreased Histological findings showed apoptosis and binuclear hepatocytes Journal Preproofshowed a disease course similar to that reported in non immunosuppressed population coronavirus the interaction of its proteins with cellular proteins and consequently the immunosuppressive therapy for autoimmune hepatitis AIH developing COVID19 Taken together both ultrastructural and histological evidence indicated a typical lesion of viral infection [] All these findings by different reports demonstrates that SARSCoV2 infection in liver is a crucial cause of hepatic impairment in COVID19 patients However alteration of cellular metabolism that give rise to systematic alterations and metabolic Report from Alessio Gerussi et al[] demonstrated that patients under today the cellular and molecular mechanisms that are altered by infection with this changes are still unknown 0cMolecular biology of SARSCov2 Coronaviruses are enveloped viruses that contain a positively polarized unsegmented RNA genome belonging to the Coronaviridae family and the order of Nidovirales they are distributed in humans and other mammals[] The size of the SARSCoV2 virions is approximately to nm in diameter [“] SARSCoV2 has a genome that consists polymerase RdRp which is nsp12 and is responsible of the replication and transcription of the virus[] which are encoded by the various genetic loci on the genome [] At the center of the virion lies a nucleocapsid composed of the genomic RNA and the nucleocapsid protein[] The virus glycoprotein S consists of two subunits S1 which is at the amino terminus and that encodes for structural proteins and a larger region that encodes two reading frames ORF 1a and 1b which together encode for the nonstructural virus proteins from nucleocapsid protein which is within the phospholipid bilayer and nonstructural proteins nsp1 to nsp16 []The virions have a structural Sspike protein outer spiky glycoprotein Mmembrane protein a type III transmembrane glycoprotein Nof nucleotides [] encoding amino acids and it is composed of a region Journal Preproofvirus [] as well as protein M which is a type III transmembrane glycoprotein[] and participates in the cellular membrane rearrangements for the replication and transcription complexes[] Among the encoded proteins is an RNAdependent RNA provides the receptor binding site and S2 which is at the carboxyl terminus responsible for membrane fusion [] The envelope protein E has a role in the assembly and release of the Nonstructural proteins have several functions during de viral cycle For example nsp 0cThe virus enters the cell by endocytosis through the interaction between envelope glycoprotein S with the cell receptor ACE2 and with the participation of the type II transmembrane serine protease TMPRSS2 [] Once it enters the cell the N protein with viral genome are released within the cytoplasm then cellular proteases degrade the capsid and the virus genome is left free Next the polyprotein containing the viral proteins that are How does the virus select which cells to infect Viruses can infect only certain species of hosts and only permissive cells within that host Permissive cells make all the necessary proteins and viral factors to allow virus to replicate Once a virus gets inside a cell it hijacks the cellular processes to produce virally encoded proteins that will replicate the virus™s genetic material []Viral replication may cause exocytosis[] will translate into viral proteins this entire process will occur in the cell cytoplasm The processed to form the replication complex is translated and then the complementary strand of negative sense pregenomic RNA is synthesized to be used as a template to replicate the structural proteins that will be synthesized in the endoplasmic reticulum membrane to assembly the viral p and finish the cycle through the release of the viruses by positive sense viral genome[] Furthermore the replication and transcription complex will lead to a series of smaller positive sense subgenomic RNAs these are the ones that Journal PreproofBoth SARSCoV and the new SARSCoV2 are very similar in structure and pathogenicity but the major structural protein S protein is slightly different between them[] Compared to other beta coronaviruses the presence of a furinlike cleavage site in SARSCoV2 enables the S protein priming and facilitates an increase on the efficiency of the spread of SARSCoV2 as is reported wide world [] 0cbiochemical changes producing cell damage called cytopathic effects Like other coronaviruses SARSCoV2 requires cellular receptors to initiate its internalization to the cells that carry these factors []Li SARSCoV2 uses the angiotensinconverting enzyme ACE2 as a host cell receptor SARSCoV2 spike S protein binds ACE2 with significantly high affinity[] In addition the main host protease that suggested to promote the pathogenesis of this coronavirus [] program httpsportalgdccancergov They compared ACE2 expression levels across human tissues between males and females and between younger and older persons in these individual tissues Furthermore other reports have analyzed the correlations between ACE2 In order to provide insights into the mechanism of SARSCoV2 infection Li [] analyzed the expression of ACE2 in various normal human tissues using the datasets from the GenotypeTissue Expression GTEx project and The Cancer Genome Atlas TCGA transmembrane serine protease[] Other host proteases such as furin have also been mediates Sprotein activation on primary target cells and initial viral entry is the type II Journal Preproofreported by Li ACE2 expression levels showed no significant difference between have no significant association with sex age or race Is the liver a direct target for SARSCoV2 males and females between younger and older persons or between Asian and nonAsian races This finding suggests that the infection risk of SARSCoV2 and SARSCoV may expression levels and immune signature enrichment levels in individual tissues As As we expected because the systemic manifestations of COVID19 it has been reported that SARSCoV2 has an anotropism beyond the respiratory tract including the kidneys 0cliver heart and brain and possibly that anotropism influences the course of Covid19 disease and aggravates preexisting conditions The ACE2 protein is found at high levels in the GI tract as the colon biliary system and liver [] On the other hand it is well documented a SARSCoV2 RNA shedding in the GI tract [] supporting its tropism for architecture express ACE TMPRSS1 receptors [] The presence of these two host factors in the liver suggests that a direct viral cytopathic effect occurs Also in SARS infection the presence of viral RNA in liver tissue was documented but not as extensively as the new coronavirus Data published by Gordon [] suggest that mitochondrial proteins interact directly with the virus which helps to understand the potential mechanism by which elevated AST the GI tract and liver cells and these may be sites of active viral replication and either direct or indirect tissue injury Indeed a large part of the cells distributed in the liver Journal Preproofeffect the exacerbated inflammatory response in COVID19 may play a central role in profiles are detected in these patients [] Furthermore in addition to this intracellular More recently [] identified the clinical and laboratory characteristics of COVID19 patients with abnormal liver transaminases and they reported that SARSCoV2 is able to which high levels of IL6 have been reported [] which are involved in both infect liver cells and cause liver impairment by direct cytopathic effect inflammatory and repair responses in liver disease Mechanisms of liver pathogenicity 0cIf SARSCoV2 replication has direct adverse effects on liver function it is still unknown Findings in liver biopsy of patients killed by COVID19 showed moderate micro vesicular steatosis and mild portal and necroinflammatory activity[] This seems to indicate that a direct injury occurred while the infection that could have been directly caused by SARSCoV2 Another possibility is that a druginduced liver injury occurred To date there are the following possible mechanisms Figure infection The massive release of cytokines by the immune system in response to the viral infection can result in a cytokine storm and symptoms of sepsis that are the cause of death in of fatal COVID19 cases [] In these cases uncontrolled inflammation induces multian damage leading including liver failure Biomarkers of inflammation such as Creactive protein PCR serum ferritin LDH Ddimer IL6 IL2 are have been found to be significantly elevated in Immune damage from exacerbated inflammation in response to SARSCoV2 Journal Preproofpathogenesis of SARS CoV “related liver disease more studies should be liver is a potential target for direct infection with this virus To understand the performed for evidence of viral mechanisms of replication in different cell anelles as cytoplasm endoplasmic reticulum Golgi apparatus and lipidrafts CoV2 enters cells through the ACE2 molecule which is abundantly expressed in the liver and in particular in bile epithelial cells [] Based on this expression the Direct cytopathic effect due to active viral replication in various liver cells SARScritically ill patients with COVID19 [] into hepatocytes and liver histology characterization It is also important to know in cells their capacity to efficiently produce both infectious and defective non 0cinfective whole virions There are not yet enough data to know the viral dynamics in the different tissues and the associated pathogenesis Anoxia respiratory failure is one of the main characteristics of COVID19 Anoxic hypoxic hepatitis is common in patients with severe symptoms [] Reactivation of preexisting liver disease Patients with preexisting chronic liver medications such as tocilizumab and baricitinib used to combat the adverse immune cholestatic liver disease various studies such as lopinavir ritonavir remdesivir chloroquine tocilizumab uminefovir traditional Chinese medicine so it is important to consider that they could be potentially hepatotoxic in some patients [] Druginduced liver damage DILI Initial clinical guidelines recommended antiviral agents for COVID19 so a variety of drugs have been administered in disease may be more susceptible to liver damage from SARSCoV2 Biological Journal Preproof Genetic factors Genetics may well be one of the determining factors in some reaction may also cause HBV reactivation [] and induce eventual impairment of liver function in those patients with HBV On the other hand it is still unknown whether SARSCoV2 infection exacerbates cholestasis in people with underlying patients who become seriously ill with COVID19 but until now we cannot be sure It is possible for example that the genetic variation that makes an individual more susceptible to high blood pressure or diabetes also makes him more vulnerable to the virus It will be important to find out what role genetic factors predisposing to liver steatosis have and their sensitivity to severe symptoms of COVID19 Ji and 0ccolleagues showed that subjects with metabolicassociated fatty liver disease MAFLD have a higher risk of COVID19 severity disease and abnormal liver blood tests than patients without MAFLD [] In contrast Louise Biquard [] demonstrated that MAFLD is not associated with changes in liver expression blood test abnormalities reported by Ji and colleagues is thus likely not explained by Concluding remarks The scenario is not yet complete which does not allow us to establish or understand the natural history of the disease and the participation or commitment of the liver in this disease Certainly the application of new technological platforms such as singlecell increased hepatic SARSCoV2 uptake Still several contradictory reports will help of genes implicated in SARSCoV2 infection The observed persistence of liver to find the real role of genetic factors in the evolution of this disease Journal Preprooftranscriptomic assays will allow us to quickly know the commitment of each cell type in affected ans and the meaning of viral dynamics in the various affected systems including the liver However as we have already learned from the old hepatotropic viruses history still is ongoing and we have much to learn and understand about the virologic characteristics of this emerging RNA virus that allow us to develop specific antivirals such as the case of HCV and the vaccine to decrease the impact of this œacute infection Declarations of interest none Ethical approval Not required 0c References [] Chen N Zhou M Dong X Qu J Gong F Han Y Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China httpsdoi101002path1570 [] Wang D Eraslan B Wieland T Hallstr¶m B Hopf T Zolg DP A deep proteome and transcriptome abundance atlas of healthy human tissues Mol Syst [] Hamming I Timens W Bulthuis MLC Lely AT Navis GJ van Goor H Tissue distribution of ACE2 protein the functional receptor for SARS coronavirus A first step in understanding SARS pathogenesis J Pathol “ a descriptive study Lancet “ httpsdoi101016S0140Journal Preproofa manifestation of COVID19 Rev Gastroenterol Mxico English Ed Biol 201915e8503 httpsdoi1015252msb20188503 Patients with COVID19 J Am Coll Cardiol httpsdoi101016jjacc202005001 [] Paranjpe I Fuster V Lala A Russak A Glicksberg BS Levin MA Association of Treatment Dose Anticoagulation with InHospital Survival Among Hospitalized [] Schmulson M D¡valos MF Berumen J Beware Gastrointestinal symptoms can be httpsdoi101016jrgmxen202004001 [] Cai Q Huang D Yu H Zhu Z Xia Z Su Y COVID19 Abnormal liver function tests J Hepatol httpsdoi101016jjhep202004006 0c[] Siddiqi HK Mehra MR COVID19 illness in native and immunosuppressed states A clinicaltherapeutic staging proposal J Hear Lung Transplant Off Publ Int Soc Hear Transplant “ httpsdoi101016jhealun202003012 [] Feng G Zheng KI Yan QQ Rios RS Targher G Byrne CD COVID19 and [] between markers of liver injury and mortality in COVID19 in China Hepatology httpsdoi101002hep31301 [] de la Rica R Bes M Aranda M del Castillo A Socias A Payeras A Low Transl Hepatol “ httpsdoi1014218JCTH202000018 albumin levels are associated with poorer outcomes in a case series of COVID19 patients in Spain a retrospective cohort study MedRxiv Liver Dysfunction Current Insights and Emergent Therapeutic Strategies J Clin Lei F Liu YM Zhou F Qin JJ Zhang P Zhu L Longitudinal association Journal Preproofmortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet “ httpsdoi101016S0140liver directly contributes to hepatic impairment in patients with COVID19 J Hepatol httpsdoi101016jjhep202005002 httpsdoi1011012020050720094987 [] Zhou F Yu T Du R Fan G Liu Y Liu Z Clinical course and risk factors for [] Wang Y Liu S Liu H Li W Lin F Jiang L SARSCoV2 infection of the [] Gerussi A Rigamonti C Elia C Cazzagon N Floreani A Pozzi R Coronavirus Disease COVID19 in autoimmune hepatitis a lesson from 0cimmunosuppressed patients Hepatol Commun 2020na httpsdoi101002hep41557 [] Richman D Whitley R Hayden F Clinical virology 4th ed ASM Press [] Ksiazek TG Erdman D Goldsmith CS Zaki SR Peret T Emery S A Novel “ httpsdoi101056NEJMoa030781 [] Kuiken T Fouchier RAM Schutten M Rimmelzwaan GF van Amerongen G van respiratory syndrome Lancet “ httpsdoi101016S0140[] Drosten C G¼nther S Preiser W Van der Werf S Brodt HR Becker S Identification of a novel coronavirus in patients with severe acute respiratory Riel D Newly discovered coronavirus as the primary cause of severe acute Coronavirus Associated with Severe Acute Respiratory Syndrome N Engl J Med Journal Preproofsyndrome N Engl J Med “ httpsdoi101056NEJMoa030747 outbreak associated with a new coronavirus of probable bat origin Nature “ httpsdoi101038s4158602020127 [] Mortola E Roy P Efficient assembly and release of SARS coronaviruslike ps by a heterologous expression system FEBS Lett “ httpsdoi101016jfebslet200409009 [] Fehr A Perlman S Coronaviruses Methods and Protocols Methods in Molecular Biology Chapter Coronaviruses an overview of their replication and [] Zhou P Yang XL Lou Wang XGG Hu B Zhang L Zhang W A pneumonia 0cpathogenesis Springer Berlin Heidelberg [] Belouzard S Millet JK Licitra BN Whittaker GR Mechanisms of coronavirus cell entry mediated by the viral spike protein Viruses “ httpsdoi103390v4061011 [] Vennema H Godeke GJ Rossen JW Voorhout WF Horzinek MC Opstelten DJ et [] Snijder EJ Decroly E Ziebuhr J The Nonstructural Proteins Directing Coronavirus [] Neuman BW Buchmeier MJ Supramolecular Architecture of the Coronavirus P Adv Virus Res “ httpsdoi101016bsaivir201608005 [] van der Hoeven B Oudshoorn D Koster AJ Snijder EJ Kikkert M Barcena M [] Neuman BW Kiss G Kunding AH Bhella D Baksh MF Connelly S A structural analysis of M protein in coronavirus assembly and morphology J Struct Biol “ httpsdoi101016jjsb201011021 expression of viral envelope protein genes EMBO J “ al Nucleocapsidindependent assembly of coronaviruslike ps by coJournal PreproofBiogenesis and architecture of arterivirus replication anelles Virus Res “ httpsdoi101016jvirusres201604001 RNA Synthesis and Processing vol 1st ed Elsevier Inc httpsdoi101016bsaivir201608008 [] Rabi F Al Zoubi M Kasasbeh G Salameh D AlNasser A SARSCoV2 and Coronavirus Disease what we know so far Pathogens [] Masters P The molecular biology of coronaviruses Adv Virus Res “ 0c [] Shang J Ye G Shi K Wan Y Luo C Aihara H Structural basis of receptor recognition by SARSCoV2 Nature “ httpsdoi101038s415860202179y [] Rabaan AA AlAhmed SH Haque S Sah R Tiwari R Malik YS SARSCoV“ [] Li W Moore MJ Vasilieva N Sui J Wong SK Berne MA Angiotensin“ httpsdoi101038nature02145 [] Hoffmann M KleineWeber H P¶hlmann S A Multibasic Cleavage Site in the [] Cohen FS How Viruses Invade Cells Biophys J “ httpsdoi101016jbpj201602006 SARSCoV and MERSCOV A comparative overview Le Infez Med converting enzyme is a functional receptor for the SARS coronavirus Nature Journal PreproofEM structure of the 2019nCoV spike in the prefusion conformation Science “ httpsdoi101126scienceabb2507 Spike Protein of SARSCoV2 Is Essential for Infection of Human Lung Cells Mol Cell 202078779784e5 httpsdoi101016jmolcel202004022 [] Ziegler CGK Allon SJ Nyquist SK Mbano IM Miao VN Tzouanas CN SARSCoV2 Receptor ACE2 Is an InterferonStimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues Cell “ httpsdoi101016jcell202004035 [] Wrapp D Wang N Corbett KS Goldsmith JA Hsieh CL Abiona O Cryo 0c[] Follis K York J Nunberg J Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell“cell fusion but does not affect virion entry Virology “ httpsdoi101016jvirol200602003 [] Millet JK Whittaker GR Host cell proteases Critical determinants of coronavirus [] Li MYY Li L Zhang Y Wang XSS Expression of the SARSCoV2 cell receptor httpsdoi101186s4024902000662x [] Xu H Zhong L Deng J Peng J Dan H Zeng X High expression of ACE2 receptor of 2019nCoV on the epithelial cells of oral mucosa Int J Oral Sci httpsdoi101038s413680200074x tropism and pathogenesis Virus Res “ httpsdoi101016jvirusres201411021 gene ACE2 in a wide variety of human tissues Infect Dis Poverty 20209NA Journal PreproofCoV2 protein interaction map reveals targets for drug repurposing Nature Infection of SARSCoV2 Gastroenterology 202015818311833e3 httpsdoi101053jgastro202002055 httpsdoi101038s4158602022869 [] Xu L Liu J Lu M Yang D Zheng X Liver injury during highly pathogenic human coronavirus infections Liver Int Off J Int Assoc Study Liver “ httpsdoi101111liv14435 [] Coomes EA Haghbayan H Interleukin6 in COVID19 A Systematic Review and [] Xiao F Tang M Zheng X Liu Y Li X Shan H Evidence for Gastrointestinal [] Gordon DE Jang GM Bouhaddou M Xu J Obernier K White KM A SARS 0cMetaAnalysis MedRxiv httpsdoi1011012020033020048058 [] Xu Z Shi L Wang Y Zhang J Huang L Zhang C Pathological findings of COVID19 associated with acute respiratory distress syndrome Lancet Respir Med “ httpsdoi101016S221326002030076X [] Zhang B Zhou X Qiu Y Feng F Feng J Jia Y Clinical characteristics of [] Chen G Wu D Guo W Cao Y Huang D Wang H Clinical and [] Chai X Hu L Zhang Y Han W Lu Z Ke A Specific ACE2 Expression in Invest “ httpsdoi101172JCI137244 death cases with COVID19 MedRxiv httpsdoi1011012020022620028191 immunological features of severe and moderate coronavirus disease J Clin Journal PreproofCholangiocytes May Cause Liver Damage After 2019nCoV Infection BioRxiv patients MedRxiv httpsdoi1011012020040120047381 httpsdoi10110120200203931766 [] Herold T Jurinovic V Arnreich C Hellmuth JC von BergweltBaildon M Klein M Level of IL6 predicts respiratory failure in hospitalized symptomatic COVID[] Grein J Ohmagari N Shin D Diaz G Asperges E Castagna A Compassionate Use of Remdesivir for Patients with Severe Covid19 N Engl J Med “ httpsdoi101056nejmoa2007016 [] U S Food and Drug Administration Fact sheet for health care providers emergency 0cuse authorization EUA of hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of COVID19 in certain hospitalized patients [] Varona Prez J Rodriguez Chinesta JM Riesgo de reactivacin de la hepatitis B asociado al tratamiento con corticoides frente a SARSCoV2 COVID19 Rev Cl­nica Espa±ola httpsdoi101016jrce202004012 [] Ji D Qin E Xu J Zhang D Cheng G Wang Y Nonalcoholic fatty liver httpsdoi101016jjhep202003044 SARSCoV2 in metabolicassociated fatty liver disease J Hepatol diseases in patients with COVID19 A retrospective study J Hepatol Journal Preproof[] Biquard L Valla D Rautou PE No evidence for an increased liver uptake of httpsdoi101016jjhep202004035 0cFigure legends Journal PreproofFig1 Proposed mechanisms of liver pathogenicity of SARSCoV2 in infected cells SARS CoV2 Infection2 Cytokinestorm3 Drugeffects4 Hypoxia5 PreviousliverdamageBiochemicallabmarkersWhite bloodcellsGenomereleaseReplicationTranslationVirionassemblyViral proteinsMaturevirus release\uf0e9AaDO2MitochondrialproteinsHypoxicisquemicliverinjuryLIVER DAMAGELopinavirRitonavirRemdesivirChloroquineTocilizumabOxidativeimbalanceSteatosisACE2S proteinCytopathiceffect\uf0e9GMCSF\uf0e9IL6\uf0e9IL1β\uf0e9IL2\uf0e9IL8\uf0e9CCL2\uf0e9CCL3\uf0e9CCL5\uf0e9CXCL \uf0e9ALT\uf0e9AST\uf0eaAlbumin\uf0e9PCR\uf0e9LDH\uf0e9Ddimer\uf0e9Ferritin\uf0e9Bilirubin 0c"

Thyroid_Cancer

Lung cancer has high mortality often accompanied with systemic metabolicdisorders The present study aimed at defining values of transnodules crossclinical phenomic and lipidomic network layers in patients with adenocarcinoma ADC squamous cell carcinomas or small cell lung cancer SCLCWe measured plasma lipidomic profiles of lung cancer patients and found thataltered lipid panels and concentrations varied among lung cancer subtypes genders ages stages metastatic status nutritional status and clinical phenomeseverity It was shown that phosphatidylethanolamine elements and were SCLC specific whereas lysophosphatidylcholine and snposition1 and phosphatidylcholine and were ADCspecific There were statistically more lipids declined in male ages latestage metastasis or body mass index Clinical transomics analyses demonstrated that one phenome in lung cancer subtypes might be generated from multiple metabolic pathways and metabolites whereas a metabolic pathway andmetabolite could contribute to different phenomes among subtypes althoughthose needed to be furthermore confirmed by bigger studies including larger population of patients in multicenters Thus our data suggested that transomic profiles between clinical phenomes and lipidomes might have the value to uncoverthe heterogeneity of lipid metabolism among lung cancer subtypes and to screenout phenomebased lipid panels as subtypespecific biomarkersK E Y WO R D Slipidomics lung cancer phenomes subtypes transomicsINTRODUCTIONLung cancer is a systemic and aggressive disease withhigh morbidity and mortality and it is often accompanied with systemic metabolic disorders for exampleup or downregulated expression of mechanismassociatedgenes or activation of metabolismdependent enzymes Forexample metabolismassociated genes of small cell lungThis is an access under the terms of the Creative Commons Attribution License which permits use distribution and reproduction in any medium provided theoriginal work is properly cited The Authors Clinical and Translational Medicine published by John Wiley Sons Australia Ltd on behalf of Shanghai Institute of Clinical BioinformaticsClin Transl Med 202010e151101002ctm2151wileyonlinelibrarycomjournalctm2 of 0c of ZHU et alcancer SCLC cells altered after mitogenactivated proteinkinase MAPK kinase module MEK5ERK5 was blockedaccompanied by dysfunctions of several lipid metabolismpathways like the mevalonate pathway for cholesterolsynthesis1 Lipids mainly including subclasses of phosphatidic acid PA phosphatidylcholines PC phosphatidylethanolamine PE phosphatidylglycerol PGphosphatidylinositol PI and phosphatidylserine PShave multiple important biological functions such asbiomembrane composition vesicular trafficking adhesion migration apoptosis energy storage neurotransmission signal transduction and posttranslational modification They have alterations under circumstance of lungcancer Circulating levels of PCs and PEs in patients withnonsmall cell lung cancer NSCLC differed from thosewith noncancer lung diseases or health and were suggested as diagnostic biomarkers of early NSCLC2 Theheterogeneity of circulating lipidomic profiles was foundto exist among patients with squamous cell carcinomasSCC adenocarcinoma ADC or SCLC and there was aclear correlation between genomic and lipidomic profilesof lipidassociated proteins and enzymes3 As the part ofclinical transomics the lung cancerspecific and subtypespecific lipidomics in the circulation were defined and evidenced by integrating lipidomic data with genomic expression of lipid proteins among lung cancer subtypesClinical transomics is defined as a new subject tointegrate clinical phenomes with molecular multiomicsfor understanding molecular mechanisms of diseases inmultiple dimensions4 Clinical transomics becomes moreimportant as a new and novel approach for the discovery of diseasespecific biomarkers and therapeutic targetsalthough there are still many obstacles to be overcomefor example specificity and decisive role of transnodulesamong multiomic networks for intra and intercellular communication56 Recent studies applied the transomics among phosphorproteomics transcriptomics genesequencing and genomics for new molecular category ofliver cancer to provide a new therapeutic strategy7 As thepart of clinical transomics clinical lipidomics was considered as one of major metabolic profiles for identificationand validation of lung cancerspecific biomarkers by integrating clinical phenomes with lipidomic profiles89 Clinical lipidomics could demonstrate the complexity of thelipidome in metabolic diseases and lung cancer and presented the variation among diseases and subtypes of lungcancer1012Our previous study demonstrated the difference oflipidomic profiles among patients with different lungcancer subtypes and the potential association betweenlipidomic phenotypes and gene expression oflipidmetabolismassociated proteins and enzymes as a conceptevaluation3 The present study furthermore investigatedthe values of transnodules crossclinical phenomic andlipidomic network layers in the recognition of lung cancersubtypes ADC SCC and SCLC in order to understandclinical phenomeassociated lipid changes or lipidomicphenotypeassociated clinical phenomes We also evaluated the differences of lipidomic profiles between maleand female various ages early and late stages with orwithout metastasis body mass index BMI or and digital evaluation scores less or more than METHODS AND MATERIALSChemical agentsThe internal standard cocktails were subscribed fromAvanti Lipids Polar Alabaster AL USA the acetone acetonitrile ammonium bicarbonate dithiothreitol formicacid iodoacetamide and Tris base Analytical Gradefrom SigmaAldrich St Louis MO USA and ammonium acetate NH4OAc hexane isopropyl alcohol IPAmethanol and highperformance liquid chromatographygrade chloroform CHCl3 from Merck Millipore Billerica MA USAPatient populationThe study designed as a casecontrol approach wasapproved by the Ethical Evaluation Committee of Zhongshan Hospital ethical code B2018187 The subjectsgave informed consent for clinical data collection andlipids analysis before all the other procedures The studyincluded lung cancer patients diagnosed according topathology of whom were ADC SCC SCLC and other healthy people The stage and severity of lung cancerwere defined according to the Eighth Edition of TNMClassification for Lung Cancer13 Patients were recruitedduring October to March Healthy controlsparticipated were blood donors in Zhongshan HospitalSubjects with other respiratory diseases or family historyof lung cancer were excluded Fasting blood was drawnfrom healthy controls and lung cancer patients on theday of entering hospital to harvest plasma All the clinicaldata including symptoms signs laboratory tests imagespathologic information and survival status years laterwere collected and followed upDigital evaluation score systemThe Digital Evaluation Score System DESS is a scoreindex system by which clinical descriptive information of 0cZHU of each phenome can be translated into clinical informatics14When the severity of each component was scored as or of which represented the most severe condition whereas indicated normal physiological range Thegross DESS scores ranged from to points the higherthe score the severer the condition A total of clinical phenomes were collected and scored in each of threelung cancer group including histories symptoms signs laboratory measurements image features and pathologic indexes as listed in Table S1spectrometry analysisLipid extraction for massAbout µL plasma was collected into a glass tubeinto which µL internal standard was added and then mL of methanolchloroformformic acid asreported previously315 This mixture was incubated at “—¦C overnight after vigorous shaking Two milliliters ofHajra™s reagent M H3PO4 M KCl were droppedblended and centrifuged at rpm for min Afterstratification chloroform in the lower layer was pipettedto another glass tube and concentrated to µL withthe nitrogen flow where the liquid with isopropyl alcoholhexane100 mM ammonium acetate at the ratio of was added till mL The sample was then centrifugated at rpm at —¦C for min The normalphaseliquid chromatography coupled TripleQuadrupole massspectrometer QTRAP SCIEX Framingham MAUSA was used for lipid extraction by the positive and negative electrospray ionization mode In the multiple reactionQTrap was utilized to scan the precursorproduct ion andexamine the mode operation Each test was repeated threetimes The peak area of each pair was quantified with multiple reaction monitoring data by the software MultiQuantAB SCIEXPurification of plasma lipidsLipid samples were derived through Ultimate SiO2 mm × mm µm Agilent Technologies Santa ClaraCA USA with mLmin flow rate highpurity heliumIn the meanwhile µL was added with the split ratio of at the ignition chamber temperature of —¦C and theinjection port temperature of —¦C It was started at temperature —¦C which gradually increased —¦Cmin to—¦C and kept for min The mass spectrometry was subjected to liquid chromatographymass spectrometer analysis FOCUS DSQTM II Thermo Fisher Scientific mainlyunder the following conditions Electron Ionization EI asionization source ion source temperature at —¦C ionization voltage at eV multiplier voltage at kV minsolvent delaying and amu of scan rangeIdentification of lipidomic profilesLipid extracts were loaded onto an Ultremex silica column mm × mm µm which was fitted with a mm× mm silica guard cartridge Phenomenex TorranceCA USA and then eluted The sample was enriched ata gradient of nLmin In the min™s run B phasewas from to min then rose to from to min linearly ramped for min as to return from to min until the end The QTrap was conductedin the multiplereaction monitoring mode and the different precursorproduct ion pairs were scanned in thepositivenegative electrospray ionization mode Up to lipids of plasma samples were carried out to get possiblelipids chemical structureslipidomic profilesComprehensive analyses ofMultiQuant software AB SCIEX was used to process dataafter lipids were identified by mass spectrometry Further MetaboAnalyst software wwwmetaboanalystcawas utilized for conducting multivariate statistical analysis cluster analysis dimensionality reduction and makingheat mapphenome and lipidome network layersTransnodule analyses crossThe typespecific lipids were identified as more than twotimes elevated or declined significantly compared withother lung cancer subtypes fold change and Pvalue whereas the coexpression lipids were identified as those similarly changed in all lung cancer subtypesas compared with healthy controls The expression quantitative trait locus eQTL model was utilized to evaluatetransnodules between lipidomic profiles and clinical phenomesStatistical analysisData were presented as mean ± SE The means of eachgroup were used for calculation and comparison Statistical significance of differences between two groupsor among multiple groups was determined by Student™sttest or oneway ANOVA test respectively Statistical 0c of ZHU et alsignificance was affirmed when Pvalue We alsoseparately calculated mean values of each phenome™sDESS score in different lung cancer subtypes which wereranked to obtain top clinical phenomes of those threegroups of patients Volcano maps showed the significantlyelevated or declined lipids in ADC SCC or SCLC patientsA VIP plot was further exploited to sort the lipids according to their importance to differentiate the four groups Toexplore the correlation between lipid elements and clinical phenomes we applied the lipidquantitative trait locimodel modified from eQTL model Besides MatrixlQTL Rpackage was used to acquire the significant phenomelipidpairs and corresponding Pvalues Moreover GraphPadPrism was utilized to make the receiver operating characteristic curve to evaluate the earlydiagnostic value andaccuracy of clinical phenomespecific lipid elements inADC SCC or SCLC The present study furthermore analyzed the significant differences of lipids among differentages eg and between female and maleearly and late stage metastasis and nonmetastasis highand low DESS scores ‰¤ and and high and lowBMI ‰¤ and RESULTSwith lung cancer subtypesClinical phenomes of patientsEighteen female and male lung cancer patients wereenrolled in the present study aged from to ± years old including ADC SCC and SCLC The totalscores of DESS were ± ± and ± in patientswith ADC SCC and SCLC respectively The DESS values of SCLC group were significantly higher than those ofhealthy control group P Top clinical phenomesof ADC SCC or SCLC patients as well as patients survivedor nonsurvived during study period were listed in Table Stages at primary diagnosis and recruitment period for thestudy lymphatic metastasis N12 in ipsilateral paratracheal hilum or mediastinum and enhanced images egfocus enhanced in CT or hypermetabolism in PETCTwere shown in all three subtypes of lung cancers In addition thyroid transcription factor1 TTF1 Napsin A keratin and location of tumor were noticed in ADC obscureboundary emphysema tumor size the cycle number offirst line chemotherapy obstructive pneumonia atelectasis and pulmonary nodule in SCC as well as number ofmetastatic lymph nodes in SCLC separately Top clinicalphenomes were similar between survived and nonsurvivedpatients but the total amounts of DESS of nonsurvivedpatients were significantly higher than those of survivedpatients Table Of total clinical phenomes hadthe statistical significance of each two groups inbetweenTable P or lesswith lung cancer subtypesLipidomic profiles of patientsTotal lipid elements of plasma were identified qualitatively and quantitatively mainly including PAs PCs PEs PGs PIs PSs lysophosphatidylcholineslysoPC lysophosphatidylethanolamines lysoPE lysophosphatidylglycerols lysoPG lysophosphatidylinositols lysoPI lysophosphatidylserines lysoPS ninediacylglycerides and triacylglycerols TAG Levels ofsome lipid elements in ADC SCC or SCLC patients weresignificantly higher Table S2 or lower Table S3 as compared with healthy control twofold P The majority of those elevated lipid elements were PC PA and lysoPC in ADC PE PC PS and PG in SCC or PS PE PG lysoPS and lysoPI in SCLC Of those declinedlipid elements were PS in ADC whereas and were PA in SCC and SCLC respectively Table demonstrates lung cancer subtypespecific lipid elements identified by those lipid elements elevated or declined exclusively in each lung cancer subtype for example some oflysoPC and PC in ADC whereas lysoPI lysoPS PE andPA in SCLC By partial least squares discrimination analysis PLSDA analysis top lipid elements were definedon the basis of variable import in project VIP score of eachgroup TAG565 lysoPG182 and PG and increased in ADC lysoPG181 PA140245 PI384180PA and and PE385PE180 increased inSCLC and PI362PI180 and lysoPG182 decreased in SCCdetail in Figure 1A There was a clear distribution oftop lipid elements among lung cancer groups as compared with the healthy control Figure 1B Of those significantly increased lipid elements in patients with lungcancers top six lipids of each group were identified Figure levels of LysoPC sn2 sn1 and sn1 in ADC Figure 2A PS363 in SCC Figure 2B andPA and and PI and inSCLC Figure 2C were significantly higher than in otherthree groups PLSDA component analysis demonstratedthat five principal components selected were and Figure 3A In the atom map whichwas based on the expression of major C atom numbersin various lipid types levels of lipids with carbons and increased whereas those with carbons decreased as compared with healthy control Figure 3BAs compared with the healthy control Figure 4A wenoticed that PI mainly declined in ADC Figure 4B PA 0cZHU of TA B L E carcinoma SCLC as well as lung cancer patients survived or nonsurvivedTop clinical phenomes of patients with adenocarcinoma ADC squamous cell carcinomas SCC or small cell lungPatients with ADCStage at primarydiagnosis ± Stage at recruitmenttime ± TTF1 ± N2 ipsilateralmediastinum ± Napsin A ± Enhanced image ± Location ± N1 ipsilateralparatracheal ± N1 ipsilateral hilum ± CK7Patients with SCCN1 ipsilateral hilum ± Enhanced image ± Stage at recruitmenttime ± Stage at primarydiagnosis ± obscure boundary ± Emphysema ± T tumor ± L1 cycle ± Obstructivepneumoniaatelectasis ± pulmonary nodulePatients with SCLCStage at recruitmenttime ± Stage at primarydiagnosis ± N1 ipsilateralParatracheal ± T tumor ± N1 ipsilateral hilum ± N2 ipsilateralmediastinum ± Enhanced image ± pulmonary nodule ± N LN ± MaintenancetreatmentPatients survivedStage at primarydiagnosis ± N2 ipsilateralmediastinum ± Enhanced image ± Stage at recruitmenttime ± N1 ipsilateral hilum ± TTF1 ± Napsin A ± Location ± N1 ipsilateralparatracheal ± LobularPatientsnonsurvivedStage at recruitmenttime ± Stage at primarydiagnosis ± N1 ipsilateralparatracheal ± N2 ipsilateralmediastinum ± N1 ipsilateral hilum ± Enhanced image ± N2 below carina ± TTF1 ± N LN ± T tumor ± ± Abbreviations N degrees of lung cancer metastasis to lymph nodes of TNM category N1 degree that has metastatic lymph nodes near pulmonary center andside of main bronchia N2 degree that has metastatic lymph nodes in the same side of the mediastinum as lung cancer TTF1 thyroid transcription factor1 asan immunohistochemical biomarker for adenocarcinoma CK7 keratin as an immunohistochemical biomarker for epithelial cells L1 cycle number of the firstline chemotherapy cycles ± ± ± in ADC and SCC Figure 4C and lysoPG in SCLC Figure 4D whereas PG and TAG increased in ADC and SCCPE in SCLC and PC in SCC The volcanic map demonstrated the clear patterns of lipid elements significantlyincreased or declined between heathy controls with ADCFigure 4E SCC Figure 4F or SCLC Figure 4G and varied among different subtypes of lung cancerspatient gendersDifferent lipidomics betweenAbout or lipid elements significantly increased and or declined more than twofold in male or femalelung cancer patients as compared with male or femalehealthy controls respectively Tables S4 and S5 Of thosePC and PE mainly elevated in male and female patientswhereas PA declined in both although the number ofPA in male patients was more than in female patientsTable demonstrates genderspecific lipid elements identified by those lipid elements elevated or declined exclusively in either male or female lung cancer patients forexample some of lysoPS PC and PS elevated in malepatients whereas lysoPI and PE in female patients Therewere about or increased or declined lipid elementsdiffered between male and female lung cancer patientsTable 0c of ZHU et alTA B L E adenocarcinoma ADC squamous cell carcinoma SCC or small cell lung cancer SCLC patientsComparisons of clinical phenomes in increased folds and statistical significance Pvalues between each two groups ofTTF1Napsin ABullaeP40HemoptysisEmphysemaSputumCK7HbCoughEGFRVacuole cavityCEAN2 ipsilateral mediastinumNew metastasisP63Cyfra211Obstructive pneumonia atelectasisSmokingPleural pullThirdlineWBCL1 cyclePDL1 tumorPTBronchiectasisPD1 tumorL2 chemo regimenSynMaintenance treatmentNSEN1 ipsilateral ParatrachealCD56CHGT tumorPD1 interstitialSum of all tumors mmN LNKi67Bronchial stenosisN3 opposite sideBurrNeuL2 cyclePulmonary noduleCK56ADC vs SCCFoldsNANANANANANANANAPvaluesNANASCC vs SCLCFoldsNANANANANANANANANAPvaluesNANANAADC vs SCLCFoldsNANANANANANANANAPvaluesNANANANA 0cZHU of Lung cancer subtypeassociated lipid elements significantly elevated or declined alone in patients with adenocarcinomaFoldsPvalues LipidsFolds PvaluesSquamous cell carcinomad181SoC1P240d181S1PPS363TA B L E squamous cell carcinoma or small cell lung cancer more than twofold as compared with healthy control PvaluesSmall cell lung cancerAdenocarcinomaLipidsLipidsElevated twofoldlysoPC sn2lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPE lysoPG lysoPG lysoPS lysoPS lysoPS PA PA PA PC PC PC 332e PC 161e181PC 352e PC 160e202PC PC lysoPG lysoPI sn1lysoPI sn2lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS PA PA PE PE orFoldsPvaluesPC PC or PC PC PC PC orPE PE Declined twofoldPG PS PS SM240PG PS401PE PE PI PI PI PI PI PI PIP PS PS TAG PA PA PA PA PA PA PA PA PA 0c of ZHU et alF I G U R E Scores of altered lipid elements in variable import in project VIP chart A where top lipid elements were defined amongpatients with adenocarcinoma ADC squamous cell carcinomas SCC small cell lung cancer SCL and healthy controls CON The xaxisrepresents the VIP score and the yaxis represents the lipid elements corresponding to the VIP score The right color grid stands for the relativeconcentration of lipid elements in four groups The degree of altered concentrations increased from green to red The heatmap B describesthe top lipid elements at the high concentration and the degree of lipid elements increased from blue low to brown highF I G U R E less than and respectively as compared with the healthy controlTop six significantly increased lipid elements in patients with ADC A SCC B and SCLC C and stand for the Pvalue 0cZHU of F I G U R E Histography of five component distributions and percentages A measured by partial least squares discrimination analysisPLSDA and the carbon atom map B in healthy controls red and patients with ADC green SCLC orange and SCC blue Each ofselected five principal components represents as the model to interpret that values of abscissa and ordinate represents the distance from thesample nodule to the origin of the center after projecting to a plane in multidimensional space A The atom map describes the expression ofmajor carbon atom number between and in various lipid types BF I G U R E The proportion of main lipid elements of healthy controls A ADC B SCC C and SCLC D and volcanic mapbetween heathy controls with ADC E SCC F or SCLC G respectively The lipid elements were identified on the basis of statistical significance The abscissa represents log values of fold changes where the left side of the first dotted line perpendicular to the abscissa represents fold changes and the right side of the second dotted line represents 2fold changes The vertical coordinate represents “log10 Pvalue Theupper side of the dotted line perpendicular to the ordinate stands for Pvalue less than as compared with healthy controls 0c of ZHU et alFoldsPvaluesPvaluesFemale patients with lung cancerLipidsFoldsTA B L E Genderassociated lipid elements significantly elevated or declined alone in male or female patients with lung cancer morethan twofold as compared with healthy control PvaluesMale patients with lung cancerLipidsElevated twofoldlysoPI sn1lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS PC PC or PC PC PC 375ePC 160e225180e205PC PC PC PC PC PC PC PC PC PC PC PC PC C1P120 MeanC1P160 MeanC1P240 MeanCer120d171Sod180Sa1Pd181S1Pd181SolysoPC sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS PC PC PE 355p PE 160p204PE 356p PE 160p205PE PE PE 376p PE 180p205 orPE PE PG PG PI 311p PI 160p160PS PS PS PS PS PS Declined twofoldlysoPS PA PA PA PA PA PA PA PA PA 181p204 160e226PE 377p PE 160p226PE PE PE PE orPI PI or or PS PS PA PA Continues 0cZHU et alContinuedTA B L E Male patients with lung cancerLipidsPG PG PS PS PS FoldsPvalues of Female patients with lung cancerLipidsFoldsPvaluesDifferent lipidomics among patientages stags metastases and survival statusAbout and lipid elements significantly elevatedor and declined in lung cancer patients at years old respectively as compared with healthycontrols P We noticed that elements of PG andPS mainly increased in lung cancer patients at all agegroups for example and at 60year group and at to 70year group and and at year group lysoPC and PC increased at 60year group and and at 70year group and PEincreased at to 70year group and at 70yeargroup as detailed in Table S6 Elements of PA mainlydeclined in lung cancer patients at all ages Table S7 Ofthose significantly altered lipid elements and appeared only at 60year to 70year and 70yeargroups respectively and considered as agespecific lipidelements Table LysoPC and lysoPI mainly increasedin 60year and to 70year old patients whereas lysoPEdeclined in 60year group We also compared lipidomicprofiles between patients at early and late stages of lungcancer and found and lipid elements significantlyincreased at early and late stages respectively of whichPE PG ad PS increased in both stages lysoPI in earlystage and PC in late stage Table S8 About and elements declined at early and late stages where the majority was PA Table S9 Table demonstrates stagespecificlipid elements identified by those lipid elements elevatedor declined exclusively at early and late stages of lung cancer for example some of lysoPI and PE elevated at earlystage and lysoPC and PE at late stageWe noticed about or lipid elements significantlyincreased in patients without or with metastasis of whichlysoPI mainly elevated in patients without metastasiswhereas PC and PE in patients with metastasis Table S10The declined number of lipid elements especially PA inpatients with metastasis was significantly higher than inpatients without metastasis Table S11 There were about or elevated or declined lipid elements in patients withmetastasis of which PA was majority of declined elementsin patients with metastasis Table Lipidomic panel also differed between survived andnonsurvived patients There were only eight lipids exclusively elevated in nonsurvived patients that is lysoPS140PC PC PE PE or PE PE PS330 PS372 andPS387 However far more lipids31 elevated alone in survived patients mainly elements of lysoPC lysoPG lysoPIlysoPS and PS On the contrary there were no lipidsdeclined alone in survived patients while lipids in nonsurvived patients of which were PA elements Table clinical phenomes and lipidomesTransomic profiles betweenWe also compared the difference of lipidomic profilesbetween general metabolism statuses of patients indicatedby BMI and between degrees of clinical phenomes measured by DESS scores Levels of lysoPC or lysoPI mainlyelevated in patients with BMI ‰¤ or respectivelyTable S12 whereas the number of declined PA in patientswith BMI ‰¤ was higher than that in patients withBMI Table S13 About BMIassociated lipid elements significantly elevated or declined exclusively inpatients with BMI ‰¤ and about in patients withBMI Table Levels of lysoPC and PE or PG and PSmainly increased in patients with DESS ‰¤ or TableS14 The number of declined PA n in patients withDESS ‰¤ was lower than that in patients with DESS n Figure demonstrates the variation of transomicprofiles among lung cancer subtypes indicated by transomic nodules cross significant networks of clinical phenome and lipidome layersDISCUSSIONThe present study preliminarily found the differencesof lipidomic profiles among patients of different lungcancer subtypes genders ages stages metastatic statusbody qualities and clinical phenome severities Besides itinitially demonstrated clinical phenomeassociated lipid 0c of ZHU et alFolds Pvalues LipidsPatient age Folds Pvalues LipidsPatient age TA B L E Ageassociated lipid elements significantly elevated or declined alone in each age group of patients with lung cancer morethan twofold as compared with healthy control PvaluesPatient age LipidsElevated twofoldC1P120 MeanlysoPC sn2lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPG lysoPG d181S1PlysoPC sn1lysoPE190lysoPE191PC PC PC PE 356p PE160p205PE PE PE PE orlysoPG140lysoPI sn2lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1Folds PvalueslysoPS lysoPS150lysoPS161lysoPS170lysoPS201lysoPS202lysoPS220PA PE PE orPE PE PE PE PE PE orPI 311p PI160p160PI PI PI PI PI361TAG PA PA PG393lysoPS lysoPS lysoPS PC PE PE PG PG PG PS Declined twofoldlysoPE sn1lysoPE sn1lysoPE sn1lysoPE sn2PA PA PS PS PE PE orPE PE PG351PS354PS372PA elements and lipid elementassociated clinical phenomesusing clinical transomics Studies on lipidomic profilesof lung cancer patients have experienced three phasesto detect the difference of lipidomic profiles betweenhealthy and lung cancer patients16 the association ofmultiomics among lung cancer subtypes3 and themolecular mechanism of clinical lipidomicsbased targetlipid elements17 Of those lipidomicsbased data limitedinformation could be adopted to understand the diseaseoccurrence and development phone progression and 0cZHU of Stageassociated lipid elements significantly elevated or declined alone in patients with lung cancer at the early stage or lateFolds Pvalues LipidsPatients at late stageFolds Pvalues LipidsTA B L E stage more than two folds respectively as compared with healthy control PvaluesPatient at early stageLipidsElevated twofoldlysoPE lysoPG lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPG lysoPS lysoPS lysoPS lysoPS PC PC orlysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS lysoPS lysoPS lysoPS PC PC PE PE PE PE PE 356p PE 160p205PE PE PE PE PE PE PI PI 311p PI 160p160PI PI or or PS PS PC PC PC 375e PC 160e225 or180e205PC PC PC PC PC PC PC PC PC PC PC PC PC PC orPC PC PC PE 355p PE 160p204PE 376p PE 180p205 or181p204 or 160e226PE 377p PE 160p226PE PE PE PE orPE PE PE PE PG PG PG PG PG PG PG PG Patients at late stageFoldsDeclined twofoldlysoPS PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PG PG PS PS PvaluesContinues 0c of ContinuedTA B L E Patient at early stageLipidsPatients at late stageFolds Pvalues LipidsPG PS PS PS ZHU et alPvaluesPatients at late stageFoldsFolds Pvalues Lipidsresponse to therapy due to the lack of link between omicsdata and clinical phenomes Like other omics investigations most genomic data were not tied with clinicalinformation so that with little values to be understoodand applied for clinical precision medicine18 In orderto face the major challenge that most clinical information was descriptive and unmatched with the digitalquantity of omics data clinical phenomes were scoredby DESS and integrated with genomic and proteomicdata of patients with acute respiratory distress syndromeand chronic obstructive pulmonary disease19“ Clinicalphenomes were furthermore integrated with lipidomicprofiles in patients with pulmonary embolism acutepneumonia and acute exacerbation of chronic obstructive pulmonary diseases based on clinical transomicsprinciple15Lipidomic profiles difference between health and lungcancer has been defined and it depends upon methodologies of measurement and analysis sample preparationsand sources and patient populations and status8 Forexample serum levels of lysoPC C260 and C261 and PCC424 and C344 were different between stage I NSCLCand healthy patients22 Some elements and pathwaysof serum PC and PE profiles increased in patients withlung benign disease and earlystage NSCLC as comparedwith healthy whereas few eg PC significantlyelevated in earlystage NSCLC patients alone2 It seemsthat patterns of lipid elements may be associated with thespecificity of lung cancer and stage rather than the intactlipid pathways We performed

Thyroid_Cancer

"widely usedcancerspecific questionnaire assessing domains of healthrelated quality of life HRQoL Our aim was to facilitatethe interpretation of scores on this questionnaire by providing Austrian normative data based on a general populationsampleMethods The calculation of normative data was based on the EORTC QLQC30 data collected from an Austriangeneral population sample that was part of an international online panel study on the development of Europeannormative data Data reported herein were stratified and weighted by age and sex Normative data were calculated forall HRQoL domains of the EORTC QLQC30 For precise predictions of EORTC QLQC30 scores a regression modelbased on sex age and the presence of health conditions was builtResults The Austrian sample comprised Austrian participants female when weighted by age andsex based on United Nation statistics The mean age was years weighted years and weighted reported at least one health condition Men reported better physical Cohen™s d and emotional Cohen™s d functioning as well as less fatigue Cohen™s d and insomnia Cohen™s d compared with womenYounger individuals years reported less dyspnea Cohen™s d and pain Cohen™s d whereas olderindividuals ‰¥ years reported better emotional functioning Cohen™s d Conclusions We present Austrian normative data for the EORTC QLQC30 Differences by age and sex are mostly inline with the findings of other European normative studies The Austrian population sample shows higher HRQoL andlower morbidity compared with other European countries The normative data in this study will facilitate theinterpretation of EORTC QLQC30 scores in oncological practice and research at a national and international levelincluding crosscultural comparisonsKeywords Normative data EORTC QLQC30 Quality of life Oncology Patientreported outcome measures AustriaGeneral population Correspondence jenslehmannimedacat1University Hospital of Psychiatry II Medical University of InnsbruckAnichstrasse Innsbruck AustriaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLehmann Health and Quality of Life Outcomes Page of BackgroundPatientreported outcomes PROs are the gold standardof evaluating the impact of cancer and its treatment fromthe patient™s perspective The importance of PROs isreflected in their widespread use as study endpoints incancer clinical trials [ ] and in the steadily increasing integration of routine PRO assessments in daily clinicalpractice [ ] Regulatory authorities such as the UnitedStates Food and Drug Administration [] and the European Medicines Agency [] have published guidance documents to foster the collection of highquality PRO datain clinical trials Guidelines from international associationssuch as the European anisation for Research andTreatment of Cancer EORTC [] and the InternationalSociety for Quality of Life Research [] deal with the integration of PRO measures into clinical routine and provideinformation on how to collect process and use PRO dataIn the present study we calculate reference values basedon data assessed with EORTC QLQC30 [] a questionnaire that is among the most widely used PRO measuresin cancer clinical trials [“] This questionnaire coversimportant functional health domains eg physical andemotional functioning and key cancer symptoms such asfatigue pain and nauseaIn the literature different methods have been proposedto support the interpretation of results from PRO measuresanalyzing not only individual patients and patient groups ata single time point but also group differences and changesover time One approach is to use minimally important differences to assess or compare changes in PRO results at thegroup or patient level [] To interpret scores obtained ata single time point thresholds for clinical importance canbe used [] Another important approach to interpretPRO data are normative data if comparisons are to bemade with for example a specific disease group or the general population on an individual or grouplevel Whenusing normative data it is important to note that these canvary considerably between countries [“] Focusing oncountryspecific values allows for more precise interpretation which becomes even more accurate when regressionmodels that take age and sex distributions into account areused to generate PRO predictionsThe EORTC Quality of Life Group conducted a largescale international panel study that collected normativedata for the EORTC QLQC30 for European countriesCanada and the United States [] While the EORTCQLQC30 has widely been used in studies in Austria eg[“] no normative studies have so far been publishedIn the current study we present age and sexspecific normative data for the health domains of the EORTCQLQC30 from the Austrian general populationInaddition we provide a regression model defined by agesex and the presence of health conditions to calculate normative data for specific groupsMethodsSamplingAs part of an international study conducted by Nolte [] data from the Austrian general populationwere collected by GfK SE wwwgfkcom apanel research company which contacted panel members who had voluntarily registered and agreed to participate in panelbased studies An equal number ofparticipants was recruited for each sex and age groupmalefemale “ “ “ “ ‰¥ yearsOnce a quota was met for a specific group the recruitment for this group was stopped Response rates topanel studies conducted by GfK are between and as participants are registered voluntarily and usually participate when contactedEORTC QLQC30The EORTC QLQC30 consists of items covering fivefunctioning scales physical social emotional role andcognitive nine symptom scales fatigue nauseavomiting pain dyspnea sleep disturbances appetite loss constipation diarrhea and financial impact and a globalhealth status scale [] Referring to a recall period ofoneweek except for physical function which does notrefer to a recall period at all patients indicate their answers on a 4point Likert scale Linear converted scalescores range from to Higher scores on the functioning scales and on the global health status scale indicate better functioning whereas higher scores on thesymptom scales indicate greater symptom burden fordetails on the scoring and scale structure see [] TheEORTC QLQC30 has been validated in a large European samples and has been widely used in Germanspeaking patients with cancer and the general populationin Germany [ ]Statistical analysisWeights were established following Nolte [] andwere based on official population distribution statisticspublished by the United Nations [] We weighted theresponses to correct for over or underrepresentation ofsex and agestratified subgroups [] We report normative values as means and standard deviations for each subgroup In addition we established a regression model topredict EORTC QLQC30 scores with the following independent predictors sex age age2 a twoway interactionage by sex and the presence of health conditions none orat least one health condition We chose the predictors asthey are linked to HRQoL and have been applied in previous studies [“] The basic model can be expressed asfollows Intercept Sex male female Age Age2 Interaction of Age and Sex age sex Presence ofHealth Conditions none at least one 0cLehmann Health and Quality of Life Outcomes Page of ResultsSampleIn April and May we collected online survey datafrom individuals from the Austrian general population with an equal distribution of participants in the predefined age and sex groups Participants were on average years old years when weighted and ofparticipants when weighted reported at least onehealth condition Some participants reported more thanone health condition when weighted reported two and when weighted reportedthree or more health conditions The most frequently reported health conditions were chronic pain when weightedand arthritis whenweighted Regarding relationship status the percentageof respondents in our sample who said that they were ina longterm relationship was and whenweighted The majority of respondents or when weighted reported at least some postcompulsoryeducation and or when weighted obtained auniversity degree ie bachelors or higher The full descriptive data are reported in Table Normative data for the EORTC QLQC30Table shows a summary of mean scores and standarddeviations for the EORTC QLQC30 across all analyzedsexage groups Men and women differed in several domains the three largest differences were observed forthe scales measuring insomnia points for womenvs points for men Cohen™s d fatigue points for women vs points for men Cohen™s d and emotional functioning points for womenvs points for men Cohen™s d The three largest differences between age groups wereobserved for the scales measuring dyspnea pointsfor those aged “ years vs points for those aged‰¥ years Cohen™s d pain points for thoseaged “ years vs points for those aged ‰¥years Cohen™s d and insomnia points forthose aged “ vs points for those aged “years Cohen™s d See Table for the normativedata stratified by age groupFor functioning scales ceiling effects ie achieving themaximum score were lowest for emotional functioning n and most prevalent for social functioning n Floor effects ie achieving theminimum score were observed most frequently in nauseavomiting n and least common for fatigue n Regression model predicting EORTC QLQC30 scoresTable shows the regression model predicting individual EORTC QLQC30 scores using the weighted normative data This model predicts EORTC QLQC30 scoresusing the individual™s sex age and presence of healthconditions An easy to use spreadsheet for the calculation of predicted values for individuals and groups isavailable online see Supplementary For example fora woman aged years with at least one health condition the predicted physical functioning score is calculated as follows Intercept Sex Age Age2 ˆ’ Interactionof Age and Sex ˆ’ Presence of HealthConditions ˆ’ DiscussionIn this study we present age and sexspecific normativedata for the EORTC QLQC30 from a sample of the Austrian general population Men and women differed in several domains most notably insomniafatigue andemotional functioning In general men reported a higherfunctioning except for social functioning and less symptom burden except for diarrhea than women did Olderparticipants ‰¥ years reported higher symptom burdeneg pain and dyspnea and lower physical and role functioning compared to younger participants We observedboth ceiling effects for functioning scales and floor effects for symptom scales for some scales of the EORTCQLQC30 However floor and ceiling effects were not unexpected considering that we administered a cancerspecific questionnaire to a general population sampleA potentiallimitation of panel studies is that thismethod of data collection may be prone to underrepresenting specific groups of individuals eg those whoare older or less educated Therefore it should be considered whether the assessment of general population datacollected via online surveys are truly representative Acomparison of our data with Statistics Austria™s report suggests that the data obtained from the online survey are representative in terms of most basic individualcharacteristics age sex and marital status [] For example regarding the relationship status in our sample of participants stated to be in a longterm relationship Statistic Austria reported a similar rate with ofAustrian adults being in a longterm relationship The unemployment rate of the Austrian sample was whilethe unemployment rate in the report of the StatisticAustria institute was for individuals older than years Furthermore the prevalence rates for commonhealth conditions found in our data match other data onthe Austrian population The prevalence of selfreportedchronic pain in our sample is close to the prevalence of chronic pain in Austria [] as diagnosed by adoctor which is for chronic back pain and for chronic neck pain The percentage of participants withdiabetes in our sample is in line with the “prevalence rate of diagnosed diabetes among adults estimated in the latest Austrian diabetes report published by 0cLehmann Health and Quality of Life Outcomes Page of Table Sample characteristics N Sex N Age yearsFemaleMaleM SDMedian IQREducationa N Less than compulsory no or some primary educationCompulsory about years of schoolingSome postcompulsory above years of schooling withoutreaching university entrance certificatePostcompulsory below universityCollege bachelor™s or equivalent levelUniversity degree Master Doc or equivalentPrefer not to answer aMarital statusa N SingleEmployment statusa N Health statusab N Married or in a steady relationshipSeparated divorced widowedPrefer not to answer aEmployed full timeEmployed part timeHomemakerStudentUnemployedRetiredSelfemployedOtherPrefer not to answer aNo health conditionAt least one health conditionChronic painHeart diseaseCancerDepressionCOPDArthritisDiabetesAsthmaAnxiety disorderObesityDrugalcohol use disorderOtherPrefer not to answerMissingM mean SD standard deviation IQR interquartile rangea For calculating percentages the category œprefer not to answer was treated as missing datab The sum of all health conditions is larger than the sample size as respondents could choose multipleUnweighted data Weighted data “ “ 0cLehmann Health and Quality of Life Outcomes Page of Table EORTC QLQC30 normative data for the Austrian general population stratified by age and sex N Physical functioningM TotalFemalesAll““““ ‰¥MalesAll“““““ ‰¥“SD Role functioningM SD Emotional functioning M SD Cognitive functioning M SD Social functioningM SD Global healthQoLM FatigueNauseavomitingPainDyspneaInsomniaSD M SD M SD M SD M SD M SD Appetite lossM SD ConstipationM SD DiarrheaM SD Financial difficultiesM M Mean scores SD standard deviation QoL quality of lifeSD the Austrian Health Ministry [] For cancer the prevalence rate was which is only slightly lower than the prevalence rate found in the latest Statistics Austriacancer report [] Notable differences of our data to theStatistics Austria data [] were observed only regardingthe level of education More than onethird of individualstaking part in the online survey reported at least auniversitylevel education while in the Statistic Austriadata only of the sample report a university or comparable degree In our publication of international normative data for the EORTC CAT CORE based on theinternational dataset the relationship between educationand HRQoL scales was investigated in depth [] Highereducation some postcompulsory vsless than postcompulsory education was linked to more favorableHRQoL scores However differences were of low practicalrelevance as indicated by the small effect sizes alleta2 ‰¤ A strength of our study is the consistent data collectionmode used by Nolte [] which allows comparisonwith other European normative data Comparisons ofHRQoL ratings across country borders can be made providing insight into international differences [ ] Atthe nationaltwo differentstudy offerslevel our 0cLehmann Health and Quality of Life Outcomes Page of Table EORTC QLQC30 normative data for the Austrian general population stratified by age N “Physical functioning“MTotal““Role functioningEmotional functioningCognitive functioningSocial functioningGlobal healthQoLFatigueNauseavomitingPainDyspneaInsomniaAppetite lossConstipationDiarrheaFinancial difficultiesSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMM Mean scores SD standard deviation QoL quality of lifeSD“‰¥approaches for interpreting EORTC QLQC30 data Firstthe normative data allow interpretation and comparisonfor different age and sex groups Second the regressionmodel permits the calculation of expected EORTC QLQC30 normative scores using sex age and the presence ofhealth conditions This regression model can be used to generate more precise predictions than those made throughcomparing different age and sex groups using normative dataCompared with other score interpretation approachesnormative data in particular when relying on regressionmodels offer the advantage of not reducing the amount ofinformation For examplein an alternative approachthresholds for clinical importance are used to condense theinformation into severity categories eg clinically important vs not clinically important which can ease interpretation but also decreases the amount ofinformationconveyed The data we present in this study can be used tocompare HRQoL among patients with cancer and HRQoLin the general population matched by sex and age groupSuch comparisons can be useful at any stage of the cancerjourney as patients™ HRQoL is likely to be compromised atthe time of diagnosis [] and they can be used to determine whether cancer survivors return to population levelsor whether problems and impairments persist [ ]Ideally such comparisons use countryspecific normativedata which most accurately reflect the average level of 0cLehmann Health and Quality of Life Outcomes Page of Table Regression model for predicting EORTC QLQC30 scores using age sex and the presence of health conditionsPhysical functioningRole functioningEmotional functioningCognitive functioningSocial functioningGlobal healthQoLFatigueNauseavomitingPainDyspneaInsomniaAppetite lossConstipationDiarrheaFinancial difficultiesInterceptˆ’Sexˆ’ˆ’ˆ’Age in yearsˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’Age in years squaredˆ’ˆ’ˆ’ ˆ’Agebysexˆ’ˆ’ˆ’ ˆ’ˆ’ˆ’Health conditions yesnoˆ’ˆ’ˆ’ˆ’ˆ’Coding is sex male female age in years above age by sex age in yearssex health conditions no health conditions at least onehealth condition QoL quality of lifeHRQoL within a particular national context Of coursenormative data need a certain amount of currentness to berelevant For the years to come this publication can serveas a reference but data should be updated in due timeSeveral observed results merit in depth discussionThe sex differences observed across various domains especially physical functioning are in line with other normative studies on the EORTC QLQC30 that havereported differences by sex In studies in Germany [] Slovenia [] Denmark [] Sweden [] and theNetherlands [] men tended to report higher functioningand lower symptom burden on most scales than womendid A similar pattern can be observed in the presentedAustrian normative data though both point differencesand effect sizes were rather small Our data allow for better interpretation of these differences among patients withcancer through the comparison of the magnitude of impairment in both groups of individuals patients with cancer and members of the Austrian general population Ashas been discussed previously [] sex differences amongpatients with cancer do not necessarily reflect a sexspecific impact of disease or treatment rather these differences may reflect more general factors including sexspecific response styles that also affect the general population as we observed in the present sampleRegarding age a European sample [] as well assinglecountry studies in Denmark Sweden and Slovenia[ ] report similar deterioration with lower physical and role functioning in older people ‰¥ or ‰¥ years compared to younger people or years asfound in our sample Those studies also found emotionalfunctioning to be independent of age [ ] or to evenincrease with age ‰¥ years [ ] showing a similarpattern of emotional functioning and age as observed inour sample However our sample differed from two studysamples in Germany which did not show an increase inemotional functioning with age and reported much higherdisparity between age groups on the global health statusscale with a difference of up to points oldest vs youngest group in favor of younger people [ ]Compared with the European sample reported byNolte [] fewer respondents in our sample reported health conditions or diseases in the European sample vs in our sample reported having nohealth conditions and our sample showed higher functioning and lower symptom burden on most scales According to the EU Statistics on Income and LivingConditions the Austrian life expectancy is slightly abovethe EU average years vs years and ofthe Austrian adult population report a good or verygood perceived health which ranks higher than mostother European countries measured [] Both of thesefindings support our result of generally high HRQoL interms of fewer health conditions and high functioningamong the Austrian general populationConclusionsThe normative data for the EORTC QLQC30 generatedin this study will ease and foster a more meaningful interpretation of scores obtained from patients with cancer 0cLehmann Health and Quality of Life Outcomes Page of and cancer survivors allowing comparisons of patientlevel and grouplevel data with the sex and agematched general population sample from Austria Usingour regression model precise predictions for individualsbased on sex age and presence of health conditions canbe generatedSupplementary informationSupplementary information accompanies this paper at doi101186s12955020015248Additional file AbbreviationsEORTC European anisation for Research and Treatment of Cancer GfKSE Gesellschaft für Konsumforschung Societas Europaea HRQoL HealthRelated Quality of Life PRO PatientReported OutcomesAcknowledgementsThe authors thank the European anization for Research and Treatment ofCancer for permission to use the data from an EORTC study grant number for this research We thank Jennifer Barrett PhD from EdanzGroup enauthorservicesedanzgroupcom for languageediting adraft of this manuscriptAuthors™ contributionsJL analyzed the data interpreted the data and wrote the manuscript JMGinterpreted the data and helped to draft the manuscript LMW and MSinterpreted the data and edited the manuscript SN GL and MR generatedthe data and edited the manuscript BH analyzed and interpreted the dataassisted in generating the data and edited the manuscript All authors readand approved the final manuscriptFundingThis research was partly funded by the European anisation for Researchand Treatment of Cancer Quality of Life Group grant number Availability of data and materialsThe datasets analyzed in the study at hand are available upon reasonablerequest from the EORTC Please use the Data Sharing form available throughthe EORTC website wwweortcdatasharingEthics approval and consent to participateNo ethics approval was sought as the study is based on panel dataAccording to the NHS Health Research Authority and the EuropeanPharmaceutical Market Research Association EphMRA panel research doesnot require ethical approval if ethical guidelines are followed The survey wasdistributed via the GfK SE member of EphMRA and obtained informedconsent by each participant before the study All data were collectedanonymously and identification of the respondents through the authors isimpossibleConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1University Hospital of Psychiatry II Medical University of InnsbruckAnichstrasse Innsbruck Austria 2Division of PsychosomaticMedicine Medical Department Charité Universitätsmedizin BerlinCorporate Member of Freie Universität Berlin HumboldtUniversität uu Berlinand Berlin Institute of Health Berlin Germany 3School of Health and SocialDevelopment Population Health Strategic Research Centre DeakinUniversity Burwood VIC AustraliaReceived June Accepted July ReferencesBlazeby JM Avery K Sprangers M Pikhart H Fayers P Donovan J Healthrelated quality of life measurement in randomized clinical trials in surgicaloncology J Clin Oncol “Cella D Grunwald V Nathan P Doan J Dastani H Taylor F Bennett BDeRosa M Berry S Broglio K Quality of life in patients with advancedrenal cell carcinoma given nivolumab versus everolimus in CheckMate a randomised label phase trial Lancet Oncol “LeBlanc TW Abernethy AP Patientreported outcomes in cancer care hearing the patient voice at greater volume Nat Rev Clin Oncol “Austin E LeRouge C Hartzler AL Segal C Lavallee DC Capturing the patientvoice implementing patientreported outcomes across the health systemQual Life Res “US Food and Drug Administration PatientReported Outcome Measures Usein Medical Product Development to Support Labeling Claims Guidance forIndustry wwwfdagovregulatoryinformationsearchfdaguidancedocumentspatientreportedoutcomemeasuresusemedicalproductdevelopmentsupportlabelingclaims Accessed Oct European Medicines Agencies Appendix to the guideline on theevaluation of anticancer medicinal products in man The use of patientreported outcome PRO measures in oncology studies wwwemaeuropaeudocumentsotherappendix2guidelineevaluationanticancermedicinalproductsman_enpdf Accessed Feb Wintner LM Sztankay M Aaronson N Bottomley A Giesinger JM GroenvoldM Petersen MA van de PollFranse L Velikova G Verdonckde Leeuw I The use of EORTC measures in daily clinical practicea synopsis of anewly developed manual Eur J Cancer “Chan E K H Edwards TC Haywoods K Mikles S Newton L ImplementingPatientReported Outcome Measures in Clinical Practice A CompanionGuide to the ISOQOL User™s Guide wwwisoqolwpcontentuploads201909ISOQOLCompanionGuideFINALpdf Accessed Mar Aaronson NK Ahmedzai S Bergman B Bullinger M Cull A Duez NJ FilibertiA Flechtner H Fleishman SB de Haes JC The European anization forResearch and Treatment of Cancer QLQC30 a qualityoflife instrument foruse in international clinical trials in oncology J Natl Cancer Inst “Smith AB Cocks K Parry D Taylor M Reporting of healthrelated quality oflife HRQOL data in oncology trials a comparison of the Europeananization for Research and Treatment of Cancer quality of life EORTCQLQC30 and the functional assessment of Cancer therapygeneral FACTG Qual Life Res “ Howell D Molloy S Wilkinson K Green E Orchard K Wang K Liberty JPatientreported outcomes in routine cancer clinical practice a scopingreview of use impact on health outcomes and implementation factorsAnn Oncol “ Gnanasakthy A Barrett A Evans E D'Alessio D Romano CD A review ofpatientreported outcomes labeling for oncology drugs approved by theFDA and the EMA Value Health “ Giesinger JM Blazeby J Aaronson NK Sprangers M Fayers P Sparano FRees J Anota A Wan C Pezold M Differences in patientreportedoutcomes that are most frequently detected in randomized controlled trialsin patients with solid tumors a pooled analysis of trials Value Health“ Musoro ZJ Hamel JF Ediebah DE Cocks K King MT Groenvold M SprangersMAG Brandberg Y Velikova G Maringwa J Establishing anchorbasedminimally important differences MID with the EORTC qualityoflife measuresa metaanalysis protocol BMJ 20188e019117 Giesinger JM Loth FLC Aaronson NK Arraras JI Caocci G Efficace FGroenvold M van Leeuwen M Petersen MA Ramage J Thresholds forclinical importance were established to improve interpretation of theEORTC QLQC30 in clinical practice and research J Clin Epidemiol Janssen MF Szende A Cabases J RamosGoni JM Vilagut G Konig HHPopulation norms for the EQ5D3L a crosscountry analysis of populationsurveys for countries Eur J Health Econ “Fayers PM Interpreting quality of life data populationbased reference datafor the EORTC QLQC30 Eur J Cancer “ 0cLehmann Health and Quality of Life Outcomes Page of consecutive annual assessments of approximately representativeDutch men and women Acta Oncol “ Giesinger J Kemmler G Mueller V Zabernigg A Mayrbaeurl B Thaler JOfner D Pegger C Rumpold G Weber B Are genderassociateddifferences in quality of life in colorectal cancer patients diseasespecificQual Life Res “Eurostat EU Statistics on Income and Living Conditions EU SILC eceuropaeueurostatdatabrowserviewsdg_03_20defaultmaplangen Accessed Mar Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsSchwarz R Hinz A Reference data for the quality of life questionnaire EORTCQLQC30 in the general German population Eur J Cancer “ Nolte S Liegl G Petersen MA Aaronson NK Costantini A Fayers PMGroenvold M Holzner B Johnson CD Kemmler G General populationnormative data for the EORTC QLQC30 healthrelated quality of lifequestionnaire based on persons across European countriesCanada and the unites states Eur J Cancer “Loth FL Holzner B Sztankay M Bliem HR Raoufi S Rumpold G GiesingerJM Cancer patients' understanding of longitudinal EORTC QLQC30 scorespresented as bar charts Patient Educ Couns “ Giesinger JM Kuijpers W Young T Tomaszewski KA Friend E Zabernigg AHolzner B Aaronson NK Thresholds for clinical importance for four keydomains of the EORTC QLQC30 physical functioning emotionalfunctioning fatigue and pain Health Qual Life Outcomes Kuijpers W Giesinger JM Zabernigg A Young T Friend E Tomaszewska IMAaronson NK Holzner B Patients' and health professionals' understandingof and preferences for graphical presentation styles for individuallevelEORTC QLQC30 scores Qual Life Res “EORTC Quality of Life Group EORTC QLQC30 scoring manual wwweortcappuploadssites2201802SCmanualpdf Accessed July Hinz A Singer S Brahler E European reference values for the quality of lifequestionnaire EORTC QLQC30 results of a German investigation and asummarizing analysis of six European general population normative studiesActa Oncol “ United Nations Department of Economic and Social Affairs PopulationDivision World population Prospects the revision DVD Edition Liegl G Petersen MA Groenvold M Aaronson NK Costantini A Fayers PMHolzner B Johnson CD Kemmler G Tomaszewski KA Establishing theEuropean norm for the healthrelated quality of life domains of thecomputeradaptive test EORTC CAT Core Eur J Cancer “ Hjermstad MJ Fayers PM Bjordal K Kaasa S Using reference data on qualityof lifethe importance of adjusting for age and gender exemplified by theEORTC QLQC30 Eur J Cancer “ Quinten C Coens C Ghislain I Zikos E Sprangers MA Ringash J Martinelli FEdiebah DE Maringwa J Reeve BB The effects of age on healthrelated quality of life in cancer populations a pooled analysis ofrandomized controlled trials using the European anisation for Researchand Treatment of Cancer EORTC QLQC30 involving cancer patientsEur J Cancer “ Husson O Haak HR Buffart LM Nieuwlaat WA Oranje WA Mols F KuijpensJL Coebergh JW van de PollFranse LV Healthrelated quality of life anddisease specific symptoms in longterm thyroid cancer survivors a studyfrom the populationbased PROFILES registry Acta Oncol “Statistik Austria Statistiken Arbeitsmarkt wwwstatistikatweb_destatistikenindexhtml Accessed Mar Bundesministerium für Gesundheit Zivilisationskrankheit DiabetesAusprägungen “ Lösungsansätze “ Herausforderungen ÖsterreichischerDiabetesbericht wwwsozialministeriumatdamjcr630d810ce02b4c62a2d95913ae0314edDiabetesbericht202017pdfAccessed Mar Statistik Austria Krebserkrankungen in Österr

Thyroid_Cancer

Lasting and SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaMarissa R Keever1 Pan Zhang2 Courtni R Bolt1 Adrienne M Antonson1Haley E Rymut1 Megan P Caputo1 Alexandra K Houser1 Alvaro G Hernandez3Bruce R Southey1 Laurie A Rund1 Rodney W Johnson14 andSandra L RodriguezZas12456 Department of Animal Sciences University of Illinois at UrbanaChampaign Urbana IL United States Illinois InformaticsInstitute University of Illinois at UrbanaChampaign Urbana IL United States Highthroughput Sequencingand Genotyping Unit Roy J Carver Biotechnology Center University of Illinois at UrbanaChampaign Urbana ILUnited States Neuroscience Program University of Illinois at UrbanaChampaign Urbana IL United States Departmentof Statistics University of Illinois at UrbanaChampaign Urbana IL United States Carl R Woese Institute for GenomicBiology University of Illinois at UrbanaChampaign Urbana IL United StatesThe prolonged and sexdependent impact of maternal immune activation MIA duringgestation on the molecular pathways of the amygdala a brain region that ‚uencessocial emotional and other behaviors is only partially understood To address thisgap we investigated the effects of viralelicited MIA during gestation on the amygdalatranscriptome of pigs a species of high molecular and developmental homology tohumans Gene expression levels were measured using RNASeq on the amygdalafor 3weekold female and male offspring from MIA and control groups Amongthe genes that exhibited significant MIA effect a prevalence of differentiallyexpressed genes annotated to the neuroactive ligand“receptor pathway glutamatergicfunctions neuropeptide systems and cilium morphogenesis were uncovered Genesin these categories included corticotropinreleasing hormone receptor glutamatemetabotropic receptor glycoprotein hormones alpha polypeptide parathyroidhormone receptor vasointestinal peptide receptor neurotensin proenkephalinand gastrinreleasing peptide These categories and genes have been associatedwith the MIArelated human neurodevelopmental disorders including schizophreniaand autism spectrum disorders Gene network reconstruction highlighted differentialvulnerability to MIA effects between sexes Our results advance the understandingnecessary for the development of multifactorial therapies targeting immune modulationand neurochemical dysfunction that can ameliorate the effects of MIA on offspringbehavior later in lifeKeywords immune activation pigs RNAseq neuropeptides glutamatergic pathway GABAergic pathwayINTRODUCTIONThe maternal immune response triggered by pathogens and other environmental stressors duringgestation can also elicit an indirect response by the fetal immune cells Kroismayr Odorizzi and Feeney Prins Viral infection during gestation for exampleactivates a cytokinerelated signaling cascade and molecules from this process can cross theEdited byNo¨lia Fern ndezCastilloCentre for Biomedical NetworkResearch CIBER SpainReviewed bySilvia PellegriniUniversity of Pisa ItalyTewarit SarachanaChulalongkorn University ThailandCorrespondenceSandra L RodriguezZasrodrgzzsillinoiseduSpecialty sectionThis was submitted toNeurogenomicsa section of the journalFrontiers in NeuroscienceReceived May Accepted July Published August CitationKeever MR Zhang P Bolt CRAntonson AM Rymut HE Caputo MPHouser AK Hernandez AGSouthey BR Rund LA Johnson RWand RodriguezZas SL Lastingand SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaFront Neurosci 103389fnins202000774Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaplacenta and reach the fetal brain The resulting maternalimmune activation MIA can impactfetal developmentalprocesses and exert longterm postnatal eï¬ects in the oï¬springRutherford The relationship between MIAand neurodevelopmental disordersincluding schizophreniaspectrum disorders SSD and autism spectrum disorders ASDand neurodegenerative disorders such as Alzheimer™s diseaseAD in oï¬spring has been established Knuesel Canetta Mattei These diseases sharesome behavior symptoms comorbidities such as eating disordersand genetic and environmental ie MIA agents Canitanoand Pallagrosi The previous neurological disorders havebeen associated with abnormal structure and dysregulationof the amygdala Schumann FernandezIrigoyen and share genes and molecular mechanismsincluding histocompatibility complex MHC genes Andersand Kinney glutamatergic and GABAergicassociatedgenes Bourgeron Marin Li andmitochondrial activity processes Pieczenik and Neustadt Sragovich socialinteractionThe fetal amygdala is susceptible to ‚ammatory signals andthe plasticity of this brain structure to MIA can lead to alterationsof the developmental trajectory These disruptions may havelonglasting and maladaptive consequences for the oï¬springdue to the significant role that the amygdala plays in manyneurological pathways Located in the forebrain the amygdala‚uencescognition neuroendocrinebehavior learning memory emotion and autonomic systemsThe amygdala also modulates the response of these processesto stressors including pathogenic infections and those resultingfrom management practices such as weaning Tian The amygdala experiences high uptake of gonadalhormones and is anatomically connected to other sexuallydimorphic nuclei Therefore this brain region is involved inregulation of several dimorphic functions such as aggressionsexual behavior gonadotropin secretion and integration ofolfactory information Hines Evidence supports thediï¬erential activation of the amygdala to stimuli between malesand females Killgore and YurgelunTodd includingdiï¬erences in the sexual responses and emotional memoryHamann and diï¬erential vulnerability to insult Baird Due to the interconnected and multiregulatorynature of this brain structureinsults to the amygdala canimpact the individual™s social locomotor and feeding behaviorPetrovich and Gallagher growth and reproductivephysiology health status and immunological response tosecondary stressorsRecent studies lend support to the link between MIA andaltered amygdala function Carlezon In miceMIA elicited by polyinosinicpolycytidylic acid [PolyIC]increased the synaptic strength of glutamatergic projectionsfrom the prefrontal cortex to the amygdala Li In field tests mice exposed to MIA spentless timein the center and traveled a higher distanceindicativeof a higher anxiety behavior incidence than the controlcounterparts These findings suggest that the change in thebalance between excitation glutamatergic and inhibitiontherefore aï¬ecting brain circuitsspike output offeedforward GABAergic modified theamygdala neuronsthatcould regulate behavior in SSD and ASD A candidate genestudy of the eï¬ects of social stress during gestation reportedthatthe expression of a corticotropinreleasing hormonereceptor in the amygdala of 10weekold pigs was higherin females than in males Rutherford Thisstudy concluded that prenatal stress substantially increasedanxietyrelated behaviorstheimpact of maternal stressors during gestation on specificamygdala molecular profiles and associated neurological orbehavioral disorders in the oï¬spring later in life highlightthe complexity of the molecular mechanisms underlying thepathophysiology of MIAin female pigs Studies ofetalvirusadvantages ofrodents when consideringstudying a pig modelto pigsResearch on the lasting eï¬ects of MIA in pigs complementsAntonson et althe insights oï¬ered by rodent modelsstem Theratherfrom the greater homology of humansan physiologythan toin particular brain growth andsize development anddevelopment processesMurphy A pigmodel that has oï¬ered insights into MIA employs porcinereproductivePRRSVtothein the brain andmicroglia ie macrophagelike cellsisin neonatal pigsAntonson associated with behavioralelicit MIA Thisand respiratorysyndromechallengeactivatesimmunechangesThe study of MIA elicited by PRRSV allows for thecharacterization of the impact of a live viral pathogen thatselfreplicates in the host evoking extended activation ofimmune pathways PRRSV challenge during gestation is a wellcharacterized replicable and eï¬ective method for inducingMIA in pigs Antonson In additionPRRSV outbreaks impose a major economic burden to thelivestock industry PRRSV is an enveloped singlestranded RNAvirus thatinfects alveolar macrophages causing interstitialpneumonia and increased serum levels ofthe cytokinesinterleukin betainterleukin and tumor necrosis factoralpha Antonson The persistent repercussionsof MIA on the molecular pathways ofthe pig amygdalaare yet to be investigated Moreover the potentially distinctvulnerability to the prolonged eï¬ects of MIA between sexesremains unknownThe overarching goal of the present study is to advancethe understanding of the impact of MIA on the molecularmechanisms ofthe amygdala Three supporting objectivesare explored a characterization of prolonged transcriptomechanges elicited by viral MIA in pigs a species that hashigh neurodevelopmental homology with humansandfood production valueidentification of molecularpathwaysthat present diï¬erential vulnerability to MIAbetween sexes and c understanding the eï¬ect of MIA onmolecular interactions assisted by gene network inferenceThe findings from these complementary analyses supportthe use of multipleto amelioratethe potential detrimental eï¬ect of MIA on the oï¬springphysiology and behaviortherapeutictargetsbFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaMATERIALS AND METHODSAnimal ExperimentsAll experimental procedures used published protocols Antonson The animal studies were approved by theIllinois Institutional Animal Care and Use Committee IACUCat the University of Illinois and are in compliance with the USDAAnimal Welfare Act and the NIH Public Health Service Policy onthe Humane Care and Use of AnimalsCamborough gilts born and raised at the University of Illinoisat UrbanaChampaign herd were inseminated at days ofage using PIC boar sperm Antonson All gilts were PRRSV negative and were moved at gestationday GD into diseasecontainment chambers maintainedat —¦C and a h lightdark cycle with lights on at AM The gilts were fed daily kg of a gestational diet andhad ad libitum water access One week after acclimation fourgilts were intranasally inoculated with live PRRSV strain P129BV School of Veterinary Medicine at Purdue University WestLafayette IN United States using mL of — median tissueculture infectious dose TCID50 diluted in sterile Dulbecco™smodified Eagle medium DMEM mL total volume Thefour gilts in the Control group were intranasally inoculatedwith an equal volume of sterile DMEM PRRSV inoculationcorresponded to the last third of gestation in pigs and humansduring initiation of rapid fetal brain growth Antonson PRRSV and Control groups were housed in separatecontainment chambersThe rectal temperatures and diet consumption of the giltswere recorded daily until farrowing Antonson The PRRSVinoculated gilts were oï¬ered the maximumfed daily and feed refusal was measured The Control giltswere fed the same amount consumed by the PRRSVinoculatedgilts on the previous day The daily body temperature andfeed intake levels were compared using a mixedeï¬ects modelanalyzed with PROC MIXED SAS Institute Inc Cary NCUnited States The model included the eï¬ects of gilt treatmentand replicate while accommodating for heterogeneity of variancebetween MIA groupsFarrowing was induced with an intramuscular injection of mg of Lutalyse dinoprost tromethamine Pfizer New YorkNY United States on GD in consideration that the averagegestation length is approximately days Antonson Gilts farrowed in individual farrowing crates ofstandard dimensions — m After farrowing thegilts were fed twice a day up to kg of a nutritionallycomplete diet for the lactating period and water remainedavailable ad libitum Pigs received intramuscular injections ofiron dextran mgpig Butler Schein Animal Health DublinOH United States and Excede for Swine mgpig ZoetisParsippany NJ United States to control for respiratory diseasesThe pigs remained with their mothers until PD The bodyweight of pigs was measured daily and analyzed using the mixedeï¬ects model in SAS PROC MIXED SAS Institute Inc CaryNC United States The model included the eï¬ect of MIA andthe random eï¬ect of gilt accommodating for heteroscedasticitybetween pig treatment and sex groups The impact of MIA wasstudied at PD because this is a common age to wean pigsThe study of transcriptome profiles from older pigs could beconfounded with changes in diet and environment associatedwith weaning while profiles from younger pigs would hinder theassessment of the prolonged eï¬ects of MIARNA Extraction and SequencingA balanced experimental design was studiedincluding pigs evenly distributed between maternal PPRSV activatedMPA group of pigs and Control gilts CON group of pigseach group encompassing males and females denoted Maand Fe respectively At PD pigs were removed fromthe farrowing crate and anesthetized intramuscularly using atelazolketaminexylazine drug cocktail mg of tiletamine mg of zolazepam reconstituted with mL ketamine gL and mL xylazine gL Fort Dodge AnimalHealth Fort Dodge IA United States at a dose of mLkgbody weight following protocols Antonson Following anesthetization pigs were euthanized using anintracardiac injection of sodium pentobarbital mgkg bodyweight Fata Plus Vortech Pharmaceuticals Dearborn MIUnited States Pig brains were extracted the amygdalae wererecognized using the stereotaxic atlas of the pig brain Felix dissected out flash frozen on dry ice and stored at ˆ’—¦Cfollowing published protocols Antonson RNA wasisolated using EZNA isolation kit following the manufacturer™sinstructions Omega Biotek Norcross GA United States TheRNA integrity numbers of the samples were above indicatinglow RNA degradation The RNASeq libraries were preparedwith TruSeq Stranded mRNAseq Sample Prep kit Illumina IncSan Diego CA United States The libraries were quantitatedby qPCR and sequenced on one lane on a NovaSeq for cycles from each end of the fragments using NovaSeqS4 reagent kit FASTQ files were generated and demultiplexedwith the bcl2fastq v220 conversion software Pairedend reads nt long were obtained and the FASTQ files are availablein the National Center for Biotechnology Information GeneExpression Omnibus GEO database experiment accessionnumber GSE149695RNA Sequence Mapping and DifferentialExpression AnalysisThe average Phred quality score of the reads assessed usingFastQC Andrews was across all read positions andtherefore no reads were trimmed The pairedend reads fromthe individual samples were aligned to the Sus scrofa genomeversion Sscrofa Pruitt using kallisto v0430Bray with default settings The normalized trimmedmean of Mvalues gene expression values were described usinga generalized linear model encompassing the eï¬ects of the MIAgroup MPA or CON levels sex Fe or Ma levels and MIAbysex interaction and analyzed using edgeR version in the R v environment Robinson Genessupported by transcripts per million TPM by each MIA“sex combination were analyzed to ensure adequate representationacross comparisonsFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaOrthogonal pairwise contrasts between MIA and sex groupswere evaluated in addition to testing for the eï¬ects of MIAbysex interaction and main eï¬ects of MIA and sex Thefour groups compared in the contrasts identified by treatmentfollowed by the sex levels are MPA_Fe MPA_Ma CON_Fe andCON_Ma The Pvalues were adjusted for multiple testing usingthe Benjamini“Hochberg false discovery rate FDR approachBenjamini and Hochberg categoriesamongtheMFand KEGG pathways The GeneFunctional Enrichment and NetworkInferenceapproaches were used to identifyTwo complementaryoverrepresented functionalgenesexhibiting diï¬erential expression across MIA and sex groupsCaetanoAnoll©s GonzalezPena et al2016ab Functional categories investigated included GeneBPs GO molecularOntology GO biological processesfunctionsSetEnrichment Analysisapproach implemented inthesoftware package GSEAP Subramanian was used to identify category overrepresentationwith gene over and underexpressed while considering allgenes analyzed The normalized enrichmentscore NESofin the Molecular Signature DatabaseMSigDB was calculated using the maximum deviation ofthe cumulative sum based on the signed and standardizedfold change The statistical significance ofthe enrichmentwas assessed using the FDRadjusted Pvalue computed from permutationscategoriesGSEAtheThe overrepresentation of functional categories was alsoevaluated among genes that exhibited a significant MIAbysex interaction or main eï¬ect using the Database forAnnotation Visualization and Integrated Discovery DAVID Huang The enrichment of Direct GOcategories in the DAVID database was assessed The Susscrofa genome was used as the background for enrichmenttesting and enrichmentis reported using the ExpressionAnalysis Systematic Explorer EASE score that was computedusing a onetailed jackknifed Fisher hypergeometric exacttest Functional categories were clustered based on geneannotation and the statisticalissummarized as the geometric mean of the log10 EASE scoresofthe categories Delfino Serao Delfino and RodriguezZas significance of clustersWeighted Gene Coexpression NetworkAnalysis and Gene Network VisualizationAn approach complementary to the identification of diï¬erentiallyexpressed genes was used to uncover coexpression networksusing Weighted Gene Coexpression Network AnalysisWGCNA version Langfelder and Horvath The input data were voomtransformed read count valuesgenerated using the limma package version Ritchie in R version Genes were filtered to removethose with low expression levels or no variation across samplesper developer recommendations The number of genes usedfor network analysis was genes Considering potentialfor interaction patterns a sexdependent softthresholdingpower was used to call for network topology analysis Thelowest power values that support a scalefree topology powerused were for the CON_MaMPA_Ma contrast and for the MPA_FeMPA_Ma contrast The Pearson correlationcoefficient ofthe normalized expression values was usedto identify modules of connected genes The minimummodule size was set to with the deepSplit set to and themergeCutHeight set to Module profiles were identifiedusing the correlation between the eigengene of each moduleand pig group Enrichment of functional categories among thegenes in each module profile was explored with DAVID using theSus scrofa genome as background and testing included an FDRmultiple test adjustmentFurther understanding of the impact of the MIAbysexinteraction was gained through the reconstruction of genenetworks using the BisoGenet package Martin inthe Cytoscape platform Shannon Information fromgene and protein interactions annotated in databases includingBIOGRID HPRD DIP BIND INTACT and MINT was usedto visualize relationships between genes Salwinski Alfarano Mishra Stark Kerrien Licata Networks highlightingdiï¬erences in gene levels associated with MIA within sex iethe contrasts MPA_MaCON_Ma and MPA_FeCON_Fe werecompared The network framework includes genes that exhibiteda significant MIAbysex interaction eï¬ect FDRadjusted P and are annotated to enriched functional categoriesThe framework genes were identified by full nodes with sizereflecting the diï¬erential expression level between the MPAand CON groups The network edges depict known molecularrelationships curated in the BisoGenet databases The frameworkgenes were connected through correlated genes listed in theBisoGenet database of molecular interactions that did not reachsignificant MIAbysex interaction eï¬ect The comparison ofthese networks oï¬ered insights into the simultaneous eï¬ect ofMIA across interacting genes and enabled the detection of sharedand distinct coregulation patterns between MPA and CONpigs across sexesRESULTSMaternal Immune Activation andSequencing MetricsThe diï¬erences between MPA and CON giltsin rectaltemperatures and daily diet consumption indicated the activationof the maternal immune system in response to PRRSV Thediï¬erence in body temperature between CON and MPA giltson GD was —¦C standard error —¦C P Thediï¬erence in feed refusal between CON and MPA gilts on GD was g standard error g P A significantincrease in rectal temperatures and decrease in feed intakeP was observed within h of inoculation and returnedto baseline levels within days for body temperature and within days for feed intake At days of age CON pigs were kgFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaheavier than MPA pigs standard error P whileno significant sex or interaction eï¬ects were detectedThe sequencing of the RNA samples produced billionsequenced reads and million pairedend reads per sampleThe number of reads was consistent across MIA and sexgroups coefficient of variation and the eï¬ects ofMIA sex and MIAbysex interaction were tested on genes that surpassed the minimum number of reads per MIA“sex combinationTranscriptome Changes Associated WithMaternal Immune Activation That AreSexDependentOverall genes exhibited a significant FDRadjusted P MIAbysex interaction eï¬ect and among these genes hada significant eï¬ect at FDRadjusted P The profile ofthese genes indicated that the eï¬ect of MIA diï¬ered betweenfemales and males Fortysix genes that presented a MIAbysex interaction eï¬ect are listed in Table together with theirexpression pattern and Pvalue The majority of the genes inTable including neurotensin NTS displayed a reversal inthe expression level between CON and MPA groups across sexesie opposite Log2[fold change] sign across sexes An extendedlist including genes that exhibited a MIAbysex interactioneï¬ect at FDRadjusted P is provided in SupplementaryFile S1 Table AAnother frequent pattern among the genes that displayed aMIAbysex interaction eï¬ect was characterized by a consistentexpression profile between CON and MPA across sexes albeitthe magnitude diï¬ered between sexes Table For exampleglycoprotein hormones alpha polypeptide CGA was overexpressed in CON relative to MPA but the diï¬erential washigher in males than in females Other genes presentingthis pattern included guanylatebinding protein GBP1transthyretin TTR aldehyde dehydrogenase family memberA2 ALDH1A2 hemoglobin subunit beta HBB and basichelixloophelix family member e22 BHLHE22GRPNotable is the significant MIAbysex interaction eï¬ecton genes associated with neuropeptides and hormones andgenes that participate in glutamatergic processes Genes underexpressed in MPA relative to CON males while presentingthe opposite pattern in females Table included NTS theneuropeptide gene proenkephalin PENK the neuropeptidegene gastrinreleasing peptidethe neuropeptiderelated gene vasoactive intestinal peptide receptor VIPR2corticotropin releasing hormone receptor CRHR2 neuronderived neurotrophic factor NDNF reelin RELN glutamatemetabotropic receptor GRM4 solute carrier family member SLC17A6 calcium voltagegated channel auxiliarysubunit alpha delta CACNA2D3 EFhand domain familymember D1 EFHD1 glutathione peroxidase GPX3parathyroid hormone receptor PTH1R thyroid hormoneresponsive THRSP and CGA The CGA gene codes for thealpha subunit protein of the hormones chorionic gonadotropinCG luteinizing hormone LH folliclestimulating hormoneFSH and thyroidstimulating hormone TSHFunctional and Network Analysis ofGenes That Exhibit SexDependentAssociations With Maternal ImmuneActivationThe genes expressing significant MIAbysex interaction eï¬ectswere analyzed for functional enrichment Table presentsthe clusters of most enriched and informative categoriesfrom the DAVID analysis and the complete list of categoriesis in Supplementary File S1 Table B The categories inTable encompass genes presenting the mostfrequentinteraction profile characterized by underexpression in CONfemales relative to males but overexpression in MPA femalesrelative to males These genes include KEGG Autoimmunethyroid diseaseCluster and BP brain developmentGO0007420 Cluster Enrichment results from GSEA complemented the findingsfrom DAVID Highly enriched informative categories amonggenes that have a MIAbysex interaction eï¬ect are presentedin Table and the extended list of categories is presented inSupplementary File S1 Table C The categories in Table in Table including ion homeostasissupport pathwaysTable and regulation of voltagegated calcium channelactivity processesenrichmentinteraction pathwayofthe neuroactiveand the hormoneactivity processesinclude genes such as CGA and VIPR2 that were identifiedin Table ligand receptorand neuropeptideTable Notablythethe diï¬erentialNetwork visualization furthered the understanding ofthe impact of MIA on the relationships among genes thatexhibited a significant MIAbysex interaction eï¬ect Thenetworks in Figures depictthe relationships betweengenes in the enriched neuroactive ligand receptor pathwaythat highlightexpression between CONand MPA in males and females ie CON_MaMPA_Maand CON_FeMPA_Ferespectively Red andrectangular nodesblueframework genes andthe known associations between genesedgesrepresentbased on curated databases of molecularinteractionsRed and blue nodes denote over or underexpressionof the gene in CON relative to MPA and the size is anthe diï¬erential expressioninverse logarithmic function ofPvalue Thediï¬erentialexpression pattern and connectivity among genes highlightsthe discrepancyelicited by MIAbetween the sexesin network modulescontrastsrepresentsimultaneousstudytheofTranscriptome Changes Associated WithMaternal Immune ActivationOverall genes exhibited diï¬erential FDRadjusted P expression between MPA and CON pigs irrespective of sexTable lists notable highly diï¬erentially expressed genesis in Supplementary File S1 Tableand the complete listD The majority of these genes were overexpressed in MPArelative to CON pigs Among the genes overexpressed in MPAcompared to CON pigs were islet amyloid polypeptide IAPPFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaTABLE Genes exhibiting significant FDRadjusted Pvalue maternal immune activationbysex interaction effectGene symbolPvalueaCON FeCON Ma MPA FeMPA MaCON FeMPA FeCON MaMPA MaCON FeMPA MaCON MaMPA FeRGS16CGAPOMCGPX3RELNVIPR2ANKRD34CGBP1GRM4CCDC136SLC17A6BTBD11TTRCACNA2D3CRHR2NDNFCXCL12USP43CCDC17KCNIP4CAMK2N2ALDH1A2GRPPENKSYT12PTH1RHBBESYT1EFHD1BHLHE22ZFP37SLC2A2THRSPNR4A3LOC396781C1QTNF1RAB27ANTSGVIN1SSTR1CCDC9BCCDC33CCDC162PPTHSYNPO2LCHGB5E115E115E115E115E115E115E115E115E1153E0911E0844E0850E0848E0728E0628E0662E0664E0671E0672E0696E0614E0515E0516E0529E0537E0567E0585E0596E0510E0412E0415E0431E0433E0444E0445E0456E0479E0479E0486E0488E0414E0314E0314E0315E0318E03ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’aLog2[fold change] between two maternal immune activationsex groups MPA PRRSVinduced maternal immune activation CON control Fe females Ma malesankyrin repeat domain ANKRD24interferoninducedtransmembrane protein IFITM1 and IFITM3 cathepsinC CTSC mitogenactivated protein kinase kinase MAP2K7heparan sulfateglucosamine 3sulfotransferase HS3ST5secreted phosphoprotein SPP1 immunoglobulin heavy chainIGHG and transforming acidic coiledcoilcontaining protein TACC1 Among the genes underexpressed in MPA relative toCON pigs are insulinlike growth factor IGF2 cellular retinoicacidbinding protein CRABP2 and aldehyde dehydrogenase family member A1 ALDH1A1Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaFunctional Analysis of Genes AssociatedWith Maternal Immune ActivationTable presents the top significant clusters of informativeenriched categoriesfrom the DAVID analysis of genesTABLE Most enriched DAVID clusters and supporting functional categoriesenrichment score ES among the genes presenting significant maternalimmune activationbysex interaction effectaCategory Category identifier and namePvaluebFDRPvalueCluster KEGGBPKEGGKEGGKEGGCluster BPBPBPBPBPCluster BPCluster BPBPES ssc05320Autoimmune thyroid diseaseGO000250ˆ¼Antigen processing andpresentation of peptide orpolysaccharide antigen via MHC class IIssc04514Celladhesion moleculesCAMsssc05323Rheumatoid arthritisssc05164Influenza AES GO0051050ˆ¼Positive regulation oftransportGO0051049ˆ¼Regulation of transportGO0050801ˆ¼Ion homeostasisGO0048878ˆ¼Chemical homeostasisGO0030001ˆ¼Metal ion transportES GO0048871ˆ¼Multicellular anismalhomeostasisES GO0061564ˆ¼Axon developmentGO0007420ˆ¼Brain development290E06190E03450E04340E01230E03320E02480E03200E02560E02170E01440E03350E01450E03130E02280E02450E02320E01360E01480E01570E01200E04370E01640E05130E03170E01310E01aBP biological process KEGG KEGG pathway bFalse discovery rate adjustedPvalueTABLE Enriched informative categories NES using GSEA among thegenes based on the overall maternal immune activationbysex interactionaCategory Category identifier and namebNES PvalueKEGGKEGGMFBPBPBPKEGGBPˆ’ 10E10ˆ’ 10E10ˆ’ 10E10ˆ’ 10E10ssc04080Neuroactive ligand receptorinteractionssc04912GnRH signaling pathwayGO0005179ˆ¼Hormone activityGO0006970ˆ¼Response to osmoticstressGO0019221ˆ¼Cytokine mediatedsignaling pathwayGO1901385ˆ¼Regulation of voltagegated calcium channel activityssc04020Calcium signaling pathway ˆ’ 12E01GO0085029ˆ¼Extracellular matrixˆ’ 18E01assemblyˆ’ 91E02ˆ’ 12E01cFDRPvalue83E0299E0222E0135E0154E0154E0154E0155E01aMF molecularfunction KEGG KEGG pathway BP biological processbNormalized enrichment score negative values indicate genes underexpressionin CON females relative to males but overexpression in MPA females relative tomales cFalse discovery rate adjusted Pvaluediï¬erentially expressed between MPA and CON groupsacross sexes the extended list of categories is presented inSupplementary File S1 Table E Some categories identifiedby the DAVID analysis are consistent with the categoriesdetected at more significant levels among the genes presentingan MIAbysex interaction eï¬ect Table and include theBP angiogenesisand KEGG autoimmunethyroid disease and Epstein“Barr virus infection pathwaysTable Also enriched Supplementary File S1 Table Ewere the BP homeostatic GO0042592 MF ion bindingGO0043167 and BP anatomical structure formation inmorphogenesis GO0048646GO0001525The GSEA enrichment results within the gene expressionpatterns of CON relative to MPA groups complemented thefindings from DAVID The most informative enriched categoriesare presented in Table and the extended list of categories ispresented in Supplementary File S1 Table F Enriched clustersof genes overexpressed in CON relative to MPA detected byGSEA were the BP enrichment of microtubule bundle formationGO0001578 and cilium morphogenesis GO0060271Transcriptome Differences BetweenSexes Independent of Maternal ImmuneActivationOverall genes were diï¬erentially expressed between malesand females FDRadjusted P These genes exhibiteda consistent diï¬erential expression between sexes irrespectiveof the MIA group The complete list of genes diï¬erentiallyexpressed between sexes at FDRadjusted P is available inSupplementary File S1 Table G and the majority were overexpressed in males relative to females Among the previousgenes excluding those that presented MIAbysex interactioneï¬ect a selection of informative genes is listed in Table Genesoverexpressed in males relative to females included eukaryotictranslation initiation factor 1A Ylinked EIF1AYleptinreceptor LEPR luteinizing hormone beta polypeptide LHBLIM homeobox LHX9 luteinizing hormone beta polypeptideLHB and immunoglobulin family member IGSF1Informative categories among the DAVID clusters ofenriched categoriesthe genes diï¬erentially expressedbetween sexes are listed in Table a complete list i

Thyroid_Cancer

Hepatitis B virus HBV infection has been associated with the risk andprognosis of many malignancies Nevertheless the association between HBV and theprognosis of breast cancer is unclear The objectives of this study were to investigate theprognostic role of hepatitis B surface antigen HBsAg and to integrate HBsAg to establishnomograms for better prognostic prediction of very young patients with breast cancerMethods This analysis was performed retrospectively in a cohort of consecutivevery young ‰ at diagnosis patients who received curative resection for breastcancer The significance of HBsAg in the prognosis of these patients was investigatedUnivariate and multivariate analyses were used to identify independent variables fordiseasefree survival DFS and overall survival OS Nomograms were built based onthose identified variablesResults Overall of the patients were seropositive for HBsAgThe median followup was CI “ months forthe entirepopulation The HBsAgpositive cohort had significantly inferior DFS HR CI “ P and OS HR CI “ P as compared with the HBsAgnegative cohort The rates of 10year DFS andOS were and in the HBsAgpositive group and and in the HBsAgnegative group respectively In multivariable analysis HBsAg statuswas identified as an independent significant unfavorable prognostic factor for DFSP and OS P in very young patients with breast cancer Nomogramswere established and displayed good calibration and acceptable discrimination TheCindex values for DFS and OS were CI “ and CI “ respectively Based on the total prognostic scores TPSof the nomograms different prognosis groups were identified for DFS and OSEdited byImtiaz Ahmad SiddiquiUniversity of Colorado AnschutzMedical Campus United StatesReviewed byAbhinit NagarUMass Memorial Medical CenterUnited StatesNidhi JainBeckman Coulter IndiaCorrespondenceLu YangyanglusysucccnWeiDong Weiweiwdsysucccn These authors have contributedequally to this workSpecialty sectionThis was submitted toCancer Epidemiology and Preventiona section of the journalFrontiers in OncologyReceived March Accepted July Published August CitationLi N Zhong QQ Yang XRWang QC Zhang DT Zheng SYang L and Wei WD Prognostic Value of Hepatitis B VirusInfection in Very Young Patients WithCuratively Resected Breast CancerAnalyses From an Endemic Area inChina Front Oncol 103389fonc202001403Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerConclusions HBsAg is an independent unfavorable prognostic factor for DFS andOS in very young patients with curatively resected breast cancer and nomogramsintegrating HBsAg provide individual survival prediction to benefit prognosis evaluationand individualized therapyKeywords HBV young breast cancer prognosis nomogram survivalINTRODUCTIONGlobally breast cancer is the most common cancer and theleading cause of cancer death for women accounting for of total cancer cases and of total cancer deaths Breast cancer has also been the top one malignancy in termsof incidence in Chinese women constituting of newlydiagnosed cases and of all deaths from breast cancer in theworld Although breast cancer occurs at a lower incidence inChinese women than in western women this disease occurs at ayounger age in China than in highincome countries and China™scontribution to global breast cancer rate is increasing rapidly The disparities between young and old breast cancer include ahigher mortality rate higher risk of recurrence poorer treatmentresponse and more aggressive phenotypes “ Thereforeunderstanding the etiology and identifying novel prognosticfactors are essential for early diagnosis prognosis evaluationearly intervention and personalized therapy in young patientswith breast cancerHepatitis B virus HBV infection is a serious public healthdilemma with ˆ¼ million chronic carriers worldwide China account for about a third of infectionassociated cancerglobally driven by high prevalence of HBV and H pylori infection Although China has made tremendous eï¬orts in controllingHBV over the past years and the prevalence of HBV ininfants and children has remarkably declined the hepatitisB surface antigen HBsAg prevalence is still high in Chineseadults ranging from to “ HBV is the leading causeof hepatocellular carcinoma and cholangiocarcinoma Inaddition there is also accumulating evidence that HBV infectionis associated with many extrahepatic malignancies includingnonHodgkin™s lymphoma pancreatic cancer gastriccancer nasopharyngeal carcinoma lung cancer esophageal cancer and ovarian cancer Thus it seemsreasonable that HBV is an important factor in the developmentof extrahepatic malignancies in endemic areasDespite the facts that HBsAg status is one of the routineexaminations in patients with operable breast cancer and severalstudies have showed that HBV is not associated with therisk of breast cancer the impact of HBV on theclinicopathological characteristics and prognosis of very youngpatients with breast cancer remains to be determined Giventhat both early breast cancer and HBV are endemic in China itis possible that HBV infection is associated with the prognosisof early breast cancer even though the precise mechanismsare yet to be determined It is crucial to address this issuesince HBV has been reported to be found in breast cancertissue We therefore performed this study to investigate theHBsAg prevalence in very young breast cancer and the impactof HBsAg on the survival of these patients and to establishnomograms to better predict prognosis for very young patientswith breast cancerMATERIALS AND METHODSPatient SelectionA retrospective review was conducted in a cohort of consecutive breast tumor women who were aged ‰ years oldand received curative resection for breast cancer at Sun Yatsen University Cancer Center between May and July This study was conducted according to the ethicalstandards of the Declaration of Helsinki Institutional ReviewBoard approval was obtained from the Medical Ethics Committeeof this cancer center All patients were restaged by the eighthinternational classification system for breast cancer Dueto the retrospective nature of this studyinformed consentwas waivedInformation was collected from electronic patient recordsand survival data were obtained from the followup registryofthis center The information collected included HBsAgstatus laterality type of breast surgery type of axillary surgeryhistological type tumor grade tumornodemetastasis TNMstage dates of surgeryrelapsedeath status of estrogen receptorER progesterone receptor PR human epidermal growthfactor receptor HER2 and Ki67 Breast cancers wereclassified as luminal Alike ER PR‰¥ HER2“ andKi6715luminal Blike ER andor PR HER2“HER2enriched ER“ PR“ HER2 or triplenegative ER“PR“ HER2“ subtypesPotentially eligible patients had to have curatively resectedbreast cancer without previous therapy other than neoadjuvanttherapy be aged years old or below and have definiteinformation of HBsAg The main exclusion criteria includedbenign tumor not having surgery having incomplete resectionprevious malignant disease hepatitis viral infections other thanHBV men patients and insufficient data of survival or HBsAgStatistical AnalysisThe main objectives of this study were to compare diseasefreesurvival DFS and overall survival OS between HBsAgpositivepatients and HBsAgnegative patients DFS was defined as theinterval from the date of being diagnosed to the date of diseaserecurrencemetastasis or death from any cause OS was defined asthe interval from the date of being diagnosed to the date of deathfrom any cause Median followup was estimated by KaplanMeier analysis with reversed meaning of status indicator Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerforindependent variablesDFS and OS were estimated by the Kaplan“Meier methodand diï¬erences were compared by the logrank test Univariateand multivariate analyses with a Cox proportional hazardsmodel were used to testforDFS and OS Covariates included laterality left vs righttype of surgery breastconserving surgery vs mastectomytype of axillary surgery sentinellymph node dissection vsaxillary lymph node dissection histological type ductal vsotherstumor grade grade III vs grade III T stageT34 vs T12 N stage N23 vs N1 HER2statustriplenegativeHER2enriched vs luminal All variables reaching asignificance of in univariate analyses were included inmultivariate analysispositive vs negative molecularsubtypesThe nomograms for predicting and 10year DFSand OS were formulated based on the results of multivariateanalysis by the œrms package of R The discriminationofthe nomogram models was estimated by the Harrell™sconcordance index Cindex The value of the Cindex rangesfrom to with implying a random chance and indicating a perfect prediction Calibration curves of thenomogram models for DFS OS were plotted to assess thepredictive value of the model In addition patients weredivided into three diï¬erent risk groups highintermediatelow according to total prognostic scores TPS The totalprognostic scores of patients were transformed into categoricalvariables based on cutoï¬ points which were determined by theminimum Pvalue from logrank × statistics with the Xtileprogram Pearson™s chisquare test was used to compare categoricaldata All Pvalues were twosided and P was consideredstatistically significant Statistical analyses were performed by theSPSS software SPSS Inc version Chicago IL USA and Rfor Windows version httpwwwrprojectData AvailabilityThe authenticity of this has been validated by uploadingthe key raw data onto the Research Data Deposit public platformwwwresearchdatacn with the approval RDD numberas RDDA2020001410 The data that support the findings ofthe study are available from the corresponding authors uponreasonable requestRESULTSPatient CharacteristicsA total of very young ‰ at diagnosis breasttumor patients were screened of whom were excludedbecause of benign tumor n not having surgeryn not having R0 resection n or unknownHBsAg status n With further exclusions forinsufficientfollowup data a total of patients withcurative resection for breast cancer and aged years orbelow were included in this study Figure The patients™FIGURE The process of patient selectionFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerTABLE Patient characteristics by HBsAg statusCharacteristicsHBsAgpositiveN No HBsAgnegativeN No Pvalue FIGURE Number of recurrences by year of entire patientsLateralityLeftRightBilateralType of breast surgeryMastectomyBreastconserving surgeryType of axillary surgeryALNDSLNDHistological typeDuctalInvasive lobularOtherTumor gradeIIIIIIUnknownT StageT1T2T3T4N StageN0N1N2N3HER2 statusPositiveNegativeUnknownMolecular subtypesLuminal ALuminal BHER2enrichedTriple negativeUnknown HBV Hepatitis B Virus ALND axillary lymph node dissection SLND sentinelnode dissectionHER2 human epidermal growth factor receptor2lymphcharacteristics are shown in Table Overall ofthe patients were seropositive for HBsAg HBsAgpositive group and HBsAgnegative group were wellmatchedfor basic characteristics including laterality type of breast andaxillary surgery histological type tumor grade T stage Nstage and molecular subtypes About in patients receivedmastectomy and most patients received axillary lymph nodedissection ALNDAssociations Between the Status of HBsAgand SurvivalThe median followup was CI “ months forthe entire population By the time of analysis December instances of disease recurrence had occurred Thenumber of recurrences in each year of the followup is shown inFigure Of note although HBsAgpositive patients had a higherfrequency of extrahepatic metastasis vs P thanHBsAgnegative patients they had a comparable frequency ofliver metastasis vs P when compared withHBsAgnegative patientsDFS was significantly shorter among those who were HBsAgpositive than among those who were HBsAgnegative HR CI “ P The rates of 10yearDFS were in the HBsAgpositive group and inthe HBsAgnegative group respectively Figure 3A A totalof death events had occurred by the data cutoï¬ HBsAgpositive group had significantly inferior OS compared withHBsAgnegative group HR CI “ P with a 10year OS of and respectivelyFigure 3B The association of HBsAg status and survival ineach molecular subtype was further analyzed As expectedDFS and OS were significantly longer among those in theluminal A subgroup and the HER2enriched and triplenegativegroups had significantly shorter DFS and OS Figures 4ABNotably HBsAgpositive status was associated with shorterDFS P and OS P in the luminalB cohort HBsAgpositive status was also associated with aslightly shorter DFS in the triplenegative cohort and shorterOS in the HER2enriched cohort but statistical significancewas not reached Supplementary Figures There was nosignificant diï¬erence in DFS between HBsAgpositive patientsand HBsAgnegative patients in luminal A and HER2enrichedcohorts and in OS in luminal A and triplenegative cohortsSupplementary Figures In the univariate analysis type of breast surgery tumor gradeT stage N stage molecular subtype and HBsAg status wereFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerindividual patient Figures 6AB The model™s explanatorycovariables consisted of HBsAg status T stage N stage andmolecular subtype Patients with higher scores correspondedto inferior survival The scatter plots for the TPS of DFSand OS and percentage of patient number were presentedin Figures 6CD The Cindex values for DFS and OS were CI “ and CI “ respectively The calibration curves for the probabilityof DFS and OS at or year presented an optimalagreement between the prediction by nomogram and actualobservation Supplementary Figure Next we divided thepatients into the following groups based on the TPS ofthe nomogram modelfor DFS using the Xtile programlowrisk group TPS “ patients intermediateriskgroup TPS “ patients and highrisk group TPS patients The 10year DFS for lowrisk groupintermediaterisk group and highrisk group were and respectively Survival analyses for DFS demonstratedsignificant discrimination between these three groups P Figure 7A Same procedures were performed for OS in theentire population and patients were divided into the following groups based on the TPS of the nomogram model for OS withthe Xtile program lowrisk group TPS “ patientsintermediaterisk group TPS “ patients and highrisk group TPS patients OS diï¬erences were alsoobserved among three subgroups with a 10year OS of and for lowrisk intermediaterisk and highrisk groupsP Figure 7BDISCUSSIONIn this study we investigated the association of HBsAg statusand very young breast cancer and to our knowledge reportfor the firsttime that HBsAgpositive status is associatedwith inferior DFS and OS from a population with a highprevalence of both HBV infection and young breast canceridentified as a significant unfavorableHBsAg status wasprognostic predictor for DFS and OSindependent of anyother clinicopathological features of breast cancer includingT stage N stage and molecular subtype We also integratedHBsAg to build nomograms to better predict prognosis foryoung patients with breast cancer The results of our studydemonstrated that the prevalence of HBsAg in young patientswith breast cancer in southern China was which wasin accordance with the “ reported in the populationof this endemic area This result suggests that unlikecervical cancer young breast cancer is not correlatedwith an increased prevalence of HBV infection Indeed breastcancer patients with HBsAg did not demonstrate a diï¬erentpattern of characteristics It is noteworthy that in our studyHBsAg did not increase the rate of liver metastases for veryyoung patients with breast cancer This results are comparableto those reported in previous studies for esophageal cancerand colorectal cancer which suggested that HBVinfection is associated with decreased risk of liver metastasis inthese malignanciesFIGURE Kaplan“Meier curves for A diseasefree survival and B overallsurvival stratified by HBsAg status in very young patients with breast canceridentified as significant prognostic factors for DFS Figure 5AWhen those variables were further analyzed in the multivariateanalysis we found that T stage P N stage P molecular subtype P and HBsAg status P remained statistically significant indicating that theyare significant independent predictors for DFS Figure 5ABy the same methods for OSthe results showed that Tstage P N stage P molecular subtype and HBsAg status P were independentprognostic factors for OS Figure 5B HBsAgpositive status isan independent negative prognostic factor for survival in veryyoung breast cancerPrognostic Nomograms For Very YoungBreast Cancer PatientsTo better assess the DFS and OS of very young breastcancer patients prognostic nomograms for DFS and OS wereestablished respectively All the independent predictors of DFSand OS in the multivariate analysis were integrated into thenomogram models and and 10year survivals weregraphically computed according to the characteristics of anFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Kaplan“Meier curves for A diseasefree survival and B overall survival stratified by molecular subtype in very young patients with breast cancerOur study shows that HBsAgpositive status is associatedwith inferior DFS and OS in young patients with curativelyresected breast cancer decreasing the 10year DFS and OS by and respectively The association between HBsAgpositive status and poor prognosis is in keeping with thatdemonstrated in nasopharyngeal carcinoma lung cancer and ovarian cancer However some studies regardingother cancers indicate that HBsAgpositive cancer patientshad a favorable survival as compared with HBsAgnegativepatients This discrepancy can be partly explainedby the diversity and heterogeneity of diï¬erent malignanciesThe genetic or biological mechanisms underlying the inferiorFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Univariate and multivariate analysis for diseasefree survival A and overall survival B for very young patients with breast cancerprognosis remain to be elucidated butthis may largelyrelate to the presence of hepatitis B Xinteracting proteinHBXIP which has been welldocumented to function asan oncoprotein in breast cancer HBXIP can act as atransactivator by activating certain genes including cMyc E2F1STAT4 and Sp1 to play a crucial role in the progression ofbreast cancer HBXIP is associated with controlling cellapoptosis and promoting cell proliferation by mTOC1 activation HBXIP can also act as a modulation factor of cellularoxidative stress by competitively binding KEAP1 to enhancethe progression of breast cancer Previously studies showedthat HBV is not associated with risk of breast cancer These results combined with the data of our study suggestthat HBV is not a risk factor but a prognostic factor forbreast cancerAnother reason for this might relate to the HBV reactivationin HBsAgpositive patients with breast cancer who werereceiving chemotherapy HBV reactivation occurs frequentlyin breast cancer patients who are HBV carriers while receivingcytotoxic chemotherapy HBV reactivation can result inliver failure and interruption of the chemotherapy scheduleOther potential mechanisms underlyingassociationtheFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE AB Nomograms predicting and 10year A diseasefree survival and B overall survival for very young patients with breast cancer CD Thescatter plots of percentage of patient number and groups of C total prognostic score for DFS and D total prognostic score for OSbetween the HBsAg and patient survival include the immunesuppression Chronic HBV infection is characterized by thefailure to elicit an eï¬ective adaptive immune response andthe immune modulation of key innate immune response Chronic HBV infection can lead to immune anergy andimpair the function of the immune system which has longbeen deemed to protect the host against the developmentof nonviral cancerstogether couldpartially explain the positive association between HBsAgpositive status and the poor prognosis in young patients withbreast cancer These reasonsInthisstudy wecharacteristicscombined HBsAgstatus withclinicopathologicaleï¬ectiveprognostic nomogram models of DFS and OS for very youngpatients with breast cancer Both nomograms showed goodcalibration and acceptable discrimination These nomogrammodels can be used for prognosis evaluation at diagnosis forvery young patients with breast cancer and may benefit patientestablishtocounseling and personalized therapy for these patients Weadopted Xtile program to divide these patients into three riskgroups based on TPS from nomograms for DFS and OS Thesurvival curves for DFS and OS separated very well Thusspecial attention should be paid to and active surveillanceshould be conducted over patients with high risk group for DFSand OSThis study nevertheless has certain limitations that shouldbe noted First this study was retrospective in nature andwe cannot rule outthe impact of selection bias Secondthe sample size was relatively small and the sample sizesof the two cohorts were unequal as only patients wereHBsAgpositive The small sample size may be insufficientto allow us to perform subgroup analysis by each molecularsubtype Anotherthat Cantonese constitutemost of our study population The monotonicity ofthestudy population confines the universality of our resultsFurthermore the information was insufficient to perform otherlimitation isFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Kaplan“Meier curves for A diseasefree survival and B overall survival stratified by risk groups based on total prognostic scores fromnomogram modelsanalysis such as that of hepatic function and HBV DNAcopy number Nevertheless the results of our study providewhat to our knowledge is the first evidence of the impactof HBsAg on the prognosis of very young patients withbreast cancerIn conclusion we have demonstrated in a retrospectivestudy that HBsAg is an independent unfavorable prognosticfactor for patients with very young breast cancer Furtherprospective studies involving varied ethnic populations arewarranted to confirm the prognostic value of HBsAg status invery young breast cancer and simultaneously other potentialclinicopathologic factors for breast cancer and HBV infection arerequired to be taken into account The mechanisms of the impactof HBV infection on the progression of breast cancer also need tobe elucidatedDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studiesinvolving human participants were reviewedand approved by the Medical Ethics Committee of SunYatsen University Cancer Center WritteninformedFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerconsentin accordance with the nationalinstitutional requirementsfor participation was not required for this studylegislation and theAUTHOR CONTRIBUTIONSNL QQZ LY and WDW designed the study NL QQZXRY QCW DTZ and SZ collected the data NL QQZ LYand WDW interpreted and analyzed the data All authors wereinvolved in writing the and approved the final versionREFERENCES Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Globalcancer statistics GLOBOCAN estimates of incidence and mortalityworldwide for cancers in countries CA Cancer J Clin “ 103322caac21492 Fan L StrasserWeippl K LiJJ St LouisJ Finkelstein DM Yu 15e279“KD et al Breast cancer 101016S1470204513705679in China Lancet Oncol Colleoni M Rotmensz N Robertson C Orlando L Viale G Renneet al Very young women years with operable breastGcancer“ 101093annoncmdf039at presentation Ann Oncolfeaturesof disease Anders CK Hsu DS Broadwater G Acharya CR Foekens JA Zhang Y et alYoung age at diagnosis correlates with worse prognosis and defines a subsetof breast cancers with shared patterns of gene expression J Clin Oncol “ 101200JCO2007142471 Narod SA Breast cancer in young women Nat Rev Clin Oncol “ 101038nrclinonc2012102 Trepo C Chan HL Lok A Hepatitis B virus infection Lancet “ 101016S0140673614602208 de Martel C Gees D Bray F Ferlay J Cliï¬ord GM Global burden of cancerattributable to infections in a worldwide incidence analysis LancetGlobal Health 8e180“ 101016S2214109X19304887 Lu FM Li T Liu S Zhuang H Epidemiology and prevention of hepatitisJ Viral Hepatitis 17Suppl “B virus infection in China 101111j13652893201001266x Liu J Zhang S Wang Q Shen H Zhang M Zhang Y et al Seroepidemiologyof hepatitis B virus infection in million men aged years in ruralChina a populationbased crosssectional study Lancet Infect Dis “ 101016S1473309915002182 Zhang Y Fang W Fan L Gao X Guo Y Huang W et al HepatitisB surface antigen prevalence among rural women of childbearingage in Hainan Province China a crosssectional study Virol J 1011861743422X1025 Cui Y Jia J Update on epidemiology of hepatitis B and C in China JGastroenterol Hepatol 28Suppl “ 101111jgh12220 Chan SL Wong VW Qin S Chan HL Infection and cancer the case ofHepatitis B J Clin Oncol “ 101200JCO2015615724 Fwu CW Chien YC You SL Nelson KE Kirk GD Kuo HS et al Hepatitis Bvirus infection and risk of intrahepatic cholangiocarcinoma and nonHodgkinlymphoma a cohort study of parous women in Taiwan Hepatology “ 101002hep24150 Kamiza AB Su FH Wang WC Sung FC Chang SN Yeh CC Chronichepatitis infection is associated with extrahepatic cancer developmenta nationwide populationbased study in Taiwan BMC Cancer 101186s1288501629185 Nath A Agarwal R Malhotra P Varma S Prevalence of hepatitis B virusinfection in nonHodgkin lymphoma a systematic review and metaanalysisInternal Med J “ 101111j14455994200902060x Luo G Hao NB Hu CJ Yong X Lu MH Cheng BJ et al HBV infectionincreases the risk of pancreatic cancer a metaanalysis Cancer Causes Control “ 101007s1055201201442FUNDINGThis work was supported by the National Natural ScienceFoundation of China No SUPPLEMENTARY MATERIALThe Supplementary Materialonline202001403fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncat Wei XL Qiu MZ Jin Y Huang YX Wang RY Chen WW et al Hepatitis Bvirus infection is associated with gastric cancer in China an endemic area ofboth diseases Br J Cancer “ 101038bjc2014406 Ye YF Xiang YQ Fang F Gao R Zhang LF Xie SHHepatitis B virusin southern China Cancer Epidemiol Biomarkers Prevent“ 10115810559965EPI150344et alinfection and risk of nasopharyngeal carcinoma Peng JW Liu DY Lin GN Xiao JJ Xia ZJ Hepatitis B virusinfection is associated with poor prognosis in patients with advancednon small cell“ 107314APJCP201516135285lung cancer Asian Pacific J Cancer Prevent Zou J Chen J Xie X Liu Z Cai X Liu Q et al Hepatitis B virus infectionis a prognostic biomarker for better survival in operable esophageal canceranalysis of patients from an endemic area in China Cancer EpidemiolBiomarkers Prevent “ 10115810559965EPI181095 Wong L Cheung TH Yim SF Lao TT Prevalence and impact of hepatitis Bvirus infection in ovarian cancer patients in an endemic areaA retrospectivecohort study J Viral Hepat “ 101111jvh13250 Song C Lv J Liu Y Chen JG Ge Z Zhu J et al Associations between hepatitisB virus infection and risk of all cancer types JAMA Netw Open 2e195718 101001jamanetworkopen20195718 Su FH Chang SN Chen PC Sung FC Su CT Yeh CC Associationrisk abetween chronic viral hepatitisinfection and breast cancernationwide populationbased casecontrol study BMC Cancer Qin B Zhao K Wei J Wang X Xu M Lang J et al Novel evidence indicatesthe presence and replication of hepatitis B virus in breast cancer tissue OncolRep “ 103892or20197393 Te HS Jensen DM Epidemiology of hepatitis B and C viruses a globaloverview Clinics Liver Dis “ vii 101016jcld200911009 Siu SS Cheung TH Chan PK Lin CK Lo KW Patients with malignant or premalignant cervical lesion have increased risk of becoming hepatitis B carrierJ Exp Clin Cancer Res “in patients with colorectal Zhao Y Lin J Peng J Deng Y Zhao R Sui Q et al Hepatitis B virus infectionpredicts better survivalliveronly metastasesundergoing liver resection J Cancer “ 107150jca24544 Liu X Li X Jiang N Lei Y Tang LL Chen L et al Prognostic value ofchronic hepatitis B virus infection in patients with nasopharyngeal carcinomaanalysis of patients from an endemic area in China Cancer “ 101002cncr28377 Zhao Y Li H Zhang Y Li L Fang R Li Y et al Oncoprotein HBXIPmodulates abnormal lipid metabolism and growth of breast cancer cells byactivating the LXRsSREBP1cFAS signaling cascade Cancer Res “ 10115800085472CAN151734 Zhang Y Zhao Y Li H Li Y Cai X Shen Y et al The nuclear import ofoncoprotein hepatitis B Xinteracting protein depends on interacting with cFos and phosphorylation of both proteins in breast cancer cells J Biol Chem “ 101074jbcM113458638 BarPeled L Schweitzer LD Zoncu R Sabatini DM Ragulator is a GEFfor the rag GTPases that signal amino acid levels to mTORC1 Cell “ 101016jcell201207032 Zhou XL Zhu CY Wu ZG Guo X Zou W The oncoproteinHBXIP competitively binds KEAP1 to activate NRF2 and enhanceFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancercancerbreast“ 101038s4138801906985growthcelland metastasis Oncogene Liu Z Jiang L Liang G Song E Jiang W Zheng Y et al Hepatitis B virusreactivation in breast cancer patients undergoing chemotherapy a reviewand metaanalysis of prophylaxis management J Viral Hepat “ 101111jvh12672 Yuen MF Chen DS Dusheiko GM Janssen HLA Lau DTY LocarniniSA et al Hepatitis B virus infection Nat Rev Dis Primers 101038nrdp201835 Dunn GP Old LJ Schreiber RD Theimmunobiology ofimmunosurveillance“ 101016jimmuni200407017immunoeditingandImmunitycancer Giuliano AE Edge SB Hortobagyi GN Eighth edition ofthe AJCCcancer staging manual breast cancer Ann Surg Oncol “ 101245s1043401864866 Schemper MSmith TL A note on quantifyingstudies 101016019724569600075Xfailuretime Control ClinofTrialsfollowup in“ Vickers AJ Elkin EB Decision curve analysis a novel method for “evaluating prediction models Med Decision Making 1011770272989X06295361 Camp RL DolledFilhart M Rimm DL Xtile a new bioinformatics toolfor biomarker assessment and outcomebased cutpoint optimization ClinCancer Res “ 10115810780432CCR040713Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Li Zhong Yang Wang Zhang Zheng Yang and Wei This is anopenaccess distributed under the terms of the Creative Commons AttributionLicense CC BY The use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in accordance with accepted academicpractice No use distribution or reproduction is permitted which does not complywith these termsFrontiers in Oncology wwwfrontiersinAugust Volume 0c'

Thyroid_Cancer

Bone metastasis classification using wholebody images from prostate cancer patientsbased on convolutional neural networksapplicationNikolaos Papandrianos1 Elpiniki PapageiouID23 Athanasios Anagnostis34Konstantinos Papageiou4 General Department University of Thessaly Lamia Greece Faculty of Technology Dept of EnergySystems University of Thessaly Geopolis Campus Larisa Greece Institute for Bioeconomy and Agritechnology Center for Research and Technology Hellas Greece Department of Computer Science andTelecommunications University of Thessaly Lamia Greece npapandrianosuthgrAbstractBone metastasis is one of the most frequent diseases in prostate cancer scintigraphy imaging is particularly important for the clinical diagnosis of bone metastasis Up to date minimalresearch has been conducted regarding the application of machine learning with emphasison modern efficient convolutional neural networks CNNs algorithms for the diagnosis ofprostate cancer metastasis from bone scintigraphy images The advantageous and outstanding capabilities of deep learning machine learning™s groundbreaking technologicaladvancement have not yet been fully investigated regarding their application in computeraided diagnosis systems in the field of medical image analysis such as the problem of bonemetastasis classification in wholebody scans In particular CNNs are gaining great attention due to their ability to recognize complex visual patterns in the same way as human perception operates Considering all these new enhancements in the field of deep learning aset of simpler faster and more accurate CNN architectures designed for classification ofmetastatic prostate cancer in bones is explored This research study has a twofold goal tocreate and also demonstrate a set of simple but robust CNN models for automatic classification of wholebody scans in two categories malignant bone metastasis or healthy usingsolely the scans at the input level Through a meticulous exploration of CNN hyperparameter selection and finetuning the best architecture is selected with respect to classificationaccuracy Thus a CNN model with improved classification capabilities for bone metastasisdiagnosis is produced using bone scans from prostate cancer patients The achieved classification testing accuracy is whereas the average sensitivity is approximately Finally the bestperforming CNN method is compared to other popular and wellknown CNN architectures used for medical imaging like VGG16 ResNet50 GoogleNetand MobileNet The classification results show that the proposed CNNbased approach outperforms the popular CNN methods in nuclear medicine for metastatic prostate cancer diagnosis in bonesa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Papandrianos N Papageiou EAnagnostis A Papageiou K Bonemetastasis classification using whole body imagesfrom prostate cancer patients based onconvolutional neural networks application PLoSONE e0237213 101371journalpone0237213Editor Jeonghwan Gwak Korea NationalUniversity of Transportation REPUBLIC OF KOREAReceived November Accepted July Published August Copyright Papandrianos This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data which areanonymized bone scintigraphy images without anyfurther information are available only after requestfor research purposes Data availability Thedataset from Diagnostic Medical CenterœDiagnosticoIatriki AE was used under thelicense of the Board Committee Director of theDiagnostic Medical Center œDiagnosticoIatriki AE Dr Vasilios Parafestas for the current studyand is not publicly available The dataset may beavailable only under request from the Director ofPLOS ONE 101371journalpone0237213 August PLOS ONE 0cthe Diagnostic Center vparafestasyahoogr andthe relevant Doctor of Nuclear Medicine DrNikolaos Papandrianos npapandrianosuthgrnikpapandrgmailcomFunding The authors received no specificfunding for this workCompeting interests The authors have declaredthat no competing interests existBone metastasis classification using convolutional neural networks IntroductionMost common tumors such as those of the breast lung and prostate frequently metastasize tothe bone tissue and so the skeleton seems to be a site with the most significant tumor burdenin cancer patients with advanced disease Statistical analysis results have shown that of allbone metastases originate from the breast in women and from the prostate in men Theremaining emanates from thyroid lung and kidney cancers [] In the case of metastaticprostate cancer diagnosis has a significant impact on the quality of patient™s life [] In mostmen the metastatic prostate cancer mainly sites on the bones of the axial skeleton causingsevere lesions that can cause pain debility andor functional impairment [] As this type ofcancer has great avidity for bone and could cause painful and untreatable effects an early diagnosis is crucial for the patient Reviews on clinical evidences and diagnostic assessments ofbone metastases in men with prostate cancer can be found in []The implementation of a properly selected diagnostic imaging can reveal the number ofmetastatic foci in the skeletal system [ ] Rapid diagnosis of bone metastases can be achievedusing modern imaging techniques such as scintigraphy Positron Emission TomographyPET and wholebody Magnetic Resonance Imaging MRIThe primary imaging method in the diagnosis of metastases that offers the highest sensitivity among all imaging methods is Bone Scintigraphy BS [“] Through the depictionof the entire skeleton in one medical examination nuclear doctor is able to detect bone abnormalities in areas where intensive radionuclide activity is present However low specificityseems to be the main drawback of this method as it cannot tell whether the causes of boneturnovers are different than those of metastatic origin leukaemia healing fracture etc Atthe same time PET has been recognized as an efficient method for detecting cancer cellsbased on recent technological advancements in medical imaging PET and Computed Tomography CT combination can produce highresolution images [“]Although PET and PETCT are the most efficient screening techniques for bone metastasisBS remains the most common imaging procedure in nuclear medicine [ ] [] Asreported in European Association of Nuclear Medicine EANM guidelines [] BS is particularly important for clinical diagnosis of metastatic cancer both in men and women At presentwhen other imaging or examination methods are unable to provide a reliable diagnosis BSimaging becomes the proper modality for making a final diagnosis of bone metastasis []To address the considerable problem of bone metastasis diagnosis artificial intelligentmethods for medical image analysis implemented with deep learning algorithms have beenadequately investigated In this direction a recent survey reveals the entire penetration of deeplearning techniques into the field of medical image analysis detection segmentation classification retrieval image generation and enhancement registration and successful application ofdeep learning to medical imaging tasks are thoroughly examined [“]Implementation of deep learning in medical imaging is mainly conducted by ConvolutionalNeural Networks CNNs [ ] a relatively new and powerful way to learn useful representations of images and other structured data Before the application of CNNs these featurestypically had to be created by less powerful machine learning models or even handcraftedWith the introduction of CNNs such features could be learned directly from the provideddata since they include certain preferences in their structure that make them powerful deeplearning models for image analysis [ ] Typical CNNs have a similar structure withArtificial Neural Networks ANN and consist of one or more filters ie convolutional layers followed by aggregationpooling layers in order to extract features for classification tasks[] Gradient descent and backpropagation are both used as learning algorithms the sameway they are used in a standard ANN Their main difference lies in the fact that CNNs havePLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkslayers of convolutions along with pooling layers in the beginning of their architecture Thefinal outputs are computed via fully connected layers located at the end of the network architecture []In recent years CNNs have gained wider recognition in medical image analysis domain aswell as in vision systems [“ ] Due to their enormous popularity several applications ofCNNs were investigated in the field of medical image analysis Two recent review studies []and [] gather all the important and most interesting applications of deep learningThe application of CNNs in medical imaging ranges from plain radiograph CT MRI andmicroscopy images to clinical photos dermatology capsule endoscopy and visual recognition[“] In addition a CNN was investigated in [] that regards automatic detection of tuberculosis on chest radiographs while in [] a brain tumor segmentation in magnetic resonanceimages was made possible with the use of a CNN More examples of CNNs™ successful application in medical domain include automated cardiac diagnosis [] detection of lesions and prediction of treatment response by PET [ ] as well as dynamic contrast agentenhancedcomputed tomography where CNN showed high diagnostic performance in the differentiation of liver masses [] Furthermore CNNs have shown outstanding performance in radiology and molecular imaging []Some models with major impact in the context of deep learning and medical image processing were introduced in several s the Unet model for biomedical data semanticsegmentation [] the GoogLeNet model introducing the inception module [] the ResNetmodel introducing the residual learning building block for extremely deep convolutional networks [] and also Deeplab which deals with the inclusion of many convolutional layersatrous for semantic segmentation of images in deep convolutional neural networks []In medical image analysis the most widely used CNN methods are the following i AlexNet [] This network has a quite deep architecture similar to GoogLeNet by YannLeCun [] incorporates more filters per layer and includes stacked convolutional layersIt attaches ReLU activations after every convolutional and fullyconnected layer ii ZFNet [] Being a rather slight modification of AlexNet this network won the ILSVRCcompetition iii VGGNet16 [ ] It consists of convolutional layers having avery uniform architecture similar to AlexNet iv GoogleNet [] It is a convolutional neuralnetwork with a standard stacked convolutional layer having one or more fully connected layers called inception modules able to extract various levels of features on the same time vResNet [] It is another efficient CNN architecture that introduced the œidentityshortcut connection to solve the notorious problem of the vanishing gradients of the deepnetworks vi DenseNet [] Being another important CNN architecture DenseNetoffers the main advantage of alleviating the gradient vanishment problem with the direct connection of all the layers Related work in nuclear medical imaging for metastatic prostate cancerdiagnosis in bonesReviewing the relevant literature for diagnosis of bone metastasis using bone scintigraphyscans the authors notice that only a couple of previous works have been adequately conductedfor metastatic prostate cancer classification using CNNs while the others are devoted to ANNsand their application in ComputerAided Diagnosis CAD These works have investigated theuse of Bone Scan Index BSI which was introduced to assess the bone scanning process andestimate the extent of bone metastasis [ ] Specifically it serves as a clinical quantitativeand reproducible parameter that can measure metastatic prostate cancer bone involvement[] The software developed for the BSIbased ANN approach was EXINI bone EXINIPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksDiagnostics AB Lund Sweden and afterwards a revised version of this software calledBONENAVI FUJIFILM Toyama Chemical Co Ltd Tokyo Japan was engineered using alarge number of Japanese multicenter training databases []The first of the reported studies was devoted to the development of a classification algorithm based on CNNs for bone scintigraphy image analysis [] It was carried out as a masterthesis in Lund University and is focused mainly on classification problems without considering any identification and segmentation tasks The used dataset was provided by Exini Diagnostics AB in the form of image patches of already found hotspots The process in whichhotspots were segmented cropped and collected from bone scans was implemented using asoftware developed at Exini BSI was calculated for wholebody bone scans by segmenting theentire skeleton from the background in both the anterior and posterior views Due to timeframe restrictions only the hotspots found in the spine have been used to train the CNN sincethey were considered to be the easiest to classify A shape model based on a mean shape of several normal wholebody scans was fitted to the skeleton using an image analysis algorithmcalled Morphon registration The outcomes of the aforementioned thesis [] have shown thatthe calculated accuracy of the validation set was whereas the calculated accuracy of thetesting set was The second study explored CNNs for classification of prostate cancer metastases usingbone scan images [] The tasks of this master project appeared to have a significant potentialon classifying bone scan images obtained by Exini Diagnostics AB too including BSI The twotasks were defined as i classifying anterior posterior pose and ii classifying metastatic nonmetastatic hotspots The outcome of this study is that the trained models produce highlyaccurate results in both tasks and they outperform other methods for all tested body regions inthe case of metastatic nonmetastatic hotspots classification The evaluation indicator of thearea under Receiver Operating Characteristic ROC score was equal to which is significantly higher than the respective ROC of obtained by methods reported in the literature for the same test setThe remaining research that concerns the same imaging modality BS is devoted to theintroduction of CAD systems with the use of ANN and other Machine Learning ML methods for bone metastasis detection in bone scintigraphy images Sadik were the first todevelop an automated CAD system as a clinical quality assurance tool for the interpretation ofbone scans [“] This bone scan CAD software was trained to interpret bone scans usingtraining databases that consist of bone scans from European patients who have the desiredimage interpretation metastatic disease or not The results showed a sensitivity of at aspecificity of These works result in certain outcomes that refer to the development of atotally automated computerassisted diagnosis system that can identify metastases after examining bone scans applying multilayer perceptron ANN techniques involving a small databaseof wholebody bone scans patients The highest sensitivity that was achieved from all thestudies and accomplished during this thesis was approximately []Horikoshi compared the diagnostic accuracy of two CAD systems one based on aEuropean and another on a Japanese training database in a group of bone scans from Japanesepatients [] The Japanese CAD software showed a higher specificity and accuracy comparedto the European Comparing the sensitivities the Japanese CAD software achieved whereas the European CAD software reached []In another study conducted by Tokuda the diagnostic capability of a completely automated CAD system which detects metastases in the images of bone scans by focusing on twodifferent patterns was investigated [] The first pattern was devoted to the detection ofmetastases per region the second one detects metastases per patient The investigated systemwas called œBONENAVI version  The produced results have shown that the new CADPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkssystem is able to decrease the number of false positive findings which depends on the primarylesion of cancerIn Aslantas proposed œCADBOSS as a fully automated diagnosis system forbone metastases detections using wholebody images [] The proposed CAD system combines an active contour segmentation algorithm for hotspots detection an advanced methodof image gridding to extract certain characteristics of metastatic regions as well as an ANNclassifier for identifying possible metastases The calculated accuracy sensitivity and specificity of CADBOSS were and respectively outperforming other state of theart CAD systemsAdditionally ML methods have been exploited and applied in CAD systems for bonemetastasis detection in bone scintigraphy images A parallelepiped classification method wasspecifically deployed in [] to assist physicians in bone metastases detection of cancer Decision Trees DT and Support Vector Machines SVM were exploited for predicting skeletalrelated events in cancer patients with bone metastases achieving higher accuracies with asmaller number of variables than the number of variables used in Linear Regression LR MLtechniques can be also used to build accurate models to predict skeletalrelated events in cancer patients with bone metastasis providing an overall classification accuracy of ± []As far as PET and PETCT imaging techniques in nuclear medicine are concerned thereare some recent and prominent studies that apply the advantageous features of CNNs In []deep learning has been applied for classification of benign and malignant bone lesions in [F]NaF PETCT images The authors in this work followed the VGG19 architecture for theirnetwork by employing × convolutional layers followed by fully connected layers and asoftmax layer as final activation The ImageNet database of natural images was further used topretrain the network™s weights In this way the network first is trained on general image features and later is tuned using the lesion images and the physician™s scores Taking a closer lookat the results it can be concluded that network™s performance was improved when it wastrained to differentiate between definitely benign score and definitely malignantscore lesions The values of prediction metrics were and concerningthe accuracy sensitivity specificity and positive predictive value respectivelyAlso a CNNbased system was examined in a recent retrospective study which included sequential patients who underwent wholebody FDG PETCT [] The main purpose ofthe study was to detect malignant findings in FDG PETCT examinations while a neural network model equivalent to ResNet24 was built Additionally GradCAM was employed toidentify the part of the image on which the neural network used the largest information Thefindings of the study showed that GradCAM reasonably highlighted the area of malignantuptake allowing physicians to make a diagnosis The same research team recently developed a CNNbased system that predicts the location of malignant uptake and furtherevaluated predictions accuracy [] A network model with configuration equivalent toResNet24 was used to classify wholebody FDG PET imagesIn the research work [] a simple CNNbased system that predicts patient sex from FDGPETCT images was proposed Specifically consecutive patients have participated in thestudy and underwent wholebody FDG PETCT The CNN system was used for classifyingthese patients by sex Another CNNbased diagnosis system for wholebody FDG PETCT wasdeveloped in [] that predicts whether physician™s further diagnosis is required or not Athorough analysis of the results shows that the accuracy considering images of patients presenting malignant uptake and images of equivocal was ± and ± respectivelyThe task of segmentation with the use of deep learning models in skeletal scintigraphyimages has been discussed in more research studies For example in [] the authors followedPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksdifferent approaches to convert convolutional neural networks designed for classificationtasks into powerful pixelwise predictors Moreover in [] a deeplearning based segmentation method was developed using prostatespecific membrane antigen PSMA PET imagesand showed significant promise towards automated delineation and quantification of prostatecancer lesions However this research domain that involves bone scintigraphy segmentationwith the inclusion of advanced deep neural networks has not been yet well establishedAlthough bone scintigraphy is extremely important for the diagnosis of metastatic cancer itis clear that a small number of research works have been carried out which presented onlysome preliminary results while the advantageous and outstanding capabilities of CNNs havenot been fully investigated Reviewing the relevant literature it appears that CNNs have notbeen sufficiently applied for the diagnosis of prostate cancer metastasis from whole bodyimages Aim and contribution of this research workCNN is an efficient deep learning network architecture that has recently found great applicability in the medical domain It has shown excellent performance on medical image applications including bone scintigraphy and nuclear medical imaging and can offer a positiveimpact on diagnosis tasksNowadays the main challenge in bone scintigraphy as being one of the most sensitiveimaging methods in nuclear medicine is to build an algorithm that automatically identifieswhether a patient is suffering from bone metastasis or not based on patient™s whole bodyscans It is of utmost importance that the algorithm needs to be extremely accurate due to thefact that patients™ lives could be at stake Deep learning algorithms whose potential lies in thefact that they can improve the accuracy of cancer screening have been recently investigatedfor nuclear medical imaging analysis Recent studies in BS and PET have shown that a deeplearningbased system can perform as well as nuclear physicians do in standalone mode andimprove physicians™ performance in support mode Even though BS is extremely importantfor the diagnosis of metastatic cancer there is currently no research paper regarding the diagnosis of prostate cancer metastasis from whole body scan images that applies robust and moreaccurate deep CNNsThis research study investigates the application of a deep learning CNN to classify bonemetastasis using whole body images of men who were initially diagnosed with prostate cancerThe proposed method employs different CNNbased architectures with data normalizationdata augmentation and shuffling in the preprocessing phase In the training phase the backpropagation technique has been used for updating the weights as part of the optimization process Finally the network architecture is finetuned and the configuration that offers the bestperformance is selected to train the CNN modelThe scope of the current work entails the following two main components First this studyintroduces the CNN method into the diagnosis of bone metastasis disease based on wholebody images In the second phase the paper deals with the improvement of the existing CNNmethod in terms of both network architecture and hyperparameter optimization Thedeployed process seemingly improves the diagnostic effect of the deep learning method making it more efficient compared to other benchmark and wellknown CNN methods such asResNet50 VGG16 GoogleNET Xception and MobileNet [ ]The innovations and contributions of this paper are well summarized as follows¢ The development and demonstration of a simple fast robust CNNbased classification toolfor the identification of bone metastasis in prostate cancer patients from wholebody scansPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networks¢ The rigorous CNN hyperparameter exploration for determining the most appropriatearchitecture for an enhanced classification performance¢ A comparative experimental analysis utilizing popular image classification CNN architectures like ResNet50 VGG16 GoogleNET Xception and MobileNetThis paper is structured in the following fashion Section presents the material and methods related to this research study Section presents the proposed solution based on CNNmethod for bone metastasis diagnosis for prostate cancer patients using whole body imagesSection shows the results of exploration analysis of CNNs in Red Green and Blue RGBmode for different parameters and configurations thus providing the best CNN model forthis case study Furthermore all the performed experiments with representative results aregathered in section whereas section provides a thorough discussion on analysis of resultsSection concludes the paper and outlines future steps Materials and methods Patients and imagesA retrospective review of consecutive whole body scintigraphy images from differentmale patients who visited Nuclear Medicine Department of Diagnostic Medical Center œDiagnostico AE in Larisa Greece from June to June was performed The selection criterion was prostate cancer patients who had undergone wholebody scintigraphy because ofsuspected bone metastatic diseaseDue to the fact that whole body scan images contain some artifacts and other nonrelatedto bone uptake such as urine contamination and medical accessories ie urinary catheters[] as well as the frequent visible site of radiopharmaceutical injection [] a preprocessingapproach was accomplished to remove these artifacts and nonosseous uptake from the original images This preprocessing method was accomplished by a nuclear medicine physicianbefore the use of the dataset in the proposed classification approachThe initial dataset of images contained not only bone metastasis presence and absencepatient™s cases suffering from prostate cancer but also degenerative lesions [] Due to thisfact as well as aiming to cope with a twoclass classification problem in this study a preselection process concerning images of healthy and malignant patients was accomplished In specific out of consecutive wholebody scintigraphy images of men from differentpatients were selected and diagnosed accordingly by a nuclear medicine specialist with years of experience in bone scan interpretation Out of bone scan images bone scansconcern male patients with bone metastasis and male patients without bone metastasis Anuclear medicine physician classified all the patient cases into categories metastasis absentand metastasis present which was used as a gold standard see Fig The metastatic imageswere confirmed by further examinations performed by CTMRI Wholebody scintigraphy Bone scansA Siemens gamma camera Symbia S series SPECT System by dedicated workstation and software Syngo VE32B with two heads and low energy highresolution collimators was used forpatients scanning The speed of scanning was cmmin with no pixel zooming Two types ofradionuclide were used for bone scintigraphy 99mTcHDP TechneScan1 and 99TcMDPPoltechMDP 5mg Whole body scintigraphy was acquired approximately hours after intravenous injection of “ MBq of radiopharmaceutical agent depending on the patientbody type The common intravenous injection was MBq of radiopharmaceutical agentPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Image samples in the dataset Label a Metastasis is present or b absent101371journalpone0237213g001In total planar bone scan images from patients with known prostate cancer werereviewed retrospectively The wholebody field was used to record anterior and posteriorviews digitally with resolution × pixels Images represent counts of detected gammadecays in each spatial unit with 16bit grayscale depthThe image data acquired were originally in DICOM files a commonly used protocol forstorage and communication in hospitals These image data were extracted from these DICOMfiles to create new images in JPEGformat instead A novel dataset of bone scintigraphyimages containing men patients suffering from prostate cancer with metastasis present andmetastasis absent two distinct classes of healthy and malignant cases was prepared for experimentation This dataset consisting of wholebody scans is available for research use afterrequestThis study was approved by the Board Committee Director of the Diagnostic Medical Center œDiagnosticoIatriki AE and the requirement to obtain informed consent was waived bythe Director of the Diagnostic Center due to its retrospective nature All procedures in thisstudy were in accordance with the Declaration of Helsinki MethodologyThe problem of classifying bone metastasis images is a complex procedure and so effectivemachine learning methods need to be exploited to cope with this diagnosis task Deep learningmethods such as CNNs are applied in order to train a classifier to distinguish images of prostate cancer patients with bone metastasis and metastasis absent on healthy patients The effective CNN method for bone metastasis classification proposed in this paper includes threeprocessing steps data preprocessing for the collected scan data normalization a trainingphase for CNN learning and validation and testing which includes the evaluation of the classification results as illustrated in Fig The proposed methodology is thoroughly presented inthe following sections Data preprocessing Step Load images to RGB The original images were saved inRGB mode All images are stored in a respective folder before loaded into the computer memory for CNN training In each image a suitable prefix was defined according to the patient™scategory for example œmalignant_ and œhealthy_ Next a small script was set up in order toPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Flowchart of the proposed CNN methodology101371journalpone0237213g002assign a numerical value as label to each image according to its prefix In our case the value ˜™was assigned for œmalignant_ prefixes whereas the value ˜™ for œhealthy_ prefixesStep Data normalization It is common to follow a normalization process feature scalingin most machine learning algorithms [] The minmax normalization processnormalizes thedataset values within the to ensure that all feature data are in the same scale for trainingand testing The data normalization process also assists the convergence of the backpropagation algorithmStep Data shuffle To avoid or eliminate unbiased sampling in machine learning anappropriate shuffling method is needed to be defined More specifically a randomnumbergenerator is used to reorder the images An image sample chosen randomly is meant to be animpartial representation of the total images An unbiased random sample is important formachine learning to provide reliable conclusions In this study Python™s randomshufflemethod is used for dataset shufflingStep Data augmentation Data augmentation is used as a method to artificially increasethe diversity of training data by a large margin by manipulating the existing data instead ofcreating new Data augmentation techniques such as cropping padding and horizontal flipping are commonly used to train large neural networks [] In this research the number ofimages used for learning processes was followed by an image augmentation processing such asrotation enlargementreduction range and flip Note that the original images used for the testwere not subjected to such an augmentation processStep Data split The dataset was split into three sections a training portion a validationportion that would allow the training process to improve and a testing holdout portionwhich is part of the dataset that is completely hidden from the training process The first datasplit takes place by removing of the total dataset and saving for later use as testing Theremaining of the dataset is then split again into an ratio where the small po

Thyroid_Cancer

"Despite recent interest in the use of ketogenic diets KDs for cancer evidence of beneficial effects islacking This study examined the impact of a randomly assigned KD on quality of life physical activity andbiomarkers in patients with breast cancerMethod A total of patients with locally advanced or metastatic breast cancer and without a history of renaldisease or diabetes were randomly assigned to either a KD or a control group for this 12week trial Concurrentwith the first third and fifth chemotherapy sessions quality of life physical activity and biomarkers thyroidfunction tests electrolytes albumin ammonia ALP lactate and serum ketones were assessed Dietary intake wasalso recorded on admission and the end of the treatmentResults No significant differences were seen in quality of life or physical activity scores between the twogroups after weeks however the KD group showed higher global quality of life and physical activityscores compared to the control group at weeks P P Also serum lactate and ALP levelsdecreased significantly in the KD group compared to the control group at the end of the intervention ± vs ± ± vs ± P and P respectively A significant inverse associationwas observed between total carbohydrate intake and serum betahydroxybutyrate at weeks r ˆ’ P No significant differences between groups were observed in thyroid hormones electrolytes albuminLDH or ammonia Compliance among KD subjects ranged from to as assessed by dietary intakeand serum ketones levels of Continued on next page Correspondence khodabakhshiadelehyahoocom6Cancer Research Center Shahid Beheshti University of Medical SciencesTehran Iran7Department of Cellular and Molecular Nutrition Faculty of Nutrition Scienceand Food Technology Shahid Beheshti University of Medical SciencesTehran IranFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cKhodabakhshi Nutrition Journal Page of Continued from previous pageConclusion According to our results besides a higher global quality of life and physical activity scores compared tothe control group at weeks KD diet combined to chemotherapy in patients with breast cancer does not bringadditional benefit about quality of life and physical activity at weeks However decreases seen in levels of lactateand ALP in the KD group suggest that a KD may benefit patients with breast cancerTrial registration This trial has been registered on Iranian Registry of Clinical Trials IRCT under the identification codeIRCT20171105037259N2 wwwirctirtrial30755Keywords Ketogenic diet Breast cancer quality of life Physical activity Lactate Alkaline phosphatase chemotherapythere are stillIntroductionKetogenic diets KDs are high in fat and very low incarbohydrate They have been used as a dietary treatment in epilepsy for nearly a century [] RecentlyKDs have gained the attention of cancer researchersdue to their potential impact on cancer cell metabolism [] Despite the growing evidence of possibleantitumor benefitssome concernsabout potential adverse effects of KDs in cancer patientsincluding micronutrient deficiencies appetitereduction nausea constipation [] fatigue [] hyperlipidemia and especially unintended weight loss [ ]KDs are perceived as restrictive in nature which mayadd to the burden of cancer patients who already suffer from considerable physical emotional and financialstress all of which are known to negativelyimpact quality of life QoL In addition alterations inphysical and cognitive function during cancer treatment are pervasive It is estimated that “ of patients undergoingfromcancerrelated fatigue [] Prior studies have foundthat KD may improve physical and mental wellbeing[] Less fatigue has been reported in healthy overweight and obese adults following lowglycemic compared to highglycemic diets [] Results ofthreestudies using the validated European anization forResearch and Treatment core QoL questionnaire tofindings [“] Aassess fatigue lacked consistentin advanced cancer patients showed imsmall trialprovementin sleep and emotionalfunction after athreemonth KD intervention [] Other studies havesuggested enhanced cognitive function [ ]cancertreatmentsufferTo date only four studies have assessed QoL in adultpatients with cancer [ “] Hunger is a reported sideeffect of restricted KDs however previous research hasfound that perceived hunger is reduced in low carbohydrate diets compared to low fat diets [] A recent systematic review has highlighted the need for additionallarger investigations on the impact of ketogenic diets onQoL [] The goal of this present trial was to assesswhether a KD had beneficial effects on QoL dietary intake physical activity and specific biomarkersinindividuals with breast cancer while also evaluating compliance to KD guidelines in these patientsThe protocol used in this trial [] and part of the resultsfrom this trial have been previously published [ ]MethodsThe study protocol was approved by the National Nutrition and Food Technology Research InstituteNNFTRI Shahid Beheshti University of MedicalSciencesIRSBMUNNFTRIREC1396187 All participants provided writteninformed consent prior to participating in the studySBMU TehranIranThis trial was a randomized controlled labelclinical trial to breast cancer patients with locally advanced or metastatic disease who were receiving chemotherapy for atleast weeks The studythe medical oncology clinic atwas conducted atShohadaeTajrish hospital Cancer Research CenterTehran Iran from July to October of Participation was to patients to years of ageExclusion criteria screened forsignificant cardiacrenal or neurologic comorbidities symptoms of malnutrition diabetes pregnancy and Karnofsky indexless than Using a block balanced randomizationmethod patients were assigned to the interventionn or controln groups Randomizationwas computergenerated by a statistician who was nota member of the medical team Blinding the participants or study personnel was not deemed feasible inthis dietintervention The project coordinator enrolled the participants and assigned them to their interventions Both the KD and the control diet werecalculated to be eucaloric using the MifflinSt Jeorformula The KD consisted of of calories fromCHO from protein from mediumchain triglyceride MCT oil and from fat A dietitianprovided specific nutritional counseling to each participantfacetoface meetings Patientsengaged in ongoing weekly counseling sessions viaphone WhatsApp or Telegram and were assessed forcompliance and possible adverse effects To furtherenhance compliance dietary recommendations werein individual 0cKhodabakhshi Nutrition Journal Page of individualized and appropriate recipes were providedto patients in the KD group were asked to refrainfrom eating any grains grain products starchy vegetables fruit or sugar Dietary carbohydrates were limited to nonstarchy vegetables and dietary proteinswere obtained primarily from egg meat poultry andfish Small amounts of lower carbohydrate berries andnuts were allowed as long as they did not exceed thecarbohydrate limit in the diet prescription Subjectswere encouraged to increase their fat intake and toselect from a variety of sourcesincluding olive oilbutter and cream cheese Patients were asked tochoose only the foods specified in the diet plan provided to them Patients were also encouraged to usemediumchain triglyceride MCT oil MCT oil anodorless and tasteless saturated fat does not requirebile or pancreatic enzymes for digestion It is easilyconverted to ketones in the liver thereby enhancingketosis Every weeks ml of MCT oilfromNutricia Erlangen Germany was provided to eachsubject in the KD group For better tolerance initialdosage of MCT was kept low and increased daily overa 6day period until maximum tolerable dosage wasachieved Dosage was reduced in a similar steppedprocessThe patients in the control group were instructed tofollow a standard diet consisting of CHO protein and fat Dietary compliance was checked byassessing blood betahydroxybutyrate levels every weeks and dietary intake at baseline and end of thestudyQoL assessmentQoL was assessed using the EORTC QLQC30 version and IORTC QLQBR23 questionnaires developed bythe European anization for Research and Treatmentof Cancer The validity and reliability of the questionnaires has previously been evaluated in Iran [ ]The questionnaires were completed at enrollment at weeks and at the end of the interventionDietary intake assessmentHospital dietitians used a 24h dietary recall 24HR toobtain a total of days intake one weekend day andtwo workdays through telephone and facetoface interviews both at the beginning and end of the study Theamount of each food consumed was estimated usingcommon household containers bowls cups and glassesand standard measuring cups and spoons as referencesThe mean quantity of total energy carbohydrate proteinand fat were estimated from the 24HRDietary intake wasanalyzed by Nutritionist IV software Version USPhysical activity assessmentPhysical activity was measured using the IPAC International Physical Activity questionnaire at baseline at weeks and at the end of the studyBiomarker assessmentFasting blood sampling for serum Na K Ca P lactate Mg LDH albumin ammonia and ALP were performed at baseline midway through the intervention weeks and at weeks T3 T4 and TSH were measuredat baseline and the end of the interventionStatistical analysisConsidering the power and α the sample sizewas calculated as individuals per group Assuming a dropout during the weeks of the study the finalnumber of participants was calculated as patients ineach groupStatistical analysis was carried out according to theintentiontotreat protocol Continuous variables weretested for normal distribution by the KolmogorovSmirnov test and then reported as mean ± standard deviation or median as appropriate Student ttest or Mann“Whitney U test was used to compare the continuousvariables between the two groups Paired sample ttestor Wilcoxon was used to compare the continuous variables within the two groups The ANCOVA test wasused to eliminate the effect of confounding factorsPearson correlation analyses were used to estimate associations between total carbohydrate intake and serumbetahydroxybutyrateData were analyzed using the SPSS version software Chicago IL USA and Stata version P was considered as statistically significantResultsDetailed patient demographics and a flow diagram werereported previously [] A total of women withbreast cancer were enrolled and randomly assigned to either the intervention n or control n groupsThree patients in the control group withdrew before beginning their assigned diet while10 patients in the KDgroup and patients in the control group withdrewfrom the study after beginning their assigned diet Ultimately patients in each group completed the studyand were included in the analysis No significant differences were seen between the two groups with regard toage cancer type metastasis and marriage or educationstatus P The intervention group included patients with locally advanced disease and patients withmetastatic disease liver bone lung liver andbone while the control group consisted of patientswith locally advanced disease and patients with 0cKhodabakhshi Nutrition Journal Page of metastatic disease bone liver lung liver andbone at other sites P Table Data related to quality of life are shown in Tables and No significant differences were seen in QoL betweenthe two groups after weeks however the KD groupshowed better global QoL compared to the controlgroup at week P Also at week diarrhea increased in the control groupcompared to the intervention P Data on week not shown Using the QoL questionnaire there was awithingroup decrease in reported hunger from baselineto weeks in the KD group P A withingroupdecrease was seen in physical performance measuresfrom baseline to weeks in both groups which was significant only in the KD group P In addition rolefunctioning and socialfunctioning scores significantlydecreased in the control group compared to the baselinebut not in the KD group P P Table Mean dietary intake is shown in Table and Fig The mean caloric and carbohydrate intake decreasedsignificantly at the end of the study compared to control P and P respectively while fatintake increased significantly in the KD group compared to the control group P After adjustingfor total energy intake this difference remained significant When data from both groups was combineda significant inverse association was observed betweentotalserum betaandr ˆ’ P hydroxybutyratealthough this effect was not seen when the KD groupwas analyzed separatelyintakeat weekscarbohydrateWithingroup analysis showed significant decreasesin energy carbohydrate and protein intake in bothgroups compared to the baseline Fat intake increasedsignificantly compared to the baseline in the KDgroup P and decreased significantly in thecontrol group P During the intervention of the subjects in the KDarm limited carbohydrates to g and of subjects consumed of calories from carbohydratesAt weeks of patients in the KD group hadserum ketones mmolL at 6weeks had ketone levels of mmolL As previously reportedserum ketone concentrations increased significantly inthe KD group ± to ± mmollP []At weeks physical activity improved in the KD groupcompared to the control group adjusted for cancer typeand baseline value P but after weeks physicalactivity did not show any significant differences in a between or withingroup analysis Fig No significant difference was observed in a betweenor withingroup analysis of thyroid hormones electrolytes albumin Ammonia and LDH Howeverlactateand ALP decreased significantly after intervention in theKD group compared to the control group P andP respectively ALP is adjusted for baseline valueand cancer type Table Data on thyroid hormones notshownDiscussionThe effect of KD on QoL physical activity dietary intake and biomarkers in patients with locally advancedand metastatic breast cancers was evaluated in thisstudy Based on our findings in the KD group globalQoL was higher at weeks perhaps in part because diarrhea was more frequent in the control group than theKD group No significant differences were seen in theQoL physical activity and biomarkers between the twogroups after the week intervention Lactate and ALPwere lower in the KD group compared to the controlEffect of diet on QoLIn our study in the KD group global QoL was higher at weeks No adverse effects were observed in thoseTable Baseline characteristics in breast cancer patients before interventionScale categoriesCancer TypeLocally AdvancedIntervention Ketogenic dietn Control Ordinaryn ERPRHER2Metastaticpositivenegativepositivenegativepositivenegative ER Estrogen receptor PR Progesterone receptorHER2 Human epidermal growth factor receptor aCalculated by chi square testbCategorical data shown as No p value008a057a043a079a 0cKhodabakhshi Nutrition Journal Page of Table Quality of life in breast cancer patients™ before andafter intervention in KD group and control group as measuredby the EORTC QLQC30aFunctioningaPhysical functioningMD CIControlKDpvalueTable Quality of life in breast cancer patients before and afterintervention in KD group and control group as measured by theEORTC QLQC30SymptomsaFatiguepvalueControlKD ˆ’Week Week pvalue“33a“Nausea and vomitingWeek Week pvalueRole functioningWeek Week pvalue ± 11a ± ± ± Cognitive functioningWeek Week pvalue ± ± Emotional functioningWeek Week pvalueSocial functioningWeek Week pvalue ± ± ± ± Global quality of lifeWeek Week pvalue ± ± ± ± ± ± ± ± ± ± ± ± ± ± ˆ’ ˆ’ ˆ’ ˆ’ˆ’ˆ’ˆ’ ˆ’ˆ’After adjusting for baseline value and chemotherapy status no significantdifferences were observedStudent ttest was used to compare the continuous variables between the twogroups Paired sample ttest was used to compare the continuous variableswithin the two groupsData shown as mean and SDaThe higher values indicate higher level of functioning and quality of lifeparticipants assigned to the KD compared to the controlgroup after weeks Withingroup analysis showed decreased hunger and physical function in the KD groupcompared to the baseline In the control group role andsocial functioning decreased significantly compared tobaselineResults of a systematic review and metaanalysis haveshown that KDs suppress appetite [] Decrease in hunger or appetite in our study may be due to the high fatcontent of the KD as it decreases the ghrelin releasewhich in turn may reduce appetite High fat intake alsoslows digestion which could also impact the perceptionof hunger Previously we have shown that the KD resultsin weight loss [] As a clinical benefit KDinduced““ ““““““““““ “ “ “ “ “ “Week Week pvaluePainWeek Week pvalueReduction in appetiteWeek Week pvalueSleep difficultiesWeek Week pvalueDyspneaWeek Week pvalueConstipationWeek Week pvalueDiarrheaWeek Week pvalueFinancial concernsWeek Week pvalue““““““““““ “ “ “ “ “ “Mann“Whitney U test was used to compare the continuous variables betweenthe two groups Wilcoxon was used to compare the continuous variableswithin the two groupsaThe higher values indicate a higher grade of symptoms Data shown asmedian and quartile 0cKhodabakhshi Nutrition Journal Page of Table Quality of life in breast cancer patients before and afterintervention in KD group and control group as measured by theEORTC QLQBR23aKDControlpvalue “ “ “ “aFunctioningFuture perspectiveWeek Week pvaluebSymptomsArmWeek Week pvalueBreastWeek Week pvalue “ “ “ “Week Systemic therapy side effects “ “Week pvalueConcerns over hair lossWeek Week pvalue “ “ “ “ “ “ “ “Table Comparison of mean ± SD macronutrient intake atbaseline and 12weeksVariableMD CIpvalueKDMean ± SDControlMean ± SDEnergy KcaldayBefore ± ± ± AfterpvalueCarbohydrate grBefore ± ± ± ± AfterpvalueProtein grBeforeAfterpvalueFat gr ± ± ± ± ± ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ 0001a ˆ’ˆ’ ˆ’041aBefore ± ± ˆ’ˆ’ ± ± AfterpvalueStudent ttest was used to compare the continuous variables between the twogroups Paired sample ttest was used to compare the continuous variableswithin the groupsMD Mean differenceCI Confidence intervalaAncova Adjusted for baseline value and energy0001aMann“Whitney U test was used to compare the continuous variables betweenthe two groups Wilcoxon was used to compare the continuous variableswithin the two groupsaThe higher values indicate higher level of functioning and quality of lifebThe higher values indicate a higher grade of symptomsData shown as median and quartile decreases in appetite weight and body fat may result infavorable changes in breast cancer patients notably inoverweight or obese women [ ]In contrast with our findings Cohen found that aKD significantly enhanced physical function scores inwomen with ovarian or endometrial cancer comparedto the control group but appetite did not change atthe end of the study compared to the baseline []Part ofstudy andCohen™s trial may be explained by the design of thestudy While only of the participants in the Cohen study were undergoing chemotherapy all of ourpatients were receiving treatmentthe inconsistency between ourAlso timing of the administration of the questionnaires and whether the participants were in positive ornegative energy balance may have influenced ourfindingsNo significant difference was reported in QoL at theend of study compared to the baseline by TanShalaby [] However a slight decrease in physical androle functioning as well as temporary constipation andfatigue were reported in the KD group in one study []In our study constipation was noted by participants inthe KD arm during the early days which was managedby dietary changesAlso after weeks in the KD group physical activityscores was higher compared to the control group but at weeks differencessignificantbetween the two groupsin scores were notDietary intake and adherenceOur study data showed a significant decrease in carbohydrate intake and a significant increase in fat intake inthe KD group compared to the control Protein intakewas not significantly different between the two groupsbut decreased overall in both groups when compared tobaseline Total daily carbohydrate intake was similar toresults in the Cohen study [] We also assessed serumbetahydroxybutyrateIn the KD group ofpatients at weeks and at 6weekshad serum ketones and patients at weeks and weeks 0cKhodabakhshi Nutrition Journal Page of Fig Mean caloric intake and distribution of macronutrients as percentage of total kilocalories before and after week intervention in breastcancer patients in two groupsFig Comparison of trend changes in physical activity in breast cancer patients in two groups 0cKhodabakhshi Nutrition Journal Page of LBsvskwleuavpitnopdmisvskwleuavpLBsviitnopdmeuavlpinorrefnoBnodesabldetauclacerewseuavlpllaerusaemdetaepeRepytlsisyanAlavretnIecnedifnoCICecnereffiDnaeMDMpuwolloftsalroskeewskeewkeewropuwolloftsitnopdMiepytrecnacdnaeuavlenilesabrofdetsudAjavocnAsnosirapmoclepitlumrofnoitcerrocˆ’±±±±ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’skeew±±itnopdMi±±enilesaB±±±±±±smArlairTDKICDMlortnoClortnoCDKlebairaVetatcaLluHDLstneitaprecnactsaerbdetaertDKdnalortnocnislevelrekramoBielbaTˆ’ˆ’ˆ’ICDM±±±±lortnoCDKldgcmianommAˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ICDMˆ’ˆ’ˆ’ˆ’ˆ’ICDM±±±±±±lortnoCDK±±±±±±lortnoCDKˆ’ˆ’ˆ’ˆ’ICDMˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ICDMˆ’ˆ’±±±±±±±±±±±±ICDMlortnoCDKlortnoCDK±±±±ˆ’±±±±ˆ’±±lortnoCDKICDMˆ’±±ICDMlortnoCDKˆ’ˆ’ˆ’ˆ’ˆ’ICDM±±±±±±lortnoCDKLdgmgMldginmubAllqemKlDgmPPLAldgmaClqemaNenilesaBLB 0cKhodabakhshi Nutrition Journal Page of had serum ketones mmoll Cohen reported that of patients had betahydroxybutyrate concentrations mmollA recent systematic study of KDs in adult cancerpatients reported a range of to with a adherence rate overall reported by [] According toour data the level of adherence to the KD interventionsuggests that the diet is a feasible option for women withbreast cancer who are receiving chemotherapyDespite the lack of any restriction in calorie intakein the study design and consistent with findings ofCohen [] the KD group showed a significant reduction in calorie intake compared to the control groupThe decrease in calorie intake may be due to reductions in appetite associated with ketosis as the subjects in the KD arm did not consume all of the fatcalculated for their diet This may also be due in partto customary practices surrounding meal preparationA decrease in appetite and subsequentinadvertentcalorie restriction most often results in weight loss inthe absence of malnutrition or cachexiathis mayhave antiinflammatory and proapoptotic propertieswhich in turn may exert a positive effect on thesehallmarks of cancer Ketosis may also enhance theeffectiveness of chemotherapy while reducing the sideeffects of treatment [ ]Effect of diet on biomarkersConsistent with the outcomes of the previous studiesour results revealed that the KD had no adverse effecton thyroid hormones electrolytes LDH urea and albumin Significant decreases were seen in serum levels oflactate KDs reduce glycolytic activity which in turn mayslow metastases by reducing the acidity of the tumormicroenvironment and lowering the availability of lactate as a substrate for biomass synthesis [] Decreaseswere also seen in ALP High levels of ALP in breast cancer patients is a negative prognostic marker often indicating progression of metastatic disease [] Moreresearch is needed to assess whether lower ALP and lactate as seen in this study contributes to slower rates ofdisease progressionTo our knowledge this is the first randomized controlled trial examining the effects of a KD on QoL inbreast cancer patientsThe primary limitation of this study was the heterogeneous nature of the sample in regards to cancer stageA secondary limitation was the small sample sizeConclusionAccording to our results besides a higher global QoLand physical activity scores compared to the controlgroup at weeks KD diet combined to chemotherapy inpatients with breast cancer does not bring additionalbenefit about QoL and physical activity at weeksWhile many blood biomarkers did not differ significantlybetween the two groups ketosis may still offer benefit tosome patients with breast cancer in part by decreasinglactate and ALPSupplementary informationSupplementary information accompanies this paper at doi101186s1293702000596yAdditional file figure Flow diagram of the patient treatmentprocessAdditional file figure Median confidence interval tyroidhormones in baseline and 12week by two trial arms in breast cancerpatientsAcknowledgmentsWe would like to thank all the patients at our clinic who took part in thisstudyAuthors™ contributionsKhodabakhshi carried out the conception Methodology performed theexperiments design of the diet and wrote the Davoodi and Seyfriedcollaborated in the design of the study Davoodi supervise on the thesisKalamian and Beheshti collaborated in the design of the diet Kalamian gavecritical review of the manuscript All authors have read and approved thefinal manuscriptFundingNot applicableAvailability of data and materialsData described in the manuscript code book and analytic code will bemade available upon request pendingEthics approval and consent to participateAll participants provided written informed consent prior to participating inthe studyConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Nutrition School of Public Health Kerman University ofMedical Sciences Kerman Iran 2Physiology Research Center KermanUniversity of Medical Sciences Kerman Iran 3Biology Department BostonCollege Chestnut Hill MA USA 4Dietary Therapies LLC Hamilton MT USA5Department of Nutrition and Dietetics Mofid children™s hospital ShahidBeheshti University of Medical Sciences Tehran Iran 6Cancer ResearchCenter Shahid Beheshti University of Medical Sciences Tehran Iran7Department of Cellular and Molecular Nutrition Faculty of Nutrition Scienceand Food Technology Shahid Beheshti University of Medical SciencesTehran IranReceived March Accepted July ReferencesNeal EG Chaffe H Schwartz RH Lawson MS Edwards N Fitzsimmons G The ketogenic diet for the treatment of childhood epilepsy arandomised controlled trial Lancet Neurol “Zhou W Mukherjee P Kiebish MA Markis WT Mantis JG Seyfried TN Thecalorically restricted ketogenic diet an effective alternative therapy formalignant brain cancer Nutr Metab 0cKhodabakhshi Nutrition Journal Page of adverse effects on blood lipids a randomized controlled trial Nutr Cancer“Fine EJ SegalIsaacson CJ Feinman RD Herszkopf S Romano MC TomutaN Targeting insulin inhibition as a metabolic therapy in advancedcancer a pilot safety and feasibility dietary trial in patients Nutrition“ Epub Zhou W Mukherjee P Kiebish MA Markis WT Mantis JG Seyfried TN Thecalorically restricted ketogenic diet an effective alternative therapy formalignant brain cancer Nutr Metab Mukherjee P Mulrooney TJ Marsh J Blair D Chiles TC Seyfried TNDifferential effects of energy stress on AMPK phosphorylation and apoptosisin experimental brain tumor and normal brain Mol Cancer Gatenby RA Gawlinski ET Gmitro AF Kaylor B Gillies RJ Acidmediatedtumor invasion a multidisciplinary study Cancer Res “Singh A Pandey A Tewari M Kumar R Sharma A Singh K Advancedstage of breast cancer hoist alkaline phosphatase activity risk factor forfemales in India Biotech “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsHuebner J Marienfeld S Abbenhardt C Ulrich C Muenstedt K Micke O Counseling patients on cancer diets a review of the literature andrecommendations for clinical practice Anticancer Res “Champ CE Palmer JD Volek JS WernerWasik M Andrews DW Evans JJ Targeting metabolism with a ketogenic diet during the treatment ofglioblastoma multiforme J NeuroOncol “Zuccoli G Marcello N Pisanello A Servadei F Vaccaro S Mukherjee P et alMetabolic management of glioblastoma multiforme using standard therapytogether with a restricted ketogenic diet case report Nutr Metab Servaes P Verhagen C Bleijenberg G Fatigue in cancer patients during andafter treatment prevalence correlates and interventions Eur J Cancer “Cohen C Fontaine K Arend R Soleymani T Gower B Favorable effects of aKetogenic diet on physical function perceived energy and food cravings inwomen with ovarian or endometrial Cancer a randomized Controlled TrialNutrients Breymeyer KL Lampe JW McGregor BA Neuhouser ML Subjectivemood and energy levels of healthy weight and overweightobesehealthy adults on highand lowglycemic load experimental dietsAppetite “TanShalaby JL Carrick J Edinger K Genovese D Liman AD Passero VA Modified Atkins diet in advanced malignanciesfinal results of a safetyand feasibility trial within the veterans affairs Pittsburgh healthcare systemNutr Metab Schmidt M Pfetzer N Schwab M Strauss I Kämmerer U Effects of aketogenic diet on the quality of life in patients with advanced cancer apilot trial Nutr Metab Klement RJ Sweeney RA Impact of a ketogenic diet intervention duringradiotherapy on body composition I Initial clinical experience with sixprospectively studied patients BMC Res Notes Schmidt M Pfetzer N Schwab M Strauss I Kämmerer U Effects of aketogenic diet on the quality of life in patients with advanced cancer apilot trial Nutr Metab Tóth C Clemens Z Halted progression of soft palate Cancer in a patienttreated with the Paleolithic Ketogenic diet alone a 20months followupAm J Med Case Rep “ Gibson AA Seimon RV Lee CM Ayre J Franklin J Markovic T Doketogenic diets really suppress appetite A systematic review and metaanalysis Obes Rev “Sremanakova J Sowerbutts A Burden S A systematic review of the use ofketogenic diets in adult patients with cancer J Hum Nutr Diet “Khodabakhshi A Akbari ME Mirzaei HR Kazemian E Kalantari K Kalamian M Effects of ketogenic diet for breast cancer treatment A protocol forrandomized controlled clinical trial J Biochem Technol “Khodabakhshi A Akbari ME Mirzaei HR MehradMajd H Kalamian MDavoodi SH Feasibility safety and beneficial effects of MCTbasedKetogenic diet for breast Cancer treatment a randomized controlled trialstudy Nutr Cancer Khodabakhshi A Akbari ME Mirzaei HR Seyfried TN Kalamian M DavoodiSH Effects of Ketogenic metabolic therapy on patients with breast Cancer arandomized controlled clinical trial Clin Nutr in press Montazeri A Harirchi I Vahdani M Khaleghi F Jarvandi S Ebrahimi M et alThe European anization for Research and Treatment of Cancer quality oflife questionnaire EORTC QLQC30 translation and validation study of theIranian version Support Care Cancer “ Montazeri A Harirchi I Vahdani M Khaleghi F Jarvandi S Ebrahimi M et alThe EORTC breast cancerspecific quality of life questionnaire EORTC QLQBR23 translation and validation study of the Iranian version Qual Life Res“ Champ CE Volek JS Siglin J Jin L Simone NL Weight gain metabolicsyndrome and breast cancer recurrence are dietary recommendationssupported by the data Int J Breast Ca

Thyroid_Cancer

"Meningiomas are the most common primary central nervous system tumors Potential risk factorsinclude obesity height history of allergyatopy and autoimmune diseases but findings are conflicting This studysought to assess the role of the different risk factors in the development of meningioma in adolescentsyoungadultsMethods The cohort included Jewish men and women who had undergone compulsory physicalexamination between and at age to years prior to and independent of actual military enlistmentTo determine the incidence of meningioma the military database was matched with the Israel National CancerRegistry Cox proportional hazard models were used to estimate the hazard ratios for meningioma according to sexbody mass index BMI height and history of allergic or autoimmune diseaseResults A total of subjects females were diagnosed with meningioma during a followup of personyears Median age at diagnosis was ± years range “ On univariate analysis female sex p and height p were associated with risk of meningioma When the data were stratified by sex heightremained a significant factor only in men Spline analysis of the male subjects showed that a height of m wasassociated with a minimum disease risk and a height of meters with a significant riskConclusions This large population study showed that sex and adolescent height in males m wereassociated with an increased risk of meningioma in adulthoodKeywords Allergy Autoimmune disease Height Meningioma SexBackgroundMeningiomas are the most common primary centralnervous system tumors They originate from the meninges which are the membranous layers surrounding thebrain Most meningiomas “ are grade I benign“ are grade II atypical and “ are grade IIIanaplastic [] Benign meningiomas have a female predominance or which is not found in the more Correspondence matanbe4gmailcom1NeuroOncology Unit Davidoff Cancer Center Rabin Medical Center “Beilinson Hospital Petach Tikva IsraelFull list of author information is available at the end of the aggressive types [] In the USA meningiomas werefound to be more common in blacks than in whites witha ratio of [] The risk of acquiring a meningiomaincreases with age The median age at diagnosis is years []The only established external nongenetic risk factorfor brain tumors is exposure to ionizing radiation []An Israeli study revealed abnormally high rates of meningioma in patients treated with lowdose radiation tothe scalp for tinea capitis during the 1950s [] Otherpotential risk factors include obesity height history ofallergyatopy and history of autoimmune diseases but The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cBenZion Berliner BMC Cancer Page of the results are conflicting [“] Establishing risk factors for meningioma can help identify individuals whomight benefit from risk reduction strategies and possiblyearly screening methodsThe aim of the present study was to assess potentialrisk factors for the development of meningioma in adolescence and early adulthoodMethodsStudy populationIsraeli adolescents undergo a compulsory medicalexamination at age to assess their fitness for military service regardless of whether they are drafted ornot Arab and Orthodox Jewish females and malesand Druze females are exempted Together with thephysical examination sociodemographic and psychobehavioral data are collected and the medical historyis thoroughly reviewed using documents provided byeach subject™s primary care physician At the end ofthe process recruits are assigned a Functional Classification Code FCC that describes their medical status and occupational medical ranking The medicaldata and FCC are stored in the army™s main databasewhich was computerized in []The population for the present study was derived from subjects born in “ who underwentpreenlistment medical examination between whenthe database was computerized and at age “years Subjects with missing data on height and weightwere excluded n We also excluded subjects of North African and Asian origin born before many of whom had been exposed to radiation forthe treatment of tinea capitis after immigrating to Israelduring the 1950s [] These communities were laterfound to have a particularly high rate of meningioma []The final cohort consisted of subjects Fig Records were reviewed from the date of the initialmedical examination for military fitness to the date of afirst diagnosis of any cancer death or the predeterminedend of followup December Study variablesAtthe preenlistment medical examination demographic variables for each recruit were recorded in thearmy database as follows date of birth age at examination country of origin education socioeconomic status height weight and body mass index BMI Originwas defined by father™s country of birth or if the examinee™s father was born in Israel by paternal grandfather™scountry of birth and categorized as Europe includingNorth and South America Australia and SouthernAfrica Asia predominantly the Middle East Africaoverwhelmingly North Africa and Israel third or latergeneration Education was categorized as ‰ Fig Selection of the study populationand ‰¥ years of schooling Socioeconomic status wasdetermined by place of residence at the time of examination coded on a scale of “ and categorized intolow score “ middle score “ and high score “ [] Height and weight were measured by trainedmedics using a stadiometer and a beam balance with examinees barefoot and in underwear BMI was calculatedas weight in kilograms divided by height squared inmeters and categorized according to the WHO asunderweight kgm2 healthy weight “kgm2 overweight “ kgm2 and obese ‰¥kgm2 Height was categorized according to the Centersfor Disease Control and Prevention below 25th percentile 25th to 50th percentile 50th to 75th percentile and75th percentile and aboveCognitive function including language skills and intellectual performance [] was assessed by a general 0cBenZion Berliner BMC Cancer Page of intelligence test administered by trained personnel Thetest is scored on a 90point scale that is adjusted fromtime to time scores are categorized as low “medium “ and high “ []inflammatory bowel disease pemphigusMedical history was assessed according to the FCCslupus vascuAutoimmune diseases diabetes mellituslitisthyroiddisease celiac rheumatoid arthritis Addison disease andidiopathic thrombocyt ia purpura and allergic diseases asthma urticaria eczema allergic rhinitis atopicdermatitis allergic conjunctivitis and anaphylaxis weregrouped together for the present analysisAscertainment of meningioma incidenceTo determine the incidence of meningioma we matchedthe subjects who underwent the preenlistment medicalexamination during the study years to the Israel National Cancer Registry INCR a national populationbased registry established in In Israeli lawmandated the reportage of all diagnoses of malignant insitu and invasive borderline and certain benign brainand central nervous system tumors The estimated rateof reportage for solid tumors is which meets thestandards of the International Association of CancerRegistrieswwwhealthgovilPublicationsFilesICDC_365_EN_summarypd The INCR data includethe date of diagnosis site affected the InternationalClassification of Diseases code and the histologic description of the tumor according to the third edition ofthe International Classification of Diseases for OncologyICDO3 codes and At the time ofmatching the INCR had been updated until the end ofStatistical analysisCategorical variables are presented as number and percentage and continuous variables as mean and standarddeviation SD median 25th and 75th percentiles minimum and maximum were also calculated The association between risk factors and time to meningiomadiagnosis was assessed using Cox proportional hazardmodels hazard ratios HR confidence intervals CI and pvalues were calculated Log minus logfigures were inspected to confirm the proportionality ofthe hazard Crude rates were also determined Independent variables were initially entered individually into theCox model After sex and the interaction of sex andheight were found to be statistically significant separatemodels were established for men and women A Cox regression cubic spline function with three equally spacedknots positioned between the minimum and maximumvalues of height was fit to the data to estimate the heightvalue associated with minimum risk of meningioma inmen SASSTAT and SASGRAPH software version SAS Institute Inc Cary NC USA Other statistical analyses were performed with SPSS Statistics for Windowsversion IBM Armonk NY USA Twosided pvalues of ‰ were considered statistically significantResultsStudy populationThe baseline characteristics of the study population arepresented in Table The mean age at initial examination was ± years of the cohort was female The mean duration of followup was ± years median which represent in this study population a follow up of personyears The characteristics of the medical history of the subjects arepresented in the supplementary table Table 1SLinkage of the military database with the INCR yieldeda diagnosis of meningioma in of the subjects who underwent medical examination in to at age “ years grade I atypical anaplastic not specified and one patient with meningiomatosisTable The mean age at diagnosis ofmeningioma was ± years range “ andat the end of followup ± years median Univariate analysisOverall as expected meningiomas were more common infemales cases crude rate per personyears than in males cases crude rate per personyears p HR CI “ However there was no sex difference in the incidence for themore aggressive meningiomas atypical and anaplasticcrude rate per personyears for males andfemales On univariate analysis only sex and height weresignificantly associated with the risk of meningioma in thewhole study population p for both variables Afterstratification by sex height remained significant only inmales Table The risk of meningioma was minimalwhen height was up to m and statistically significantwhen height was greater than m Fig BMI was notassociated with an elevated risk of meningioma even whenanalyzed separately by sex Table Past medical history of asthma diabetes and otheratopic or autoimmune diseases was not associated withrisk of meningioma Even when autoimmune and allergic diseases were analyzed as a group there was no association with lower risk of meningioma Table andSupplemental Table When the subjects of African and Asian origin whowere excluded from the main analysis were included inthe cohort there was a significant interaction betweenperiod of birth “ vs “ and Asianand African origin representing the Middle East andNorth Africa as opposed to European and Israeli origin 0cBenZion Berliner BMC Cancer Page of Table Baseline characteristics of the study population total and by sexMaleCharacteristicsNumberBirth yearTotal““““LowMediumHigh years years years years“““ 25th percentile25th“50th percentile50th“75th percentile 75th percentileEuropeAsiaAfricaIsraelEuropeAsiaAfricaIsraelSocioeconomic statusEducationCognitive indexaBMI category Kgm2Height category CDC percentileCountry of birthOriginAge at time of medical examination yearsBMIHeight metersaRated on a 90point scaleFemaleNumberSDMeanSDTotalNumberMeanSDMeanThe conjoined effect of birth year and origin showedthat origin North Africa and Asia was significant onlyfor subjects born between and SupplementalTable DiscussionIn this nationwide populationbased study we analyzedthe association of the development of meningioma insubjects born between and with baseline variables obtained for the subjects at the average age of years As expected meningiomas were found to be associated with sex female and taller stature None of theother sociodemographic and medical variables assessedincluding BMI and a diagnosis of asthma or diabetes atage years was associated with an increased risk ofmeningioma 0cBenZion Berliner BMC Cancer Page of Table Meningioma type and rate total and by sexMeningioma typeMeningioma NOSMeningiomatosisGrade Atypical AnaplasticTotalPersonyearsNOS Not otherwise specifiedMalesNumberPer FemalesNumberPer TotalNumberPer It is well accepted that benign meningioma is morecommon in females than males but the sex predilectiondisappears with the more aggressive meningiomas []The female predominance might be explained by thefinding that meningiomas harbor receptors for estrogenand progesterone []We discovered an association between the risk ofmeningioma and height in men but not with BMI inmen or women The results of previous studies for thesetwo factors were conflicting A large Norwegian studyincluding million subjects found that height was associated with meningioma in both men and women butBMI was not [] whereas another study of postmenopausal females revealed an association of meningiomawith both BMI and height [] A metaanalysis of studies supported the correlation of BMI and meningioma It is worth noting that the Norwegian study exceeds the metaanalysis in size and power and that inthe Norwegian study a subgroup analyses for womenand men as well as different age groups was performedwithout finding convincing evidence of a strong association between overweight obesity and risk for meningioma [] In our study BMI was measured when thesubjects were years old much younger than theTable Univariate analysis association of potential risk factors with diagnosis of meningioma by sexVariablesMalesNCases Crude rate HR CILower UpperpCases Crude rate HR CILower UpperpFemalesNHeightHeight continuousBMIPercentile ““ Kgm2 Autoimmune diseasesa NoAllergic diseasesbAsthmaDiabetesYesNoYesNoYesNoYes aAutoimmune disease diabetes mellitus lupus vasculitis IBD pemphigus thyroid disease celiac rheumatoid arthritis Addison disease and idiopathic thrombocyt icpurpurabAllergic disease including asthma urticaria eczema allergic rhinitis atopic dermatitis allergic conjunctivitis and anaphylaxis 0cBenZion Berliner BMC Cancer Page of Fig Spline analysis in the men group showing the minimum risk for meningioma at a height of m and a statistically significant increase inthe risk for meningioma at heights taller than mstudies included in the metanalysis which might explainthe discordant results []Height has been associated with different types of cancer melanoma thyroid testis breast and lymphomaSuggested mechanism for the greater risk of meningioma in taller people is their higher levels of circulatinginsulinlike growth factors IGFs which may influencecell proliferation and tumor growth [] Moreoveroverexpression of IGFI and IGFII mRNA transcriptshas been demonstrated in meningioma [] Circulatinglevels of IGFs are highest during puberty They decreaserapidly in the third decade of life in the general population but seem to stay consistently higher in taller adults[] It is not clear why this association was evident onlyin males in our study maybe in women the influence ofthe hormonal status blurred the influence of the heightSeveral earlier studies reported an inverse associationbetween a history of allergic diseases including asthmaand meningioma [ ] However this finding wasnot supported by others [ ] We failed to demonstrate an association between meningiomas and allergicdiseases including asthma urticaria eczema allergicrhinitis atopic dermatitis conjunctivitis and anaphylaxisand allergy to beesSimilarly a recent study reported an inverse association between hyperglycemia and the risk of meningioma [] whereas another found a positive associationwith a history of diabetes mellitus [] In the presentstudy diabetes was not associated with the risk of meningioma This was true for other autoimmune diseasesas welllimitationThis analysis also has certain limitations The followup period in this study was limited to years such thatthe study population was still young when the studyended Subsequently the median age of those who developed meningioma in our study was younger than themedian age of patients with meningioma in the generalpopulation [] With a more extensive followup wemight find more latent tumor growths that could potentially increase or shift the incidence of intracranial neoplasms Anotherisunderreporting of meningiomas that are diagnosed onlyaccording to radiographic findings without histologicalfindings As it is well known that in some cases meningiomas diagnosed radiographically mayjust befollowed by repeat scanningcohortits prospectivepopulationbased design large sample size high degreeof completeness of the cancer registry data throughoutthe study period and the ability to carefully control forpotential confounders such as exposure to radiation Itshould be noted that in a study that was published recently and examined the same cohort the median heightremained almost stable during the study period theStrengths of ourofthestudyinclude 0cBenZion Berliner BMC Cancer Page of median height of males increased by cm and that offemales remained stable despite environmental socialand nutritional changes []ConclusionThis large populationbased study showed that sex female and tall stature in adolescent males was associatedwith an increased risk of meningioma in adulthoodSupplementary informationSupplementary information accompanies this paper at doi101186s12885020072924Additional file Supplementary Table Medical historycharacteristics of the study populationAdditional file Supplementary Table Univariate analysisassociation of potential risk factors with diagnosis of meningioma by sexAdditional file Supplementary Table Interaction between birthperiod and origin whole population adjusted for sexAbbreviationsCNS Central nervous system BMI Body mass index FCC FunctionalClassification INCR Israel National Cancer Registry ICDO InternationalClassification of Diseases for Oncology SD Standard deviation HR Hazardratio CI Confidence interval IGF Insulinlike growth factorAcknowledgmentsNot applicableAuthors™ contributionsMBZB and SYK analyzed the preliminary database extracted the relevantinformation to allow hypothesis testing and prepared the manuscript andtables Statistical analysis and figure preparation were performed by LHK andED HL LKB YL AH JM and GT participated in the preliminary preparationand conceptual design and revised the final manuscript ABA OG AK and TSreviewed the neurological proof of concept and revised the final manuscriptand supplementary materials All authors read and approved the finalmanuscriptFundingThe study was funded by the Israel Cancer Association by the Lillia andJacob Alther donation financial support without any role in the manuscriptpreparationAvailability of data and materialsThe datasets used during this study are available from the correspondingauthor on reasonable requestEthics approval and consent to participateAll procedures performed in studies involving human participants were inaccordance with the ethical standards of the institutional andor nationalresearch committee and with the Helsinki declaration and its lateramendments or comparable ethical standards The study was approved bythe IDF Israel Defense Forces Medical Corps Institutional Review Boardwhich waived the requirement for informed consent because the data usedwere obtained from medical records without patient participation referencenumber “Consent for publicationNot applicableAuthor details1NeuroOncology Unit Davidoff Cancer Center Rabin Medical Center “Beilinson Hospital Petach Tikva Israel 2Department of GastroenterologyHadassah University Hospital “ Ein Kerem Jerusalem Israel 3Sackler Facultyof Medicine Tel Aviv University Tel Aviv Israel 4Braun School of PublicHealth and Community Medicine Hadassah University Hospital “ Ein KeremJerusalem Israel 5Israel Center for Disease Control Israel Ministry of HealthRamat Gan Israel 6School of Public Health University of Haifa Haifa Israel7Department of Neurosurgery Rabin Medical Center “ Beilinson HospitalPetach Tikva Israel 8Medical Corps Israel Defense Forcesand Department ofMilitary Medicine Hebrew University of Jerusalem Faculty of MedicineJerusalem Israel 9Institute of Endocrinology and Talpiot Medical LeadershipProgramSheba Medical Center Tel Hashomer IsraelReceived April Accepted August ReferencesOstrom QT Gittleman H Fulop J Liu M Blanda R Kromer C Wolinsky YKruchko C BarnholtzSloan JS CBTRUS statistical report primary brain andcentral nervous system tumors diagnosed in the United States in NeuroOncology 201517Suppl 4iv1“iv62 doi101093neuoncnov189Claus EB Bondy ML Schildkraut JM Wiemels JL Wrensch M Black PMEpidemiology of intracranial meningioma Neurosurgery “doi10122701neu000018828191351b9Braganza MZ Kitahara CM Berrington de González A Inskip PD Johnson KJRajaraman P Ionizing radiation and the risk of brain and central nervoussystem tumors a systematic review NeuroOncology “doi101093neuoncnos208 Modan B Baidatz D Mart H Steinitz R Levin SG Radiationinduced headand neck tumours Lancet “ doi101016s0140 Wiedmann MKH Brunb C Di Ieva A Lindemann K Johannesen TBVatten L Helseth E Zwart JA Overweight obesity and height as risk factorsfor meningioma glioma pituitary adenoma and nerve sheath tumor alarge populationbased prospective cohort study Acta Oncol “ doi1010800284186X20171330554Johnson DR Olson JE Vierkant RA Hammack JE Wang AH Folsom ARVirnig BA Cerhan JR Risk factors for meningioma in postmenopausalwomen results from the Iowa Women's health study NeuroOncology“ doi101093neuoncnor081 Michaud DS Bové G Gallo V Schlehofer B Tjønneland A Olsen A OvervadK Dahm CC Teucher B Boeing H Steffen A Trichopoulou A Bamia CKyrozis A Sacerdote C Agnoli C Palli D Tumino R Mattiello A BuenodeMesquita HB Peeters PH May AM Barricarte A Chirlaque MD DorronsoroM José Sánchez M Rodríguez L Duell EJ Hallmans G Melin BS Manjer JBquist S Khaw KT Wareham N Allen NE Travis RC Romieu I Vineis PRiboli E Anthropometric measures physical activity and risk of glioma andmeningioma in a large prospective cohort study E Cancer Prev Res Phila“ doi10115819406207CAPR110014Niedermaier T Behrens G Schmid D Schlecht I Fischer B Leitzmann MFBody mass index physical activity and risk of adult meningioma andglioma a metaanalysis Neurology “ doi101212WNL0000000000002020Brenner AV Linet MS Fine HA Shapiro WR Selker RG Black PM Inskip PDHistory of allergies and autoimmune diseases and risk of brain tumors inadults Int J Cancer “ doi101002ijc10320 Wang M Chen C Qu J Xu T Lu Y Chen J Wu S Inverse associationbetween eczema and meningioma a metaanalysis Cancer Causes Control“ doi101007s1055201198086 Gal R The selection classification and placement process in a portrait ofthe Israeli soldier Westport CT Greenwood Press p “ YustKatz S Bar Oz A Derazne E Katz LH Levine H KeinanBoker L Amiel ACompeting interestsThe authors declare that they have no financial or nonfinancial competinginterestsKanner A Laviv Y Honig A Shelef I Siegal T Twig G Kark J Echoes fromthe past changing associations between brain tumors and ethnicity JNeurol Sci doi101016jjns2019116552 [Epubahead of print]Israel Central Bureau of Statistics Characterization and classification of localauthorities by the socioeconomic level of the population Jerusalem Israelcentral Bureau of Statistics 0cBenZion Berliner BMC Cancer Page of Twig G Gluzman I Tirosh A Gerstein HC Yaniv G Afek A Derazne E Tzur DKarasik A Gordon B Fruchter E Lubin G Rudich A CukiermanYaffe TCognitive function and the risk for diabetes among young men DiabetesCare Nov37112982“ doi102337dc140715 Guevara P EscobarArriaga E SaavedraPerez D MartinezRumayor A FloresEstrada D Rembao D Calderon A Sotelo J Arrieta O Angiogenesis andexpression of estrogen and progesterone receptors as predictive factors forrecurrence of meningioma J NeuroOncol “ doi101007s110600172662y Gunnell D Oliver SE Donovan JL Peters TJ Gillatt D Persad R Hamdy FCMeal DE Holly JMP Do heightrelated variations in insulinlike growthfactors underlie the associations of stature with chronic diseases J ClinEndocrinol Metab “ doi101210jc2003030507 Zumkeller W Westphal M The IGFIGFBP system in CNS malignancy MolPathol “ Crowe FL Key TJ Allen NE A crosssectional analysis of theassociations between adult height BMI and serum concentrations of IGFIand IGFBP1 and ˆ’ in the European prospective investigation intocancer and nutrition EPIC Ann Hum Biol “ doi BergBeckhoff G Schüz J Blettner M Münster E Schlaefer K Wahrendorf JSchlehofer B History of allergic disease and epilepsy and risk of glioma andmeningioma INTERPHONE study group Germany Eur J Epidemiol “ doi101007s1065400993556Schneider B Pülhorn H Röhrig B Rainov NG Predisposing conditions andrisk factors for development of symptomatic meningioma in adults CancerDetect Prev “ doi101016jcdp200507002Linos E Raine T Alonso A Michaud D Atopy and risk of brain tumors ametaanalysis J Natl Cancer Inst “ doi101093jncidjm170 Bernardo BM Orellana RC Weisband YL Hammar N Walldius G MalmstromH Ahlbom A Feychting M Schwartzbaum J Association betweenprediagnostic glucose triglycerides cholesterol and meningioma andreverse causality Br J Cancer “ doi101038bjcPublisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

"conceptualization data curation investigation project administration supervision validation visualization writing review and editing Goneim I and Ibraheim A performed data curation Kamal NM was involved in literature review writingoriginal draft writing review and editing Alsofiani F and Alawur A performed literature review and writingoriginal draft All authors had read and approved the final manuscriptInformed consent statement Written informed consent in the patient™s native language was obtained from her fatherConflictofinterest statement The authors declare that they have no conflict of interestCARE Checklist statement The authors have read the CARE Checklist and the manuscript was prepared and revised according to the CARE Laila M Sherief Department of Pediatric Hematology and Oncology Faculty of Medicine Zagazig University Zagazig EgyptLaila M Sherief Amr Ibraheim Department of Pediatrics Faculty of Medicine Zagazig University Zagazig EgyptEsmael Goneim Department of Pediatric Oncology Tanta Cancer Institute Tanta EgyptNaglaa M Kamal Department of Pediatrics and Pediatric Hepatology Faculty of Medicine Cairo University Cairo EgyptNaglaa M Kamal Fuad A alsofiani Abdulraouf H Alawur Department of Pediatrics Alhada Armed Forces Hospital Taif Saudi ArabiaCorresponding author Naglaa M Kamal MD Full Professor Department of Pediatrics and Pediatric Hepatology Faculty of Medicine Cairo University Kasralainy Cairo Egypt naglakamalkasralainyeduegAbstractBACKGROUND thalassemia intermedia TI is one of the hemoglobinopathies It constitutes of thalassemia cases yet being associated with a better quality of life than thalassemia major TMCASE SUMMARY We recently reported the first case of acute lymphoblastic leukemia ALL from Egypt in a child with TM and we herein report the first case of ALL from Egypt in a child with TI In this report literature was reviewed for cases of malignancies associated with TI and the possible factors underling the relationship between the two entities We stress that physicians should have a high index of suspicion of malignancies in thalassemia patients if they present with any suggestive symptoms or signsKey words Acute lymphoblastic leukemia Thalassemia intermedia Children Malignancies Iron overload Hydroxyurea Case reportThe Authors Published by Baishideng Publishing Group Inc All rights reservedWJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIChecklist Access This is an access that was selected by an inhouse editor and fully peerreviewed by external reviewers It is distributed in accordance with the Creative Commons Attribution NonCommercial CC BYNC license which permits others to distribute remix adapt build upon this work noncommercially and license their derivative works on different terms provided the original work is properly cited and the use is noncommercial See httpcreativecommonslicensesbync40Manuscript source Unsolicited manuscriptReceived January Peerreview started January First decision April Revised May Accepted June in press June Published online August PReviewer Fujioka K Moschovi MA SEditor Dou Y LEditor A EEditor Li JHCore tip Cases have been reported for malignancies in patients of thalassemia major However rare case reports have been reported for malignancies in patients of thalassemiaintermedia as it is a nontransfusion dependent anemia Physicians should have high index of suspicion to diagnose malignancies in patients with thalassemiaintermediaCitation Sherief LM Goneim E Kamal NM Ibraheim A Alsofiani F Alawur A Acute lymphoblastic leukemia in a thalassemia intermedia child A case report World J Clin Pediatr URL wwwwjgnetcom22192808fullv9i11htm dx105409wjcpv9i11INTRODUCTIONThalassemia represents the most common singlegene disorder worldwide The total annual incidence of symptomatic individuals with thalassemia is estimated at in throughout the world of whom nearly have thalassemia intermedia TI which is intermediate in severity between the milder thalassemiaminor and the more severe transfusiondependent thalassemiamajor TM[]We herein report the first case from Egypt with TI who developed acute lymphoblastic leukemia ALLCASE PRESENTATIONChief complaintsThe reported patient is a 15yearold girl with TI who presented at the age of years with pallor decreased growth rate and decreased activity She had severe microcytic hypochromic anemia with hemoglobin Hb of gdLHistory of present illnessPediatric hematologist workup proved the diagnosis of TI Her Hb electrophoresis showed HbA HbF and HbA2 Genetic molecular testing revealed compound heterozygosity for cd27 GT and cd39 CT mutations Hydroxyurea at a dose of mgkg per day was started in addition to folic acidShe was then followed at the pediatric hematology unit at regular intervals to monitor her tolerance to drug therapy with special attention to hematological toxicity There were no significant side effects during seven years of therapy and the patient showed good response with occasional need for blood transfusions She underwent splenectomy during her late teensHistory of past illnessAt the age of years she developed generalized bone aches abdominal pain persistent fever and dyspnea and so she was referred to our hospitalPhysical examinationOn physical examination there was severe pallor tachypnea tachycardia and hepatomegalyLaboratory examinationsInitial complete blood picture showed a Hb level gdL white blood cell count of — 109L and platelets count of — 109LSerum electrolytes cerebrospinal fluid analysis and kidney and liver function tests were normal expect for mild elevation of total serum bilirubin which was mgdLSerum ferritin was ngdL Serological studies including EpsteinBarr virus cytomegalovirus human immunodeficiency virus hepatitis C virus and hepatitis B virus were negative Lactate dehydrogenase was UL and serum uric acid was mgdLWJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIImaging examinationsHer chest Xray was normal Abdominal ultrasonography revealed hepatomegaly with calcular cholecystitis and bilateral diffuse renal enlargement Echocardiography showed mitral valve prolapse with trivial mitral regurgitationMULTIDISCIPLINARY EXPERT CONSULTATIONThe pediatric haematologistoncologist assessment requested bone marrow biopsy which was carried by the hematopathologistBone marrow examination revealed blast cells in a hypercellular marrow with depressed erythropoiesis and granulopoiesies and normal thrombopoiesis Immunophenotyping showed lymphoblasts that are CD10 positive CD19 positive CD34 positive TDT positive HLADR positive CD13 positive and CD33 positiveCytogenetic examination showed a normal karyotype with a DNA index of and negative t1221 t119 BCRABL or 11q23 translocationsmutationsFINAL DIAGNOSISA final diagnosis of Bacutelymphoblasticleukemia ALL with aberrant expression of CD13 and CD33 was achievedTREATMENTInduction chemotherapy of the total XV protocol with prednisone vincristine Lasparaginase doxorubicin cyclophosamide cytarabine 6mercaptopurine and intrathecal chemotherapy was commencedShe received multiple packed red cell transfusions which eventually led to elevation of serum ferritin to ngdL Thus she was started on oral chelation therapy with deferasirox with no complicationsThe patient eventually went into complete remission She then received consolidation chemotherapy of standard risk of the total XV protocol with times of high dose methotrexate HDMTX 6mercaptopurine and intrathecal chemotherapyShe received multiple packed red blood cell transfusions and other supportive measures during the periods of induction and consolidation The transfusions therapy was given according to the guidelines of pediatric oncologists who usually transfuse if Hb level is less than gdL and if associated with pulmonary or cardiac comorbidities or exposed to invasive procedure and hemorrhage and they transfuse with Hb less than gdL The transfusions were not associated with any complications Deferasirox was stopped in consolidation phase during infusion of high dose methotrexateThe main problem observed during the periods of induction and consolidation therapy was increased requirement of blood transfusions as well as repeated infections as during this period the child received intensive chemotherapy which caused bone marrow suppressionOUTCOME AND FOLLOWUPThe child is still in complete remission while being now in the continuation phase for standard risk week fortyDISCUSSIONWe have recently reported the first case from Egypt with thalassemia major TM who developed ALL[] herein we report similarly the first report from Egypt for a patient with TI who developed ALL to highlight that the coexistence of malignancy and beta thalassemia is not rareA thorough look in literature for previously reported cases of malignancies in WJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIpatients with TI revealed only one report in from Turkey on a years old boy with TI who developed ALL[] Other previous reports on malignancies associated with TI described nonHodjkinlymphoma[] chronic myeloid leukemia[] Hodjkin lymphoma[] hepatocellular carcinoma[] and thyroid malignancies[] Table To our knowledge our patient is the second worldwide and the first from EgyptAlthough reported cases of malignancies associated with TI are few but it raises the attention of physicians to have high index of suspicion of malignancies in this group of patients when they present with unexplained new symptoms or proposed symptoms and signs of malignancySpecial concern about management plans in these patients as they usually require more frequent blood transfusions as the chemotherapy causes suppression of the bone marrow which adds to the base line chronic hemolysisIn spite that reported cases of malignancies in TI are scarce which makes our trial to find causal relationship between TI and cancer development beyond the scope of our report but we tried to search literature foe possible contributing factors Those factors can™t rise to the level of conclusions and definitely need to be proved and validated by larger prospective cohort with large control groups multicenter worldwide studies addressing all possible hypothesesIndeed the most practical logical thinking about that underlying factors for the development of malignancy in TI is being multifactorial[]In a large multicenter study on thalassemia patients from Iran the proportion of patients with cancer was higher in those with TI than those with TM and respectively[] They explained it by the fact that bone marrow in TM patients is suppressed by the regular transfusions while it is very active with high turnover in those with TI[] They suggested that this can lead to a higher rate of DNA repair faults and mutations with subsequent higher rate of hematological malignancies[]Another potential factor is the prolonged use of hydroxyurea Conflicting data are there regarding its carcinogenic potential Hydroxyurea as an antimetabolite interferes with both DNA synthesis and repair mechanisms with later accumulation of mutations and subsequent chromosomal damage Although no studies have yet investigated the relationship between hydroxyurea and the development of cancers in thalassemia but clinically concerns have been raised regarding its potential leukemogenic potential[] Other authors were against this assumption[] The BABYHUG clinical trial which compared hydroxyurea with placebo treated controls refuted this assumption and did not suggest any increased risk of genotoxicity[]Overall there is no evidence to suggest an increased risk of carcinogenesis in patients with thalassemia with hydroxyurea and further studies will need to be designed to establish any potential relationship[]One more probable factor is that patients with TI being having milder disease than those with TM with fewer blood transfusions might lead to delayed diagnosis and even if diagnosed usually there is underestimation of their iron overload problem and sometimes the deceiving relatively mildly elevated ferritin as compared to TM which has been shown to underestimate the true iron burden in TI patient with ultimate fate that these patients accumulate iron but it usually goes unnoticed unchelated and unmonitored Anemia hypoxia and ineffective erythropoiesis suppress the expression of hepcidin by increasing expression of growth differentiation factor and hypoxiainducible transcription factors with the resultant increased intestinal iron absorption and in turn adds to the problem of iron overload[]The longstanding iron overload with its deposition in different body ans with the wellknown association between excess iron and cancer development can be a predisposing factor for all types of malignancies through direct and indirect effects[]Iron can directly damage DNA by nontransferrinbound iron with the consequent inactivation of tumorsuppressor genes such as p53 or their products The indirect effects include the formation of reactive oxygen species ironinduced lipid peroxidation and altered immune system with decreased immune surveillance suppression of tumoricidal action of macrophages and alteration of cytokine activities TI patients usually survive longer than TM patients with enough time for iron overload to develop[]Some authors suggested that improved management protocols of thalassemia patients have led to increased survival with most of them reaching adult age with the consequent occurrence of diseases associated with long life span like malignancies[] This assumption can partially explain other reports in elder patients but it can™t work in our patient and the Turkish one who are teenagersMany authors suggested that the occurrence of malignancies in thalassemia patients could be a pure coincidence or a combination of genetic and environmental factors[]WJCPwwwwjgnetcomAugust Volume Issue 0cTable Previously reported cases of thalassemia intermedia who developed malignanciesSherief LM ALL in a child with TINumber of patientsType of malignancyAcute lymphoblastic leukemiaNonHodgkin lymphoma NHLNHL Hodgkin lymphoma HLNHL HL chronic myeloid leukemia CMLCMLHLHepatocellular carcinoma HCCHCCHCCHCCHCCThyroid cancerSome patients have thalassemia major and others have thalassemia intermedia in references and Ref[][][][][][][][][][][][]We can sum up to a clear message that whatever the pathogenesis of malignancies in thalassemias the most important message is to alarm physicians to have high index of suspicion for malignancies if their thalassemia patients develop suggestive symptoms and signs Worsening anemia leukocytosis fever boneache lymphadenopathy and splenomegaly are alarming to look for leukemias and other hematological malignanciesREFERENCES Galanello R Origa R Betathalassemia Orphanet J Rare Dis [PMID ]Sherief LM Kamal NM Abdelrahman HM Hassan BA Zakaria MM First report of acute lymphoblastic leukemia in an Egyptian child with thalassemia major Hemoglobin [PMID ]Tuğcu D Karakaş Z G¶k§e M Ağaoğlu L Un¼var A Sarıbeyoğlu E Ak§ay A Devecioğlu O Thalassemia Intermedia and Acute Lymphoblastic Leukemia Is it a Coincidental Double Diagnosis Turk J Haematol [PMID 104274tjh20140068]Chehal A Loutfi R Taher A Betathalassemia intermedia and nonHodgkin's lymphoma Hemoglobin [PMID 101081hem120015025]Benetatos L Alymara V Vassou A Bourantas KL Malignancies in betathalassemia patients a singlecenter experience and a concise review of the literature Int J Lab Hematol [PMID 101111j1751553X200700929x]Karimi M Giti R Haghpanah S Azarkeivan A Hoofar H Eslami M Malignancies in patients with betathalassemia major and betathalassemia intermedia a multicenter study in Iran Pediatr Blood Cancer [PMID 101002pbc22144]Alavi S Safari A Sadeghi E Amiri S Hematological malignancies complicating thalassemia syndromes a single center experience Blood Res [PMID 105045br2013482149]Jabr FI Aoun E Yassine H Azar C Taher A Betathalassemia intermedia and Hodgkin lymphoma Am J Hematol [PMID 101002ajh20478]BnaPignatti C Vergine G Lombardo T Cappellini MD Cianciulli P Maggio A Renda D Lai ME Mandas A Forni G Piga A Bisconte MG Hepatocellular carcinoma in the thalassaemia syndromes Br J Haematol [PMID 101046j13652141200304732x]Mancuso A Sciarrino E Renda MC Maggio A A prospective study of hepatocellular carcinoma incidence in thalassemia Hemoglobin [PMID ]Restivo Pantalone G Renda D Valenza F D'Amato F Vitrano A Cassar  F Rigano P Di Salvo V Giangreco A Bevacqua E Maggio A Hepatocellular carcinoma in patients with thalassaemia syndromes clinical characteristics and outcome in a long term single centre experience Br J Haematol [PMID 101111j13652141201008180x]Fragatou S Tsourveloudis I Manesis G Incidence of hepatocellular carcinoma in a thalassemia unit Hemoglobin [PMID ] WJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TI Maakaron JE Cappellini MD Graziadei G Ayache JB Taher AT Hepatocellular carcinoma in hepatitisnegative patients with thalassemia intermedia a closer look at the role of siderosis Ann Hepatol [PMID ]Poggi M Sorrentino F Pascucci C Monti S Lauri C Bisogni V Toscano V Cianciulli P Malignancies in thalassemia patients first description of two cases of thyroid cancer and review of the literature Hemoglobin [PMID ]Halawi R Cappellini MD Taher A A higher prevalence of hematologic malignancies in patients with thalassemia Background and culprits Am J Hematol [PMID 101002ajh24682]McGann PT Flanagan JM Howard TA Dertinger SD He J Kulharya AS Thompson BW Ware RE BABY HUG Investigators Genotoxicity associated with hydroxyurea exposure in infants with sickle cell anemia results from the BABYHUG Phase III Clinical Trial Pediatr Blood Cancer [PMID 101002pbc23365] WJCPwwwwjgnetcomAugust Volume Issue 0cPublished by Baishideng Publishing Group Inc Koll Center Parkway Suite Pleasanton CA USA Telephone Email bpgofficewjgnetcom Help Desk wwwf6publishingcomhelpdesk wwwwjgnetcom Baishideng Publishing Group Inc All rights reserved\x0c"

Thyroid_Cancer

"Evidence on the association between exposure to perfluoroalkyl and polyfluoroalkyl substancesPFASs and blood glucose concentrations in pregnant women is inconsistent This study aimed to examine theassociation between PFAS exposure and the concentrations of fasting plasma glucose FPG and onehour plasmaglucose hPG after a 50g oral glucose tolerance test in pregnant womenMethods The study was based on the ShanghaiMinhang Birth Cohort in which pregnant women were recruitedAmong them women provided blood samples at “ gestational weeks for PFAS measurement FPG data collectedfrom women at “ GW and hPG data collected from women at “ GW were obtained through medicalrecords from the routine prenatal care system High FPG or hPG was defined as ‰¥90th percentile of FPG or hPG Theanalysis of eight PFASs was conducted in this study perfluorohexane sulfonate PFHxS perfluorooctane sulfonate PFOSperfluorooctanoic acid PFOA perfluorononanoic acid PFNA perfluorodecanoic acid PFDA perfluoroundecanoic acidPFUdA perfluorododecanoic acid PFDoA and perfluorotridecanoic acid PFTrDA The odds ratios ORs and associated confidence intervals CIs were estimated to determine the associations of each PFAS compound with high FPG and hPG from a logistic regression modelResults After adjustment for potential confounders most PFASs were positively associated with high hPG concentrationsThe OR for high hPG concentrations was CI “ with a one log unit increase of PFOS similar associationswere observed for PFNA OR CI “ PFDA OR CI “ PFUdA OR CI “and PFDoA OR CI “ When the PFAS concentrations were categorized into three groups by tertiles thehighest tertiles of PFOS PFOA PFNA PFDA PFDoA and PFTrDA had a statistically significant increase in the risk of high hPG concentrations compared with the lowest tertiles No statistically significant association was observed between PFASexposure and high FPGConclusion PFAS exposure was associated with an increased risk of high hPG among pregnant women but no suchassociation was observed for FPGKeywords Perfluoroalkyl and polyfluoroalkyl substances Plasma glucose Cohort study Pregnancy Correspondence miaomaohua163com Yanfeng Ren and Longmei Jin contributed equally to this work4NHC Key Lab of Reproduction Regulation Shanghai Institute of PlannedParenthood Research Fudan University Shanghai ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cRen Environmental Health Page of IntroductionPerfluoroalkyl and polyfluoroalkyl substances PFASs agroup of manmade chemicals with water stain andgreaseresistant properties are used in a wide range ofconsumer products including fast food packaging stainresistant carpets windshield washing fluid firefightingfoam insecticides and paints [] Humans are widely exposed to PFASs through the ingestion of contaminateddrinking water and food as well as the inhalation ofcontaminated indoor air and dust [] Some PFASs havebeen shown to bioaccumulate in anisms [] Themean halflives of PFASs in adult humans vary from to years [ ] The most commonly studied PFASsincluding perfluorohexane sulfonate PFHxS perfluorooctane sulfonate PFOS perfluorooctanoate PFOAand perfluorononanoate PFNA are detected in the majority of human serum samples []Animal studies have shown that PFAS exposure is associated with a wide range of adverse health effects including the disruption of endocrine hormones such astestosterone estrogen and thyroid hormones [ ] alterations in serum lipid levels [] impaired glucose metabolism and insulin hypersensitivity [] and immune systemdisturbance [ ] Human studies have also suggested theadverse effects of PFASs on the immune system [] carcinogenesis [] pregnancyinduced hypertension arterialatherosclerosis [ ] and glucose metabolism [“]Although epidemiological studies have suggested thatPFASs are associated with impaired glucose toleranceand homeostasisinsulin resistance betacell dysfunction and a higher risk of diabetes [“] the associations observed in the general population cannot begeneralized to metabolically vulnerable pregnant womenowing to their specialinsulinresistant state duringpregnancy The current evidence on the effects of PFASson glucose metabolism in pregnant women is limitedand inconclusive In the Odense Child Cohort studyPFHxS and PFNA concentrations were associated withimpaired glycemic status in pregnant women and maytherefore enhance the risk of developing gestational diabetes mellitus GDM [] In another prospective studyof women higher prepregnancy PFOA concentrations were associated with an increased risk of GDMbut the associations for six other PFASs were not statistically significant [] In contrast Valvi found noassociations between PFOA PFOS PFHxS PFNA orperfluorodecanoic acid PFDA concentrations and therisk of GDM in pregnant women []In the present study we sought to evaluate the associations between PFAS exposure and fasting plasma glucose FPG and 1h plasma glucose concentrations hPG measured after a 50g oral glucose tolerance testOGTT in pregnant women by using data from theShanghaiMinhang Birth Cohort Study SMBCSMethodsStudy participantsAll study participants were recruited from the SMBCSbetween April and December Pregnantwomen attending their first routine antenatal care at theMaternal and Child Health Hospital of Minhang districtin Shanghai were consecutively recruited if they wereat “ gestational weeks GW of pregnancy theywere registered residents of Shanghai they had nohistory of chronic disease of the liver kidney or otherans they planned to give birth in the study hospital and they were willing to participate in specifiedinterviews during pregnancy and after delivery Among pregnant women who were invited pregnantwomen were recruited corresponding to a response rateof Exposure assessment and quality controlBlood samples for PFASs measure were collected at recruitment and plasma samples were separated andstored at ˆ’ °C before they were transported to theCenter for Disease Control and Prevention in HubeiProvince for the assay of PFASHighperformanceliquid chromatographycoupledwith tandem mass spectrometry Agilent TechnologiesInc USA was used for the quantitative measurement ofPFASs The information on sample collection separation reservation transportation quantification limit ofdetection LOD and quality control has been detailedpreviously [] Among the PFASs measured in ourstudy eight PFASs with detection rates above including PFHxS PFOS PFOA PFNA PFDA perfluorododecanoic acid PFDoA perfluoroundecanoic acidPFUdA and perfluorotridecanoic acid PFTrDA wereincluded in the final analysesAn internal standard approach was used to aidquantification MilliQ water was used to performprocedural blank analysis for each batch of samplesThe concentrations of each detected congener shouldbe more than three times of that in the proceduralblank and were corrected by subtracting the procedural blank concentration in the present study LODwas defined as the concentration with a signaltonoise ratio equal to or greater than All the recoveries ranged from “ A five point calibration curve was drawn and each precursor rangedfrom “ ngmL Calibration curves presenteda linear pattern over the concentration range of theprecurslucose and covariate measurementThe information on plasma glucose concentrations inpregnant women was collected from the medical recordsof the prenatal care system and included results for FPG 0cRen Environmental Health Page of and hPG In the study hospital within the studyperiod pregnant women were asked to provide bloodafter overnight fasting for FPG testing at their earliestconveniences generally within week after their firstantenatal care It was suggested that pregnant womenunderwent a h 50g OGTT at GW in order toscreen for gestational diabetes if they were consideredto have a high risk of GDMn ie FPG ‰¥ mmolL mgdL [] or overweight and obeseieBMI ‰¥ kgm2 [] otherwise it was suggested thatthe examination of hPG was performed between and GW The 50g OGTT was performed after anovernight fasting also The distribution of gestationalweeks in which the FPG and hPG examination wasperformed is shown in Supplemental Table S1 Information on whether the women had been diagnosed withGDM was extracted through medical records at birthA structured questionnaire was used by trained interviewers to collect information on the covariates Thewomen were asked about age per capita household income education level passive smoking height prepregnancy weight parity history of abortion and stillbirth pregnancy complications etc Prepregnancy BMIkgm2 was calculated as body weight divided by bodyheight squaredStatistical analysisAmong the pregnant women recruited womendelivered singleton live births and women providedblood samples at enrollment for PFAS measurement FPGconcentrations measured at “ GWs were obtainedfor women and hPG concentrations measured at“ GWs were obtained for pregnant womenPregnant women who had data on PFASs and FPG concentrations were included in this study Fig We firstdescribed and compared the demographic characteristicsofthe included and excluded pregnant women Themeans and standard deviations SD were used to describethe distributions of FPG and hPG according to thedemographic characteristics ofthe included pregnantwomen A logistic regression model was used to examinethe association between PFAS exposure and plasma glucose with the 90th percentiles of FPG mmolL ie mgdL and hPG mmolL ie mgdL usedlogarithm lntransformedas the cutoff value NaturalPFAS concentrations were firstincluded in logisticFig Study population of the present study from SMBCS FPG fasting plasma glucose hPG hplasma glucose after a 50g oral glucosetolerance test SMBCS ShanghaiMinhang Birth Cohort Study 0cRen Environmental Health Page of regression models and those with concentrations belowthe LOD were assigned a value of LODˆš PFAS concentrations were also categorized into three groups by tertilesT1 lowest tertile T2 middle tertile and T3 highest tertile and included in the logistic regression models withthe lowest tertile as the reference group Odds ratiosORs and associated confidence intervals CIs wereestimated for the association between each PFAS and highFPG1 hPG ie ‰¥90th of FPG concentration or ‰¥ 90th of hPG concentration Based on tertiles the concentrations were transformed to ordinal data and assigned to allpersons to calculate ptrend values In addition multiplelinear regressions were used to analyze the associationbetweenglucoseconcentrationscontinuousplasmaPFASsandPotential confounders were identified a prior according to the previous literature Age of pregnant womeneducation economic income prepregnancy BMI passive smoking parity history of abortion and stillbirthand pregnancy complicationsincluding bleeding thyroid disease and pregnancyinduced hypertension [ ] were identified and a directed acyclic graphsSupplemental Figure S1 was used to evaluate the appropriation of covariates We did not adjust for alcoholconsumptionn in the final models because of thelow prevalence The statistical assumptions of logistic regressions were evaluated and met including linear relationship of independent variables with logitp outliersand colinearity of independent variablesSeveral sensitivity analyses were performed to test therobustness of the primary results1 Considering the potential effect of prepregnancy BMI on GDM [] andthe variation in PFAS concentrations across BMI we repeated the analysis in women with a prepregnancy BMIof kgm2 to eliminate the confounding effect ofBMI To test the generalizability of the results we repeated the analyses in pregnant women without GDM To examine whether the associations of PFASs withFPG1 hPG were timedependent we performed subgroup analyses for different spans of GW at glucosemeasurement for FPG at “ GWs and “ GWsfor hPG at “ GWs and “ GWs StatisticalAnalysis System SAS software version SAS Institute Inc Cary NC USA was used for statistical analysis P values of were considered statisticallysignificantResultsTable presents the characteristics of the included pregnant women are compared with those of the excludedwomen in the study The majority of women included inthe present analyses were nulliparous “years of age with a BMI between “ kgm2 with a household income per capita of Table Characteristics of the included and excluded pregnantwomenCharacteristicsPvalue of Student™sttest or Chisquare testIncludedN N Mean ± SDExcludedN N Mean ± SDMaternal age at enrollment yearsMean ± SD ± “‰¥ Prepregnancy BMI kgm2Mean ± SD ± “‰¥ Maternal education ± ± Below highschool High School College orabove Per capita household income CNY “ Passive smokingYesNo Pregnancy complicationYesNo History of abortion and stillbirthYesNoParity‰¥ CNYmonth well educated collegeleveleducation or above without pregnancy complication without history of abortion and stillbirth Approximately of women were exposed topassive smoking during pregnancy The distributions ofthese demographic characteristics were not significantlydifferent between the included and excluded womenexcept parityTable presents PFHxS PFOS PFOA PFNA andPFDA were detected in all maternal plasma sampleswhile PFUdA PFDoA and PFTrDA were detected in 0cRen Environmental Health Page of Table PFASs concentrations ngmL of the includedpregnant women N PFASLODLOD NGMGSDPercentiles5th25th 50th 75th 95thPFHxS PFOSPFOAPFNAPFDAPFUdA PFDoA LOD PFTrDA Note LOD limit of detection GM geometric mean GSD geometricstandard deviationLOD about samples PFOA and PFOS had the highestconcentrations PFOA GM ngmL PFOS GM followed by PFHxS GM ngmL ngmLPFDA ngmL PFNA ngmL and PFUdA ngmL while PFDoA and PFTrDA had the lowestconcentrationsTable presents the concentrations of FPG and hPGaccording to the demographic characteristics of the subjects The mean SD FPG and hPG concentrationswere mmolL ie mgdL and mmolL ie mgdL respectively Theconcentrations of FPG and hPG were comparableacross pregnant women with different BMI household income passive smoking status pregnancy complicationand history of abortion and stillbirth The concentrationof hPG was higher in pregnant women who were olderor had higher education levels but not in those with FPGThe concentration of FPG was lower in nulliparous pregnant women but not in those with hPGTable presents that higher concentrations of PFOSPFOA PFNA PFDA PFDoA and PFTrDA were associatedwith an increased risk of high FPG however the associationswere not statistically significant AORPFOS CI“ AORPFOA CI “ AORPFNA CI “ AORPFDA CI “AORPFDoA 95CI “ AORPFTrDA CI “ Higher concentrations of PFASs were associated with an increased risk of high hPG except for PFHxSand the associations with PFOS PFNA PFDA PFUdA andPFDoA were statistically significant after adjustment for potential confounders AORPFOS CI “AORPFNA CI “ AORPFDA CI “ AORPFUdA CI “ AORPFDoA CI “ In addition multiple linearregressions were also used to analyze the association betweenPFASs and plasma glucose Similar results were found inmultiple linear regression as in logistic regression modelalthough the association of PFDoA with hPG is not statistically significant Supplemental Table S2We further examined the associations between the categorized PFAS concentrations and FPG1 hPG Weak associations between the highest tertiles of PFASs and anincreased risks of high FPG were observed but the associations were not statistically significant Fig Comparedwith pregnant women with the lowest tertiles of PFASsthe risk of high hPG was increased in women with thehighest tertiles of PFASs with statistically significant associations observed for PFOS PFNA PFDA PFUdA andPFDoA AORPFOS CI “ AORPFNA CI “ AORPFDA CI “ AORPFUdA CI “ AORPFDoA CI “ Fig Linear trends were observed between the tertiles of PFOS PFNA PFDAPFUdA and PFDoA and high hPG P for trend and respectivelyWe repeated the analysis after excluding women withGDM The pattern of associations between PFASs andhigh FPG and hPG did not change substantially except that the association between PFDoA and high hPG was no longer statistically significant SupplementalTable S3 In addition the analysis among pregnantwomen with a BMI of kgm2 produced similar results Supplemental Table S4thatIn the subgroup analysis for different GW spans theassociations between PFASs and high FPG remainednonsignificant disregard of the timing of FPG measurements exceptthe increased concentrations ofPFNA were associated with an increased risk of highFPG at “ GWs AORPFNA CI “ The pattern of association between PFASs andhigh hPG did not substantially change disregard ofmeasurement time of hPG with the exception thatthe association with high hPG became nonsignificantfor PFOS PFUdA PFDoA at “ GWs and PFOSPFNA PFDA and PFUdA at “ GWs largely owingto the reduced sample size Supplemental Table S5DiscussionIn this prospective cohort study PFAS exposures inpregnant women were found to be associated with high hPG but not FPG and the association persisted forpregnant women without GDM or with BMI kgm2Many studies have demonstrated that PFASs wereassociated with impaired glucose homeostasis and anincreased risk of diabetes in the general population [“] However in pregnant women the associations between PFASs and glucose homeostasis have not beenwell investigated Wang et al™s study showed that severalPFAS compounds were associated with increased postpartum FPGincluding perfluoro1metylheptylsulfonat mPFOS perfluoro34metylheptylsulfonat m 0cRen Environmental Health Page of Table The distribution of FPG and hPG mmolL according to participant™s demographic characteristicsCharacteristicsFPGN Mean ± SD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± PvalueTotalMaternal age at enrollment years “‰¥ Prepregnancy BMI kgm2 “‰¥ Maternal educationBelow high schoolHigh SchoolCollege or abovePer capita household income CNY “ Passive smokingYesNoPregnancy complicationYesNoHistory of abortion and stillbirthYesNoParity‰¥ hPGN Mean ± SD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Pvalue FPG fasting plasma glucose hPG hplasma glucose after a 50g oral glucose tolerance test p compared with the first groupmPFOS perfluoro5metylheptylsulfonat mPFOSand PFHxS [] The Longitudinal Investigation of Fertility and the Environment LIFE study reported thateach SD increment in PFOA concentrations was associated with a 187fold increase in GDM risk [] In theOdense Child Cohort study in metabolically vulnerablepregnant women ie BMI ‰¥ kgm2 family history ofdiabetes mellitus previous GDM multiple pregnancy ordelivery of a macrosomic child PFHxS and PFNA concentrations were associated with impaired glycemic status however no associations were found in women withlow GDM risk [] It™s a pity that the absence of information on history of family diabetes and subjects withprevious GDM limited our ability of examining the[]association in subjects with high risk Higher concentrations of PFASs in our study may partially contributeto the differences with other studies In our studyconcentrations of most PFASs were much higher thanthose in the Odense Child Cohortthe LIFEStudy [] and Wang et al™ study [] except thatPFOS is higher in the LIFE Study compared to thecurrent study The differences in concentrations aswell as outcome indices of impaired glucose homeostasistiming of measurement and population included make the comparison between these studiesdifficult nevertheless the potential for PFAS exposureto disturb glucose homeostasis has been supported inmost studies 0cRen Environmental Health Page of Table Association between PFAS concentrations lntransformed and high FPG and hPG in pregnant womenInPFASngmlPFHxS hPGN COR CIFPGN COR CI AOR CIPFOSPFOAPFNAPFDAPFUdAPFDoA AOR CI PFTrDACOR crude odds ratio AOR adjusted odds ratio CI confidence interval FPG fasting plasma glucose hPG hplasma glucose after a 50g oral glucosetolerance testModels were adjusted for maternal age at enrollment years prepregnancy BMI kgm2 per capita household income education level passive smokingpregnancy complication history of abortion and stillbirth and parity Although the underlying mechanism linking PFASs toglucose homeostasis is not yet clear it has been suggestedthat inhibition of phosphorylation of protein kinase BAkt and the activation of peroxisome proliferator activated receptors PPARs may play a role [ ] Studies using animal models and HepG2 cells have indicatedthat PFAS compounds reduce the expression of the phosphatase and tensin homolog protein and affect the Aktsignaling pathway [ ] The inhibition of Akt a keymediator of cellular insulin sensitivity may stimulate gluconeogenesis and hepatic insulin resistance [] BothPFOA and PFOS have been certified to affect glucose metabolism by AKT signaling pathway However the otherPFASs were not investigated in these studies [ ] Inaddition studies have demonstrated that PFASs can bindto and activate the PPAR α and γ receptors [] PPAR anuclear transcription receptor is known to play essentialroles in the regulation of gene expression glucose homeostasisfatty acid metabolism and inflammation []Therefore PFASactivated PPAR could disturb glucosehomeostasis by influencing insulin resistance [] and insulin secretion [] PFOA have the highest potential ofPPARα activation than the other PFASs with a shortercarbon chain length including PFHxS PFNA PFDA andPFDoA [] Moreover PFAS exposure may interferewith secretion and function of glucocorticoids andthyroid hormones via hypothalamic“pituitary“adrenalaxis and hypothalamic“pituitary“thyroid axis whichmay further disturb glucose metabolism [ ]The physiologicaleffects of PFASs on glycemichomeostasis may depend on the potency and concentration of individual PFASs [ ]Fig Association between PFAS concentrations divided by tertiles and high FPG Notes All the ptrend values for PFASs with FPG were insignificant 0cRen Environmental Health Page of Fig Association between PFAS concentrations divided by tertiles and high hPG Notes p for trend The strengths of the present study were the prospective nature of the study design the large sample size andthe measurement of a wide range of PFAS compoundsHowever the potential limitations of the study shouldbe considered First a considerable proportion of subjects was lost to followup which may have led to selection bias However the characteristics of the includedsubjects were similar to those excluded in terms of ageeducation prepregnancy BMI and household incomeand thus a substantial selection bias was not expectedSecond the followup period from the measurement ofPFAS exposure to the endpoints FPG and hPG wasshort but the singlepoint measurement of PFAS concentration may reflect PFAS exposure long before thedate of blood collection owing to their long halflifeThirdthe relationships between PFASs and bloodglucose measures may have been confounded by unmeasured confounders such as family diabetes historyand dietary habits this should be examined in futurestudiesinformation on maternal activesmoking was not collected since the proportion of activesmoking was quite low in Chinese women [] For example only of pregnant women have been exposedto active smoking during pregnancy in a Shanghai BirthCohort [] Thus the current result is not expected tobe severely biased by the unadjustment of active smoking Fourth not all the subjects had information on hPG after the 50g OGTT which may have led to missedcases of GDM and affect the association between PFASsand outcome indicesin the sensitivity analysis ofpregnant women with GDM However the absence ofGDM cases if any would have attenuated the observedassociation Fifth data for FPG “ GWs or hPG“ GWs were collected over a long time span andIn additionthus the associations between PFASs and FPG and hPG may have been confounded by the gestational weekHowever we performed subgroup analyses using different GWs spans at glucose measurement and found thatthe results did not change significantlyConclusionExposure to certain PFASs ie PFOS PFNA PFDAPFUdA and PFDoA was associated with an increasedrisk of high hPG among pregnant women Furtherstudies are needed to clarify the effect of PFASs ongestational glycemic homeostasis and the underlyingmechanismSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12940020006408Additional file Table S1 The distribution of gestational week atglucose measurement for the included pregnant women Table S2Association between PFAS concentrations lntransformed and high FPGand hPG using multiple linear regression Table S3 Associationbetween PFAS concentrations lntransformed and high FPG and hPGin pregnant women without GDM Table S4 Association between PFASconcentrations lntransformed and high FPG and hPG in pregnantwomen with BMI kgm2 Table S5 Subgroup analysis of theassociation between PFAS concentrations lntransformed and high FPGand hPG in pregnant women by gestational age Figure S1 Assumeddirected acyclic graph for PFASs and plasma glucoseAbbreviationsPFASs Perfluoroalkyl and polyfluoroalkyl substances FPG Fasting plasmaglucose hPG Onehour plasma glucose GWs Gestational weeksPFHxS Perfluorohexane sulfonate PFOS Perfluorooctane sulfonatePFOA Perfluorooctanoic acid PFNA Perfluorononanoic acidPFDA Perfluorodecanoic acid PFUdA Perfluoroundecanoic acidPFDoA Perfluorododecanoic acid PFTrDA Perfluorotridecanoic acidORs Odds ratios CIs Confidence intervals GDM Gestational diabetesmellitus OGTT Oral glucose tolerance test SMBCS ShanghaiMinhang Birth 0cRen Environmental Health Page of Cohort Study LOD Limit of detection SD Standard deviations HOMAIR Homeostasis model of assessment for insulin resistance Akt Proteinkinase B PPARs Peroxisome proliferator activated receptorsAcknowledgementsThe authors thank fieldworkers involved in the survey for their efforts in datacollection and quality control and all the pregnant women investigatedAuthors™ contributionsWY HL and MM conceived and designed the study YR LJ and MMperformed data analysis and drafted the WY MM YR FY HL XS ZZand JD revised the manuscript and critically discussed the results All authorswere involved in interpreting the data and approved the final FundingThis work was supported by grants from the National key research anddevelopment program [grant numbers 2016YFC1000505 2018YFC1002801]Shanghai Municipal Commission of Health and Family Planning [grantnumber ] Innovationoriented Science and Technology Grantfrom NHC Key Laboratory of Reproduction Regulation [grant numbersCX2017“] and Shandong Medical and Health Science and Technology Development Project [grant numbers 2018WS060]Availability of data and materialsThe datasets used during the current study are available from thecorresponding author on reasonable requestEthics approval and consent to participateThe study was approved by the ethical review committee of ShanghaiInstitute of Planned Parenthood Research SIPPR Written informed consentwas obtained before the data collection and analysis and the survey wasconducted in accordance with the Declaration of Helsinki PrinciplesConsent for publicationNot applicableCompeting interestsThe authors declare they have no actual or potential competing financialinterestsAuthor details1Department of Health Statistics School of Public Health Weifang MedicalUniversity Weifang Shandong China 2Minhang District Maternal and ChildHealth Hospital Shanghai China 3Department of Global Public HealthKarolinska Institute Stockholm Sweden 4NHC Key Lab of ReproductionRegulation Shanghai Institute of Planned Parenthood Research FudanUniversity Shanghai ChinaReceived May Accepted August ReferencesLau C Anitole K Hodes C Lai D PfahlesHutchens A Seed J Perfluoroalkylacids a review of monitoring and toxicological findings Toxicol Sci “Tittlemier SA Pepper K Seymour C Moisey J Bronson R Cao XL DabekaRW Dietary exposure of Canadians to perfluorinated carboxylates andperfluorooctane sulfonate via consumption of meat fish fast foods andfood items prepared in their packaging J Agric Food Chem “Conder JM Hoke RA De Wolf W Russell MH Buck RC Are PFCAsbioaccumulative A critical review and comparison with regulatory criteria andpersistent lipophilic compounds Environ Sci Technol “Olsen GW Burris JM Ehresman DJ Froehlich JW Seacat AM Butenhoff JLZobel LR Halflife of serum elimination of perfluorooctanesulfonateperfluorohexanesulfonate and perfluorooctanoate in retired fluorochemicalproduction workers Environ Health Perspect “Bartell SM Calafat AM Lyu C Kato K Ryan PB Steenland K Rate of declinein serum PFOA concentrations after granular activated carbon filtration attwo public water systems in Ohio and West Virginia Environ HealthPerspect “Tian Y Zhou Y Miao M Wang Z Yuan W Liu X Wang X Wang Z Wen SLiang H Determinants of plasma concentrations of perfluoroalkyl andpolyfluoroalkyl substances in pregnant women from a birth cohort inShanghai China Environ Int “Biegel LB Liu RC Hurtt ME Cook JC Effects of ammoniumperfluorooctanoate on Leydig cell function in vitro in vivo and ex vivostudies Toxicol Appl Pharmacol “Fuentes S Colomina MT Rodriguez J Vicens P Domingo JL Interactions indevelopmental toxicology concurrent exposure to perfluorooctanesulfonate PFOS and stress in pregnant mice Toxicol Lett “Seacat AM Thomford PJ Hansen KJ Olsen GW Case MT Butenhoff JLSubchronic toxicity studies on perfluorooctanesulfonate potassium salt incynomolgus monkeys Toxicol Sci “ Yan S Zhang H Zheng F Sheng N Guo X Dai J Perfluorooctanoic acidexposure for days affects glucose homeostasis and induces insulinhypersensitivity in mice Sci Rep Goudarzi H Araki A Itoh S Sasaki S Miyashita C Mitsui T Nakazawa HNonomura K Kishi R The Association of Prenatal Exposure to Perfluorinatedchemicals with glucocorticoid and androgenic hormones in cord bloodsamples the Hokkaido study Environ Health Perspect “ Barry V Winquist A Steenland K Perfluorooctanoic acid PFOA exposuresand incident cancers among adults living near a chemical plant EnvironHealth Perspect ““Lind PM Salihovic S van Bavel B Lind L Circulating levels of perfluoroalkylsubstances PFASs and carotid artery atherosclerosis Environ Res “ Darrow LA Stein CR Steenland K Serum perfluorooctanoic acid andperfluorooctane sulfonate concentrations in relation to birth outcomes in themidOhio Valley Environ Health Perspect “ MatillaSantander N Valvi D LopezEspinosa MJ ManzanoSalgado CBBallester F Ibarluzea J SantaMarina L Schettgen T Guxens M Sunyer J Exposure to Perfluoroalkyl substances and metabolic outcomes inpregnant women evidence from the Spanish INMA birth cohorts EnvironHealth Perspect Zeng XW Lodge CJ Dharmage SC Bloom MS Yu Y Yang M Chu C Li QQHu LW Liu KK Isomers of per and polyfluoroalkyl substances and uricacid in adults Isomers of C8 Health Project in China Environ Int 133Pt A105160Lin CY Chen PC Lin YC Lin LY Association among serum perfluoroalkylchemicals glucose homeostasis and metabolic syndrome in adolescentsand adults Diabetes Car

Thyroid_Cancer

CytoskeletonAssociated Role ofPDLIM5Xiaolan Huang Rongmei Qu Jun Ouyang Shizhen Zhong and Jingxing DaiGuangdong Provincial Key Laboratory of Medical Biomechanics Department of Anatomy School of Basic MedicalSciences Southern Medical University Guangzhou ChinaRegenerative medicine represented by stem cell technology has become one of thepillar medical technologies for human disease treatment Cytoskeleton plays importantroles in maintaining cell morphology bearing externalforces and maintaining theeffectiveness of cellinternal structure among which cytoskeleton related proteins areinvolved in and play an indispensable role in the changes of cytoskeleton PDLIM5 isa cytoskeletonrelated protein that like other cytoskeletal proteins acts as a bindingprotein PDZ and LIM domain PDLIM5 also known as ENH Enigma homolog isa cytoplasmic protein with a molecular mass of about KDa that consists of a PDZdomain at the Nterminus and three LIM domains at the Cterminus PDLIM5 bindsto the cytoskeleton and membrane proteins through its PDZ domain and interactswith various signaling molecules including protein kinases and transcription factorsthrough its LIM domain As a cytoskeletonrelated protein PDLIM5 plays an importantrole in regulating cell proliferation differentiation and cellfate decision in multipletissues and cell types In this review we briefly summarize the state of knowledge onthe PDLIM5 gene structural properties and molecular functional mechanisms of thePDLIM5 protein and its role in cells tissues and an systems and describe thepossible underlying molecular signaling pathways In the last part of this review wewill focus on discussing the limitations of existing research and the future prospects ofPDLIM5 research in turnKeywords PDZ and LIM domain microfilament actin cytoskeleton cytoskeletonassociated proteinINTRODUCTIONPDZ and LIM domain also known as ENH ENH1 L9 and LIM is a cytoskeletonrelated proteinthat was first discovered by Kuroda using yeast twohybrid technology with proteinkinase C PKC as the bait protein PDLIM5 which consists of a PDZ domain and one or moreLIM domains is a PDZLIM family member whose sequence is highly conserved across speciesThe proteins of the PDZLIM family EnigmaLMP1 ENH ZASPCipher RIL ALP and CLP36have been suggested to act as linkers to direct LIM binding proteins to the cytoskeleton Vallenius PDZLIM protein can act as a signal modulator to aï¬ect actin dynamics regulate cellstructure and control gene transcription to promote the assembly of protein complexes The PDZLIM protein family which function as protein“protein interaction modules act as scaï¬olds to bindto filamentous actinassociated proteins a range of cytoplasmic signaling molecules and nuclearEdited byMarina PajicGarvan Institute of Medical ResearchAustraliaReviewed byClment ThomasLuxembourg Institute of HealthLuxembourgHongqiang ChengZhejiang University ChinaCorrespondenceJun OuyangjouyangsmueducnShizhen ZhongzhszhsmueducnJingxing Daidaijxsmueducndaijx2013163comSpecialty sectionThis was submitted toIntegrative Physiologya section of the journalFrontiers in PhysiologyReceived May Accepted July Published August CitationHuang X Qu R Ouyang JZhong S and Dai J AnOverview of theCytoskeletonAssociated Roleof PDLIM5 Front Physiol 103389fphys202000975Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5proteins allowing this family to carry out diverse functionsduring development and adulthood Krcmery Several members of the PDZLIM proteins family play aregulatory role in the invasion and migration of cancer cellsDysfunction of the proteins of the PDZLIM family is knownto aï¬ect the maintenance of an function and weaken theinvasion ability and metastatic potential of cancer cells BagheriYarmand TanakaOkamoto Accordingto recent studies PDLIM5 may be involved in the progressionof multiple tumor types Eeckhoute Edlund Heiliger Li The important role ofPDLIM5 in various anizational systems have led to a deeperunderstanding of its physiological function This review aimsto summarize the state of knowledge and progress related toPDLIM5 from multidisciplinary perspectivesTHE PDLIM5 GENE AND ITSEXPRESSION AND THE STRUCTUREAND DISTRIBUTION OF PDLIM5The PDLIM5 GeneThe PDLIM5 gene is located on the human chromosome 4q223between markers W12900 and W13273 and spans exonsUeki Te Velthuis and Bagowski PDLIM5can be categorized as long isoforms with three LIM domainsand short isoforms without LIM domains according to thepresence or lack of three LIM domains and the long and shortisoforms each contain ˆ¼ subtypes Cheng Ito Mouse PDLIM5 isoform I mENH1 encodes afulllength 591aminoacid protein containing a PDZ domainand three LIM domains two smaller transcripts called mousePDLIM5 isoform and mENH2 and mENH3 encode a aminoacid protein and a 239aminoacid protein respectivelyBoth mouse PDLIM5 isoforms and lack three LIM domainsNiederlander Zheng In humans fourPDLIM5 splice isoforms have been identified one long isoformENH1 which contains three LIM domains at its Cterminaland is widely expressed in all tissues and three short isoformsENH24 which are mainly expressed in cardiac and skeletalmuscle Kuroda Ueki Analysis of humanPDLIM5 transcripts showed that three transcripts hENH12 were similar to those of mice while the fourth transcripthENH4 encodes a 215aminoacid protein lacking three LIMdomains Niederlander The Expression Structure andDistribution of PDLIM5is a memberThe PDLIM5 protein also known as ENHofthe Enigma family which consists of an NterminalPDZ domain and three Cterminal LIM domains The mainfunction of PDZ and LIM domains is to participate inproteinprotein interactions Table The PDZ domain oneofischaracterized by a highly conserved amino acid sequenceconsisting of six antiparallel strands and two αhelicesthe most common proteinprotein binding domainsTABLE Binding partners of PDLIM5 and their functionsProteinDomain FunctionsReferencesProtein kinaseA PKAαactininMyotilinLtype calciumchannelYAPProtein kinaseC PKCProtein kinaseD1 PKD1CREBID2PDZPDZPDZPDZPDZLIMLIMLIMLIMNtype Ca2channelsAMP activatedprotein kinaseKAE1Protein kinaseLin Sarcomere Zline proteinSarcomere Zline proteinCalcium channels inmyocytesTranscriptionalcoactivatorProtein kinaseProtein kinaseCAMP relatedtranscription factorsDifferentiation inhibitorNtype calcium channelsin nervous systemProtein kinaseNakagawa Ito Ito Maturana Elbediwy Kuroda Maturana Maturana Ito Lasorella and Iavarone Nakatani et alMaenoHikichi et alYan Liu Kidney anion exchanger Su Fanning and Anderson Sheng and Sala The PDZdomain provides a proteinbinding interface that facilitates theformation of multiprotein complexes with a variety of partnersincluding membraneassociated proteins cytoplasmic signalingproteins and cytoskeleton proteins Jelen Krcmery Zheng Ito The LIM domainis approximately amino acids long and is characterized byhighly conserved and spatially defined cysteine and histidineresidues that coordinate the binding of two zinc ions to formtwo zinc fingerlike structures LIM domains can exist in proteinsalone or in combination with other domains Dawid Bach Kadrmas and Beckerle Te Velthuisand Bagowski The LIM domains can combine highlydiverse partners ranging from signaling molecules and actincytoskeletal components to transcription factors and it alsosupport cellular functions Dawid In particularthe crosslinking with actin cytoskeleton such as LIM domainprotein can maintain the functional structure of cardiomyocytesby a mechanism involving its own binding and actin filamentcrosslinking which plays an important role in the developmentof heart disease Hoï¬mann In addition LIM domainproteins have also been reported to be involved in the invasionand metastasis of cancer as components and targets of thecytoskeleton Hoï¬mann exhibitThe PDLIM5 splicing isoformstissuespecificexpression the long isoforms are widely expressed in varioustissues while the short isoforms are only highly expressedin cardiac and skeletal muscle Yamazaki Thisdiï¬erential expression of PDLIM5 may be related to theirdiï¬erent roles in diï¬erent tissues and an systems Table Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5TABLE List of different disease involved in PDLIM5Disease and developmentReferencesRelated signalingpathwaysRASERKAMPKPKCMicroRNA17ˆ¼Papillary thyroid carcinomaProstate cancerWei Koutros Liu Li Gastric cancernonsmall cell carcinomaEdlund Cancer associated with steroid use Wang Heart developmentCardiomyocyte hypertrophyTGFSmadPulmonary hypertensionDilated CardiomyopathyBipolar disorderDepressive disorderSchizophreniaAlcohol dependence type diabetes and hypertensionNakagawa Yamazaki Bang Chen Cheng Cheng Zhao Liu Numata Owusu THE DIFFERENTIAL ROLES OF PDLIM5IN VARIOUS AN SYSTEMSThe Role of PDLIM5 in the NervousSystemPDZ and LIM domain is widely expressed in diï¬erent regionsof the brain such as the hippocampus thalamus hypothalamuscerebral cortex and amygdala MaenoHikichi Incentral neurons PDLIM5 is mainly localized in the membraneand cytoplasm where it regulates neuronal calcium signaling andcolocalizes with neurotransmitterprotruding vesicles Numata these observations indicate that PDLIM5 playsa role in brain development Some studies have shown thatthe expression of PDLIM5 is associated with multiple mentaldisorders such as bipolar disorder major depression andsusceptibility to schizophrenia Liu Zhao Herrick Wang In the nervous system PDLIM5 plays an important rolein the formation of nerve growth cones and promotes thediï¬erentiation of nerve cells Some studies have shownthat PDLIM5 expression is upregulated during neuraldiï¬erentiation and it has been shown that its ectopic expressionin neuroblastoma cells leads to the translocation of ID2 whichis one of the four members of the ID protein family called adiï¬erentiation inhibitor from the nucleus to the cytoplasmresulting in the inactivation of transcriptional and cellcyclepromoting functions ofthe latter Lasorella and Iavarone Furthermore PDLIM5 can form large complexes withPKC and Ntype Ca2 channels to promote the regulationof Ntype calcium channel activity MaenoHikichi Ren showed that PDLIM5 and PKCεcoexist in the nerve growth cone Through interaction withαactinin PDLIM5 may be involved in regulating microfilamentremodeling in neurons and the formation of the PDLIM5PKCεcomplex in the nerve growth cone which acts as a scaï¬old tomediate PKCε translocation to the membrane during PMAinduced growth cone collapse It is suggested that PDLIM5participates in a variety of functions of the nervous systemas well as in a signaling pathway involving the sequestrationof the transcription factor ID2 in the cytoplasm Howeverthe precise mechanisms by which PDLIM5 regulates thefunctions of the nervous system via ID2 blockade requiresfurther elucidationPDLIM5 in the Heart and Skeletal MusclePhysiological Roles of PDLIM5 in the HeartThe heart undergoes development and begins to function in theearly stages of embryonic development PDLIM5 is expressedat high levels in the skeletal muscle and myocardium and isconsidered to be a heart and skeletalmusclespecific scaï¬oldprotein to regulates mouse heart development Mu PDLIM5 is capable of binding to αactinin throughthe PDZ domain and colocalizing in the zdisk region ofcardiomyocytesindicating that this protein plays a role incardiac development Studies have shown that PDLIM5 mRNAsare mainly expressed in the heart and skeletal muscle of adultrats and that PDLIM5 acts as a scaï¬old protein to mediatethe transmission of PKC signals in cardiomyocytes playing animportant role in development of the muscle cell in an earlydevelopmental stage Nakagawa Zheng The PDZLIM protein family is highly expressed in the striatedmuscle PDLIM5 is similar to other PDZLIM members in thestriated muscle in which the PDZ domain binds to αactininwhile the LIM domain binds to several protein kinases C andprotein kinase D Kuroda Nakagawa For example PDLIM5 traditionally activates the PKC throughthe direct binding of its LIM domain Maturana and interacts to PKA Lin Transcription factorCREB which is one of the first transcription factors activated byneurohumoral factors stimulation is a transcription factor cAMPresponse element binding protein is a known target of the PKCand protein kinase D1 PKD1 pathways Thonberg Ozgen The interaction between PDLIM5 isoform and CREB is necessary for the phosphorylation of CREB at theaminoacid residue Ser133 which promotes the transcriptionalactivation and nuclear localization of CREB the phosphorylatedCREB enters the cardiomyocyte nucleus to play the role oftranscription factor and promote the growth of cardiomyocytesIto Moreover in neonatal rat cardiomyocytesPDLIM5 interacts with PKD1 through its LIM domain and formscomplexes with PKD1 and cardiac Ltype voltagegated calciumchannelα1C subunits to regulate the activity of Ltype voltagegated calcium channels Maturana Although theformation of protein complexes such as PDLIM5PKCPKD1is well understood the downstream molecular events remainto be elucidatedThese results suggest that the localization of PDLIM5 at somesubcellular sites and its ability to interact with multiple functionalproteins play an important role in cellular and physiologicalfunctions Furthermore the role of PDLIM5 in the heart wasFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5studied using a heartspecific PDLIM5knockout mouse modelIt was found that the ablation of PDLIM5 disrupted the stabilityof the PDLIM5CiphersCalsarcin complex formed in the zdiskregion thus interfering with the connection between adjacentsarcomeres and extracellular matrix These eï¬ects were found toresult in the loss of optimal force transmission and a significantdecrease in cardiac shortening fractionleading to dilatedcardiomyopathy Cheng Novel polymorphisms inthe PDLIM5 gene encoding the Zline protein have also beenshown to increase the risk of idiopathic dilated cardiomyopathyWang that underpinsPDZ and LIM domain is additionally involved in skeletalmuscle development Myogenesis is an important biologicalprocessskeletal muscle regeneration andpostnatal growth The silencing of PDLIM5 increases the nuclearaccumulation of diï¬erentiation inhibitor Id2 which inhibits theproliferation and diï¬erentiation of myoblasts Nakatani Qiu In addition the diï¬erentiation of andmorphological changes in skeletal muscle is regulated by a groupof transcription factors known as myogenic regulators PDLIM5isoform overexpression leads to the upregulation of MyoDand myogenin while PDLIM5 isoform knockout significantlydecreases the expression of these two proteins these findingsindicate that the main eï¬ect of PDLIM5 isoform on musclecells is to stimulate the transcription of MyoD andor myogeninencoding genes Ito Although the main role of PDLIM5 is as a specific scaï¬oldprotein which to bridge the connection between cytoskeletonand membrane proteins and promote the formation of proteincomplexes it is capable of generating numerous splicing isoformsENH24 that exert various eï¬ects on the development ofheart and skeletal muscle In vitro PDLIM5 isoform hasbeen shown to promote the expression of myogenic genes andmyotube formation while the short PDLIM5 isoform hasbeen found to abrogate myotubelike morphological changeswithout altering the expression of the myogenic transcriptionfactors MyoD and myogenin Ito Furthermorethe overexpression of PDLIM5 isoform prevented ventricularcardiomyocyte hypertrophy induced by vascular stress hormonesYamazaki Western blotting analysis of muscle tissueusing a nonisoformspecific antiPDLIM5 antibody showed thatPDLIM5 isoform is only expressed in skeletal muscle witha specific distribution of PDLIM5 members between skeletalmuscle and myocardium Niederlander These results suggest that PDLIM5 plays a key role in thedevelopment of the myocardium and skeletal muscle Howeverthe signal transduction mechanisms underlying the role ofPDLIM5 in heart and skeletal muscle remain to be furtherstudied For example the specific molecular mechanisms bywhich PDLIM5 regulates the development of myocardium andskeletal muscle through binding protein kinases are unknownFurthermore the mechanisms by which PDLIM5 sequestersnuclear protein Id2 in the cytoplasm remain to be elucidatedThe Role of PDLIM5 in Cardiovascular DiseasesPDZ and LIM domain is mainly distributed on the zline ofthe sarcomere of cardiomyocytes therefore the eï¬ect of PDLIM5on myocytes may be related to the contractile function of thesecells PDLIM5knockout mice exhibit dilated cardiomyopathywhich is characterized by thinning of the left ventricular wallenlargement of the left ventricular cavityimpaired cardiaccontraction and reduced ejection function Cheng PDLIM5 can regulates vascular remodeling which canas a new proatherosclerotic factor to be a therapeuticallytargeted Huang Cardiac remodeling which isindicative of progression in many cardiovascular diseases ischaracterized by cardiomyocyte hypertrophy and myocardialfibrosis which lead to heartfailure Swynghedauw Barry and Townsend microRNA miR21 derivedfrom cardiac fibroblasts exosomes is a strong paracrine RNAmolecule that induces cardiomyocyte hypertrophy Thum Recentlyit has been reported that PDLIM5 is thedirect target of miR17ˆ¼ Bang Chen By acting on its target gene PDLIM5 miR participates in the interaction between cardiac fibroblastsand cardiomyocytesthus inducing myocardial pathologicalhypertrophy Bang PDLIM5 also plays a role invascular smooth muscle AMPactivated protein kinase is anintracellular energy receptor of AMPK which is activatedunder hypoxia ischemia glucose loss and stress Steinberg andKemp AMPK is generally considered to be an energysensor kinase and requires AMP for its activation Carling Nakano reported that AMPK controls microtubuledynamics by phosphorylating cytoplasmic connexin170 CLIPthus regulating cell migration Nakano In addition AMPK is involved in the regulation of actincytoskeleton dynamics and plasma membrane reanizationBae Kondratowicz Studies haveshown that AMPK activation plays an important role inneovascularization and metastasis HoyerHansen and Jaattela In vascular smooth muscle cells AMPK phosphorylatesPDLIM5 at Ser177inhibiting the downstream Rac1Arp23signaling pathway to mediate cell migration Yan In pulmonary artery smooth muscle cells PASMCsSMCspecific knockout of PDLIM5 enhances hypoxiamediatedvascular remodeling while overexpression of PDLIM5 inhibitsthe TGFSmad signal pathway and prevents hypoxiainducedpulmonary hypertension elevation in vivo Cheng In addition PDLIM5 silencing induces the activity of TGF3 TR1 and TGF and increases the overall expressionlevel of Smad2 The suppression of PDLIM5 has beenfound to enhance the nuclear staining of Samd23 Chen indicating that PDLIM5 participates in thedevelopment of cardiovascular disease by negatively regulatingthe TGF3Smad23 signal pathway Additionally demonstratedthat PDLIM5 plays an important role in the cardiovascularsystem through miRmediated regulation of the phenotype ofpulmonary artery smooth muscle cells miR17ˆ¼ regulatesthe diï¬erentiation of PASMCs through its target PDLIM5indicating that the miR17ˆ¼92PDLIM5TGFSmad pathwayis essential for vascular remodeling during the developmentof pulmonary hypertension PDLIM5 therefore represents apromising therapeutic target for future cardiovascular drugdiscovery eï¬ortsFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5The Role of PDLIM5 in TumorAs an actin adaptor protein PDLIM5 is not only involved incytoskeletal tissue cellular processes and an development butis also considered to play roles in tumorigenesis and developmentEeckhoute Edlund Heiliger Li PDLIM5 is expressed in many cancer celllines In a study of neurologic tumor it was found that thetranscription factor ID2 binds to the PDLIM5 LIM domains andin these cancer cells high levels of PDLIM5 sequester ID2 in thecytoplasm preventing neuronal diï¬erentiation and promotingcell proliferation Lasorella and Iavarone PDZ and LIM domain is additionally upregulated inpapillary thyroid carcinoma PTC tissues elevated PDLIM5expression promotes the migration invasion and proliferation ofPTC cells by activating the RASERK pathway Wei PDLIM5 may therefore serve as a therapeutic target in a varietyof cancers It can promote the invasion and metastasis of cancercells Genotyping chip detection experiments have shown thatPDLIM5 is overexpressed in prostate cancer tissue Koutros and some studies have proved that the utility of serumand urine PDLIM5 levels as indicators for auxiliary diagnosisof prostate cancer with potential value in predicting the risk ofprogression in advanced prostate cancer PCA Ma PDLIM5 plays a key role in regulating the proliferation invasionand migration of malignant tumor cells by binding to AMPKand regulating its activation and degradation Liu PDLIM5 may therefore act as an oncogene in the developmentand progression of PCAIn view of the important role of PDLIM5 in cancer somestudies have indicated that it is involved in the growth of gastriccancer cells and suggested that PDLIM5 silencing through theuse of small interfering RNAs siRNA may be a potentialstrategy for the treatment of gastric cancer Li In addition Edlund found that the increased expressionof PDLIM5 is related to high tumor proliferation rates innonsmall cell carcinoma Edlund Steroids suchas corticosteroid medications play an important role in thedevelopment of cancer it has been reported that singlenucleotide polymorphisms SNPs in PDLIM5 interact withsteroids thus aï¬ecting the occurrence and development of cancerWang The above findings show that PDLIM5 plays an important rolein the occurrence and development of cancer and that PDLIM5represents a candidate oncogene in various cancersThe Role of PDLIM5 in Other DiseasesreportedIn addition to playing a role in the diseasesinvolved in the link between alcoholabove PDLIM5 isdependence and diabetes Owusu found thatPDLIM5 gene polymorphism is associated alcoholdependentAD type diabetes T2D and hypertension and Severalgenetic variants of the PDLIM5 gene can aï¬ect AD T2Dand hypertension indicating that PDLIM5 is a shared geneamong the three diseases Therefore elucidation oftheunderlying molecular mechanisms and identification of hithertoundiscovered molecular functions of PDLIM5 are expectedto enable the development of eï¬ective clinicalfor these diseasestherapiesIn addition PDLIM5 plays a role in the formation of cellextensions Being a scaï¬old protein PDLIM5 is involved inpromoting the activity of microfilamentassociated proteinsMicrofilamentassociated proteins play a central role in theprocess of cell extension Lanier Some studieshave found that PDLIM5 recruitment to cell extensions and isnecessary to form these extensions and that PDLIM5 knockoutreduces the assembly of actin filaments in cell extensionsYuda THE RELATIONSHIP BETWEEN PDLIM5AND INTEGRINS AND ITS POTENTIALROLE IN THE REGULATION OF STEMCELL DIFFERENTIATIONSome studies have shown that the PDZ domain of PDZLIMprotein binds to αactinin protein at the adhesion junctionwhich is the site of integrin localization Xia Pomies Vallenius Tamura The functional interaction with integrin indicates that PDZLIM protein can participate in the adhesion signal cascadewhich transmits extracellular signals via intracellular regulatorypathwaysthereby modifying the actin cytoskeleton Thesefindings show that the PDZLIM protein plays an overall role incell“cell and cell“matrix interaction and cell migration Krcmery The regulation of PDZLIM activity plays animportant role in preventing uncontrolled actin recombinationproliferation and cell migration For example PDLIM5 canalso bind to the integrin protein kinase ILK acting as ascaï¬old bridge between renal ion exchanger KAE1 and ILKproviding a bridge between KAE1 and potential microfilamentsSu It has been reported that PDLIM5 is recruited from thecytoplasm to the integrin adhesion and Factin stress fibersand responds to stress by directly binding to the key stressfiber component αactinin Microfilaments control the nuclearand cytoplasmic localization oftranscriptional coactivatorsYAP and TAZ to regulate gene expression and mediate thediï¬erentiation of MSCs Dupont Halder The eï¬ective domains of some proteins that are recruited intothe actin structure in a forcedependent manner through theLIM domain regulate actin signal transduction Smith The action of mechanical force on integrin results in therecruitment of PDLIM5 to activate the YAP pathway duringmechanical transduction Elbediwy PDLIM5 acomponents of the integrin adhesion complex mediates therelationship between integrin and the cytoskeleton Horton et al2016ab Proteomic analysis of integrinrelated complexes fromMSCs has demonstrated the formation of significant amountsof a vinculinpositive adhesion complex on a hard substratecoated with fibronectin FN in MSCs a subset of whichcolocalized with or closely to clusters of PDLIM1 or PDLIM5Ajeian Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5PDZ and LIM domain undergoes tensiondependentrelocalization in cells Both embryonic and induced pluripotentstem cells can diï¬erentiate into derivatives of the third germlayer as a result human pluripotent cells are important toolsin regenerative medicine Thomson Takahashiand Yamanaka Studies have reported that duringcardiogenesis embryonic stem cells exhibit dramatic changes inthe expression of metabolic enzymes and cytoskeleton proteinsKonze in particular Zdisk related proteins withPDZ and LIM domain proteins including PDLIM5 are upregulated during cardiogenesisFurthermorethe expression of NKX25 an importantmyocardial transcription factor results in the generation ofspecific PDLIM5 splicing variants during the early developmentof cardiomyocytes which in turn aï¬ectsthe myogenicdiï¬erentiation of myocardium Konze At presentthere are few studies on PDLIM5 in stem cells elucidation of itsmechanism in diï¬erent stem cell types is warranted to identify itsfunctions in these cellsPROBLEMS AND PERSPECTIVESIn this we briefly reviewed the known functionsof the PDLIM5 protein the progress in elucidation ofitsroles in various cellular and physiological processes and thesignaling pathways in which it participates As a member ofthe PDZLIM protein family PDLIM5 is involved in actinbinding αactinin binding protein kinase binding acts asa scaï¬old bridge between connective proteins and plays anindispensable role in various cellular processes However so farthe specific molecular mechanisms underlying the functions ofPDLIM5 remain unclearFuture research directions in investigation of PDLIM5 shouldseek to answer the following questionsFirst research on PDLIM5 has mainly focused on its roles intumor the nervous system and the cardiovascular system As amicrofilamentassociated protein does PDLIM5 play the samerole in other physiological systems or cell lines Is the PDLIM5gene expressed in multiple systemsREFERENCESAjeian J N Horton E R Astudillo P Byron A Askari J A and MillonFrmillon A Proteomic analysis of integrinassociated complexes frommesenchymal stem cells Proteomics Clin Appl “ 101002prcaBach I The LIM domain regulation by association Mech Dev “ 101016S0925477399003147Bae H B Zmijewski J W Deshane J S Tadie J M Chaplin D D andTakashima S AMPactivated protein kinase enhances the phagocyticability of macrophages and neutrophils FASEB J “ fj11190587BagheriYarmand R Mazumdar A Sahin A A and Kumar R LIMkinase increases tumor metastasis of human breast cancer cellsvia regulationof the urokinasetype plasminogen activator system Int J Cancer “ 101002ijc21650Bang C Batkai S Dangwal S Gupta S K Foinquinos A and HolzmannA Cardiac fibroblastderived microRNA passenger strandenrichedSecond although the role of PDLIM5 in diseases hasbeen studied eg its expression is upregulated during tumordevelopment the specific mechanisms by which it exerts theseeï¬ects are unknown and further elucidation of the underlyingmechanisms and other functions is warrantedThird PDLIM5 has four splicing isoforms which performdiï¬erent functions what are the mechanisms by which they playdiï¬erent roles Are these diï¬erences in their roles attributable tothe presence or absence of LIM domains Additionally what isthe mechanism by which they play diï¬erent rolesFourth in regard to strategies used for the inhibition ofPDLIM5 expression only viral transfection has been reportedto date no pharmaceutical compounds that inhibit PDLIM5expression have been identified Therefore additional research onPDLIM5 inhibitors is also criticalFinally although PDLIM5 plays a crucial role in binding actinand has attracted much attention as a connecting protein thereare few studies on its eï¬ects on the actin cytoskeleton or othercytoskeleton such as binding to cytoskeletonrelated proteinsbridging the connection with the cytoskeleton Does PDLIM5aï¬ect the shape and location of the cytoskeleton in the processof participating in the biological functions of cellTheeï¬ects of PDLIM5mediated modulation ofthecytoskeleton on cell diï¬erentiation proliferation and othercellular functions remain to be explored in detail in future studiesAUTHOR CONTRIBUTIONSAll authors participated in the conception and writing of themanuscript SZ JO and JD reviewed and suggested modificationsto the content JD designed the structure of the reviewFUNDINGThis work wassupported by the National Key RDProgram of China grant number 2017YFC1105000 andthe Sanming Project of Medicinein Shenzhen grantnumber SZSM201612019exosomes mediate cardiomyocyte hypertrophy J Clin Invest “ 101172JCI70577Barry S P and Townsend P A What causes a broken heart“molecularinsights into heart failure Int Rev Cell Mol Biol “ S1937644810840031Carling D Mayer F V Sanders M J and Gamblin S J AMPactivatedprotein kinase nature™s energy sensor Nat Chem Biol “ nchembio610Chen T HuangJ B DaiJ Zhou Q RajJ U and Zhou Gthe miR17ˆ¼ signaling that PAI1 is a novel component ofregulates pulmonary artery smooth muscle cell phenotypes Am J PhysiolLung Cell Mol Physiol L149“L161 101152ajplung00137Chen T Zhou G Zhou Q Tang H Ibe J C and Cheng H Loss of microRNA17 approximately in smooth muscle cells attenuatesexperimental pulmonary hypertension via induction of PDZ and LIM domain Am J Respir Crit Care Med “ 101164rccm2014050941OCFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5Cheng H Chen T Tor M Park D Zhou Q and Huang J B A highthroughput screening platform targeting PDLIM5 for pulmonary hypertensionJ Biomol Screen “ Cheng H Kimura K Peter A K Cui L Ouyang K and Shen T Lossof enigma homolog protein results in dilated cardiomyopathy Circ Res “ 101161CIRCRESAHA110218735Dawid I B Breen J J and Toyama R LIM domains multiple roles asadapters and functional modifiers in protein interactions Trends Genet “ 101016s0168952598014243Dupont S Morsut L Aragona M Enzo E Giulitti S and Cordenonsi M Role of YAPTAZ in mechanotransduction Nature “ 101038nature10137Edlund K Lindskog C Saito A Berglund A Pontn F and G¶ranssonKultima H CD99 is a novel prognostic stromal marker in nonsmallcell lung cancer Int J Cancer “ 101002ijc27518Eeckhoute J A celltypespecific transcriptional network required forestrogen regulation of cyclin D1 and cell cycle progression in breast cancerGene Dev “ 101101gad1446006Elbediwy A Vanyai H DiazdelaLoza M Frith D Snijders A P andThompson B J Enigma proteins regulate YAP mechanotransductionJ Cell Sci 131jcs221788 101242jcs221788Fanning A S and Anderson J M PDZ domains fundamental buildingblocks in the anization of protein compl

Thyroid_Cancer

"Pressure sores are sometimes refractory to treatment often due to malnutrition Small intestinalbacterial overgrowth SIBO obstructs absorption in the digestive tract and causes malnutrition However little isknown about the association between pressure sore wound healing and SIBO Here we report a case of a patientwith a refractory sacral pressure sore and SIBOCase presentation A 66yearold woman who was spinal cord injured years before visiting our hospitalpresented with the chief complaint of a sacral pressure sore — cm in size which was refractory totreatment Physical examination showed abdominal distension and emaciation with a body mass index of Further examination revealed elevated serum alkaline phosphatase UL bilateral tibial fracture multiple ribfracture and osteoporosis We diagnosed the patient with osteomalacia with vitamin D deficiency Despite oralsupplementation serum levels of calcium phosphorous and vitamin D remained low Also despite concentrativewound therapy for the sacral pressure sore by plastic surgeons no wound healing was achieved Due to asuspicion of disturbances in nutrient absorption we performed bacterial examination of collected gastric andduodenal fluid which showed high numbers of bacteria in gastric content E coli Streptococcus speciesand Neisseria species and duodenal content E coli Candida glabrata Therefore we diagnosed thepatient with SIBO and started selective decontamination of the digestive tract using polymyxin B sulfate andamphotericin B After starting treatment for SIBO the sacral pressure sore began to heal and was nearly healed after days The patient™s serum levels of calcium phosphorous vitamin D and other fatsoluble vitamins alsogradually increased after starting treatment for SIBOContinued on next page Correspondence 2m2hy4gmailcom1Department of Plastic Surgery Chiba University Inohana ChuokuChibacity Chiba JapanFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cKubota BMC Gastroenterology Page of Continued from previous pageConclusion We report a case of a patient with a refractory sacral pressure sore that healed after starting treatmentfor SIBO We conclude that SIBO may be an overlooked cause of malnutrition and poor wound healing in patientswith chronic pressure soresKeywords Pressure wound Small intestinal bacterial overgrowth Spinal cord injury Malnutrition Wound healingCase reportBackgroundPressure sores in patients with spinal cord injury SCIare sometimes refractory to treatment Chronic gastrointestinal symptoms are also frequently seen in patientswith SCI [ ] and malnutrition caused by decreasedgastrointestinal motility in SCI patients is a major causeand exacerbating factor of pressure sores Evaluation ofnutritional status in patients with pressure sores is essential [] as nutritional intervention can be a valuabletreatment option for pressure sores However small intestinal bacterial overgrowth SIBO is rarely consideredin the evaluation of malnutrition in SCI patients withpressure sores SIBO is defined as the presence of morethan — colony forming units CFUmL of bacteriaor any amount of E coli in the proximal small bowelcontent [] Here we report the case of an SCI patientwith a refractory sacral pressure sore that healed afterstarting treatment for SIBO To the best of our knowledge this is the first report of an association between apressure sore and SIBOCase presentationA 66yearold woman visited our hospital for the purpose of treating her sacral pressure sore day whichshe developed months prior due to bed rest duringtreatment of a left humeral fracture in another hospitalShe had paraplegia as well as bladder and rectal disturbance due to SCI at the fourth lumbar level L4 causedby a suicidal jump in response to paranoid delusions at years of age Spinal fusion surgery and cystostomywere performed early after SCI Otherwise she had ahistory of hysterectomy due to uterine cancer at yearsof age lymphaticovenular anastomosis as a treatment forposthysterectomy lymphedema in the bilateral lower extremities at years of age and cholecystectomy at years of ageWhen she visited our hospital she was taking the following oral medicines propiverine hydrochloride vitamin B12 etizolamflunitrazepam sodium bicarbonateanhydrous monobasic sodium phosphate mixture Clostridium butyricum tablets sodium risedronate hydraterebamipide sodium ferrous citrate fursultiamine hydrochloride alfacalcidol and potassium Lasparate She didnot take proton pump inhibitors PPI Her vital signswere as follows body temperature of °C low bloodpressure of mmHg pulse rate of bpm and respiratory rate of per min Physical examinationshowed abdominal distension emaciation with a bodymass index of and a sacral pressure sore — cm in size including a pocket entrance of — cmFig 1a Most of the surface of the pressure sore wascovered by granulation Our evaluation of the pressuresore with DESIGNR [] was D3 e3 s8 i0 g3 N3 P24with a total score of Table Bacterial culture examination ofthe pressure soreshowed Corynebacterium striatum and methicillinresistant Staphylococcus aureus Laboratory data showedan elevated serum alkaline phosphatase level of UL and low serum levels of hemoglobin gdL albumin gdL calcium mgdL and zinc μgdL Onday we observed a sudden decrease of hemoglobin to gdL with a positive fecal occult blood test bilateralpleural effusion on chest xray and serum albumin levelof gdL Upper gastrointestinal endoscopy showed agastric ulcer at H2 stageAs a result of searching for the cause of alkaline phosphatase elevation bilateral tibial fracture multiple ribfracture and osteoporosis were found Fig 1e and fFemoral bone density was of the young adult meanA low serum inanic phosphorous level was foundTable along with a low serum level of 25hydroxyvitamin D3 25OHVitD3 below the detection limit andelevated level of parathyroid hormone Table Levelsof other fatsoluble vitamins were also low vitamin A μIUdL vitamin K1 ngdL and vitamin E mgdL Examination using ultrasound and computedtomography showed normalthyroid and parathyroidglands Basing on these finding we diagnosed osteomalacia with vitamin D deficiencyOn day oral supplementation of calcium phosphorous and vitamin D was started Despite supplementationserum levels of calcium phosphorous and 25OHVitD3on day showed poor improvement calcium mgdLphosphorous mgdL and 25OHVitD3 below the detection limitOn day we performed bacterial examination ofcollected gastric and duodenal fluid with suspicion of adisturbance in absorption which showed elevated num E coli bers of bacteria in gastric contentStreptococcus species and Neisseria species and 0cKubota BMC Gastroenterology Page of Fig See legend on next page 0cKubota BMC Gastroenterology Page of See figure on previous pageFig Patient images a b c and d Sacral pressure sore a Day sore — cm in size with an entrance of — cm DESIGNR score wasD3 e3 s8 i0 g3 N3 P24 with a total score of b Day ie days after starting SDD for treating SIBO reduced size of sore DESIGNR scorewas D3 e3 s3 i0 g1 n0 p0 with a total score of c Day ie days after staring SDD healed sore DESIGNR score was d0 e0 s0 i0 g0 n0p0 with a total score of d Day ie days after staring SDD no recurrence of the sore e and f Osteoporosis and multiple fractures eXray showing left tibial fracture f Tc99 m bone scan showing accumulation in multiple ribs vertebrae and right ulna g h and i Endoscopicexamination and results of bacterial culture of the upper digestive tract All stomach duodenum and proximal jejunum samples were positive forE coli g Stomach Food residue can be seen Acid level was decreased to pH h Duodenum Food residue is evident i Proximal jejunum Flatvilli and a jejunal ulcer are observedTable DESIGNR assessment tool for pressure sore Reprinted with permission from John Wiley and Sons In Matsui et alDevelopment of the DESIGNR with an observational study an absolute evaluation tool for monitoring pressure ulcer woundhealing Wound Repair Regen “Depthd No particular skin lesion and no rednessD Lesion extends into the subcutaneous tissuePersistent rednessLesion extends into dermisExudatee NoneSlight does not require daily dressing change Moderate requires daily dressing changeSizes NoneSmaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2Lesion extends to the muscle tendon and boneLesion extends into the articular or body cavityU It is impossible to measure the depthE Heavy requires dressing change more than twice a dayS cm2 or largerInflammationInfectioniNoneSigns of inflammation fever redness swelling and pain around thewoundIClear signs of local infection eg inflammation pus and foulsmellSystemic impact such as feverGranulation tissueg Granulation cannot be assessed because the wound is healed or tooshallowG Healthy granulation tissue occupies or more but lessthan Healthy granulation tissue occupies or moreHealthy granulation tissue occupies or more but less than Healthy granulation tissue occupies less than No healthy granulation tissue existsNecrotic tissuen NonePocketp NoneN Soft necrotic tissue existsHard and thick necrotic tissue is attached to the woundP Smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger 0cKubota BMC Gastroenterology Page of Table Laboratory data before starting supplementation withvitamin DWBCμL104μLgdL104μLgdLgdLULULULULULULULULmgdLmgdLmLmin173 m2mEqLmEqLmgdLmgdLmgdLμgdLμgdLμgdLμgdLμIUmLmgdLmgdLmgdLmgdLsecondpgmLμgdLμIUmLpgmLngdLof woundirrigationtreatmentdebridementstarting SDD the pressure sore was refractory to multiple methodsincludingdepressurizationointmentbasic fibroblast growth factor and negative pressurewound therapy After starting SDD the pressure sorebegan to heal On day ie days after startingSDD the pressure sore DESIGNR score was D3 e3 s3i0 g1 n0 p0 with a total score of Fig 1b Regardingthe nutritional status the serum albumin level increasedfrom gdL just before starting SDD to gdL at days after starting SDD Also the hemoglobin levelincreased to gdL and the serum zinc level increasedto μgdL Serum levels of calcium phosphorous vitamin D and other fatsoluble vitamins also gradually increased Fig and Table A repeat diagnosticbacterial examination of the upper digestive tract contents for SIBO was not performed because of obviousimprovements in most of the laboratory data Therewere no adverse effects of SDD such as antibioticassociated diarrhea In contrast the patient presentedwith constipation that was noted before starting SDDThe sacral pressure sore was completely healed on day ie days after starting SDD with a DESIGNRscore of d0 e0 s0 i0 g0 n0 p0 and total score of Fig1c In addition the patient showed improved nutritionalstatus and had a serum albumin level of gdL Wesuccessfully reduced the dose of polymyxin B from to million units daily similarly the dose of amphotericin B was reduced from to mg daily on day ie days after starting SDD without any signs ofSIBO recurrence There was no recurrence of the sacralpressure sore with a serum albumin level of gdL onday ie days after staring SDD Fig 1d Onday we successfully ended the use of amphotericinB however the use of polymyxin B at million unitsper day continued On day ie days afterstarting SDD while still using polymyxin B at million per day the serum albumin level was gdL thehemoglobin level was gdL and the serum zinc levelwas μgdL There were no signs of SIBO recurrenceor the sacral pressure soreDiscussion and conclusionsWe report the case of a patient whose sacral pressuresore and osteoporosis were improved by treatment forSIBO Although nutrition status is known to be important for the healing of pressure sores SIBO is rarelychecked as a cause of malnutrition in patients with pressure sores However SIBO is a potential cause of malnutrition in patients with SCI due to decreased intestinalmotility resulting from autonomic disturbances and reduced physical activity [] SCI is also a risk factor forpressure sores [] However to the best of our knowledge there are no previous reports of an associationRBCHbPltTotal ProteinAlbuminASTALTγGTPLDHALPChECKAmyBUNCreatinineeGFRNaKCaiPMgFeZnUIBCFerritinErythropoietinTotal CholesterolTriglycerideHDLCholesterolLDLCholesterolPTINRAPTTACTHCortisolTSHFT3FT4duodenal content E coli Candida glabrataFig 1g h and i Therefore we diagnosed SIBO Onday we started selective decontamination of the digestive tract SDD using oral administration of polymyxin B sulfate million units daily and oraladministration of amphotericin B mg daily Before 0cKubota BMC Gastroenterology Page of Table Vitamins and bone metabolism markers before starting supplementation of vitamin DVitamin AVitamin K1Vitamin K2Vitamin E125OH2 Vitamin D25OH Vitamin D3Retinol binding proteinVitamin B1Vitamin B12Nicotinic acidFolic acidTRACP5bNTxBone type ALPIntact P1NPOsteocalcinintact PTHPTHrPFGF23TRPTmPGFR Below the detection limitIUdLngmLngmLmgdLpgmLpgmLmgdLngmLpgmLμgmLngmLμUmLnmolBCELμgLμgLngmLpgmLpmolLpgmLmgdLnormal range““ “““““““ ““““““ “““between pressure sores and SIBO Thus our case drawsattention to the fact that SIBO can be an overlookedcause of poor wound healing during the treatment ofpressure soresSIBO was first reported by Vantrappen as an increased concentration of 14CO2 in a bile acid breath testfor patients with an absent interdigestive motor complex[] Today consensus diagnostic criteria for SIBO arethe presence of more than — CFUmL of bacteriaor any amount of E coli in the proximal small bowelcontent [] Relatively little is known about commensalsinhabiting the small intestine mainly due to the limitedaccessibility of this environment for microbiological analysis [] In the healthy state the numbers of intestinalbacteria range from to CFUmL and mainly include gramnegative and grampositive aerobes such asStreptococcus Lactobacillus and Bacteroidesspecies[] Regarding the amount of bacteria in proximal jejunal aspiration Khoshini report that normal subexceed — CFUmL and thereforejectsproposed more than — CFUmL coliform bacteriaas the threshold for SIBO [] In our case CFUmLE coli existed in duodenal content and CFUmL Ecoli existed in gastric content which met the traditionaldiagnostic criteria of SIBOrarelyOther diagnostic methods for SIBO are breath testsusing hydrogen or hydrogen methane with lactulose lucose [] Breath tests have clinical utility for diagnosing SIBO because they are less invasive than obtaining proximal small bowel content However there areno standardized criteria for diagnosing SIBO usingbreath tests [] We did not perform a breath test inour case studyDespite no previous reports of an association betweenunhealed pressure sores and SIBO nutritional status isknown to be important for the healing of pressure sores []In our case the sacral pressure sore which was initially refractory began to heal after starting treatment for SIBOAmong intrinsic factors related to the healing of pressuresores blood levels of hemoglobin albumin and zinc are especially important [] Our patient had anemia hypoalbuminemia and a low zinc concentration which graduallyimproved after starting treatment for SIBOSIBO is caused by multiple factors including disturbances in defense mechanisms of the digestive tractanatomical abnormalities surgical interventions and disturbed gastrointestinal motility [ ] Bures described several endogenous defense mechanisms thatprevent bacterial overgrowth [] including secretion ofgastric acidintestinal motility a properly functioning 0cKubota BMC Gastroenterology Page of Fig Bone metabolism markers After starting SDD levels of bone metabolism markers gradually improvedTable Vitamins and trace elements before and after supplementation and selective digestive decontamination SDDVitamin K1 ngmLVitamin K2 ngmLVitamin E mgmL125OH2 Vitamin D pgmL25OH Vitamin D3 pgmLNicotinic acid μgmLMg mgdLFe μgdLBefore supplementationAt the time SDD started days after starting SDD days after starting SDDNANANA 0cKubota BMC Gastroenterology Page of ileocecal valve production of secretory immunoglobulinson the surface of the gastrointestinal mucous membraneand the bacteriostatic properties of pancreatic juice andbile In our case the patient™s history of cholecystectomyand hysterectomy were possible causes or exacerbatingfactors of SIBO Disturbed gastrointestinal motilitycaused by paraplegia below the L4 level due to SCI is another possible cause of SIBO in our case as well as decreased physical activity due to fracture ofthe lefthumerus and bilateral tibiaChronic gastrointestinalinvolvement is seen in “ of patients with SCI [ ] SCI patients lackcentral nervous system control over the gastrointestinalsystem [] Liu report that bowel problems in SCIpatients are related to high levels of cord injury completeness of cord injury and postinjury durations of years or more [] Moderate or severe grade depressivestatus is also associated with neurologic bowel dysfunction in SCI patients Of these risk factors our patienthad complete cord injury that had occurred more than years ago Also many bowel symptoms appear in patients with SCI eg constipation distension incontinence abdominal pain bowel accidents nausea diarrheastrainingautonomichyperreflexia headaches or sweat relieved by a bowelmovement [ “] However our patient showedno appetite loss a sufficient amount of food intake andnonsevere bowel symptoms Thus the presence of malnutrition despite adequate food intake and low levels oflipidsoluble vitamins that were unresponsive to supplementation led us to suspect SIBOrectal bleeding hemorrhoidsAlthough gastrointestinal symptoms are frequently observed in patients with SCI there are few reports ofSIBO in SCI patients Cheng report that of of SCI patients were diagnosed with SIBO basedon the glucose hydrogenmethane breath test [] However the prevalence of SIBO among SCI patients as confirmed by the consensus diagnostic criteria ofthepresence of more than CFUmL bacteria or anyamount of E coli in upper digestive tract content is unknown In patients with SCI absent central nervous system innervation of the digestive tract can change theinhabiting environment of bacteria Gungor reportdifferences in gut microbial patterns between SCI patients and control individuals as measured by bacterialgenome sequencing [] Specifically they found thatbutyrateproducing bacteria were specifically reduced inSCI patients Thus it is possible that SIBO is overlookedin patients with SCI In our caseit is unclear whenSIBO occurred relative to the time of SCI but we suspect that it arose due to gastrointestinal motility disorder caused by autonomic disturbancesDisturbances in fat absorption and deficiency in fatsoluble vitamins ie vitamins A K E and D3 areobserved in patients with SIBO [] Excess bacteria inthe small intestine promotes a change from conjugatedbile acid into deconjugated bile acid which decreasesthe micellar solubilization of dietary fat Bacterial fermented short chain fatty acid causes osmotic watermovement to the intestinal lumen which results in diarrhea and malabsorption [] Intestinal epithelial damagein SIBO also interferes with fat absorption Mucosaldamage is caused by metabolites of aerobic bacteria endotoxins of anaerobic bacteria and lithocholic acidwhich is a bacterial degradation product of unconjugatedbile acid [“] Our patient however showed constipation rather than diarrhea in spite of SIBO Whether ornot diarrhea occurs in patients with SIBO is determinedby multiple factors Constipation frequently occurs inpatients with SCI due to decreased physical activity andautonomic dysfunction De Looze reported that therate of constipation in the patients with SCI is []A certain proportion of the patients with SCI show constipation in spite of the coexisting SIBO Cheng reported that in patients with both SCI and SIBO showed constipation [] We believe that the factorsleading to constipation in our patient were stronger thanthose leading to diarrhea Vitamin D deficiency in SIBOcauses osteomalacia Our patientshowed multiplefractures and osteoporosis with serum vitamin D3 levelsbelow thetosupplementationrefractorydetectionlimitandThere is no consensus on the choice dose or durationof antibiotics for treating SIBO [] In principle antibiotics should be chosen based on the results of an antimicrobial susceptibility test but this approach cannotaddress the great diversity in microbiota of the digestivetract [ ] Metronidazole is a firstline choice forSIBO [] with other choices being rifaximin ciprofloxacin norfloxacin amoxicillinclavulanate trimethoprimsulfamethoxazole cephalexin or their combination []However these antibodies are selected based on customrather than scientific evidence [] In our case we usedoral polymyxin B and amphotericin B in accordancewith SDD which was first reported as a method of preventing ventilationassociated pneumonia and microbialtranslocation of gramnegative rod bacteria and fungi incritically ill patients treated in the intensive care unit[“] Polymyxin B administered to the digestive tractis nonabsorbent into the human body and has strongbactericidal power against gramnegative rod bacteriaexcept for naturally polymyxinresistant bacteria such asProteus Providencia Manella Burkholderia and Serratia [] Amphoteric B is an antifungal drug that isalso nonabsorbent into the human body when administered to the digestive tract In our case after startingSDD fatsoluble vitamins were increased and osteoporosis was improved No obvious adverse effects of SDD 0cKubota BMC Gastroenterology Page of such as antibioticassociated diarrhea were observed inour caseWhen and how to stop antibiotherapy for the treatment of patients with SIBO are difficult problemsFew reports are available for the method and timingfor making a decision to stop antibiotherapy in SIBOLauritano reported that the recurrence rate at months after stopping antibiotherapy in SIBO patientsis [] They also showed that an older age history of appendectomy and chronic use of PPIs areassociated with SIBO recurrence Bures reportedthat cyclical gastrointestinal selective antibiotics areneeded for SIBO treatment [] These reports indicatethat in many patients with SIBO it is actually impossible to stop antibiotherapy because of the underlyingconditions that lead to SIBO Similarly in our case itwas difficult to ameliorate the underlying condition ofdecreased motility of the digestive tract due to SCIWe were compelled to continue SDD for a long duration We did however succeed in gradually reducingthe dose of polymyxin B and end the use of amphotericin B without signs of SIBO recurrence Withcareful consideration it may be possible and feasibleto stop SDD completelyProbiotics are also a treatment approach for SIBO assome species of bacteria are thought to protect againsthigh numbers of E coli and fungi in the digestive tract[] However the role and effects of probiotics are stillunclear The digestive tract microbiome has both pathogenic potential and a protective role in maintaininghealth However metagenomic analysis reveals that “ of microanisms in the digestive tract cannot becultured under laboratory conditions [] The effects ofSDD and probiotics on the digestive tract microbiome inpatients with SIBO should be investigated to furtherunderstand the pathogenesis of the diseaseIn conclusion we treated a patient with a sacral pressure sore who also had SCI multiple fractures withosteoporosis and malabsorption especially of fatsolublevitamins Based on culture of upper digestive tract content we diagnosed the patient with SIBO and startedSDD using polymyxin B and amphotericin B which effectively ameliorated the absorbency disturbance andallowed healing of the pressure sore In light of severalcommon risk factors between pressure sores and SIBOsuch as decreased physical activity our case providesadditional information on the associations among pressure sores malnutrition and SIBOAbbreviationsCFU Colony forming units SCI Spinal cord injury SDD Selectivedecontamination of the digestive tract SIBO Small intestinal bacterialovergrowth 25OHVitD3 25hydroxy vitamin D3AcknowledgementsWe thank the many personnel involved in this interdisciplinary diagnosticworkup as their effective technical assistance enabled a comprehensiveapproach to this difficult diagnosisAuthors™ contributionsYK and TT treated the patient conceived of and wrote the manuscript KISK and TK treated the patient and collected the data SA and NMinterpreted the data HN analyzed the data and created the figures andtables All authors read and approved the final manuscriptFundingNo funding was receivedAvailability of data and materialsData on this case not reported in the manuscript are available from thecorresponding author upon reasonable requestEthics approval and consent to participateEthical approval was not necessary for the reported investigations as theywere performed in a routine clinical setting with therapeutic intentionConsent for publicationThe patient provided written consent for reporting her case in aninternational published medical journal including clinical details and imagesCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Plastic Surgery Chiba University Inohana ChuokuChibacity Chiba Japan 2Department of Molecular DiagnosisChiba University Inohana Chuoku Chibacity Chiba Japan3Department of Plastic Surgery Chiba Emergency Medical Center Isobe Mihamaku Chiba JapanReceived July Accepted August ReferencesStone JM NinoMurcia M Wolfe VA Perkash I Chronic gastrointestinalproblems in spinal cord injury patients a prospective analysis Am JGastroenterol “Liu CW Huang CC Chen CH Yang YH Chen TW Huang MH Prediction ofsevere neurogenic bowel dysfunction in persons with spinal cord injurySpinal Cord “Eglseer D Hodl M Lohrmann C Nutritional management of olderhospitalised patients with pressure injuries Int Wound J “Bures J Cyrany J Kohoutova D Forstl M Rejchrt S Kvetina J Vorisek VKopacova M Small intestinal bacterial overgrowth syndrome World JGastroenterol “ Matsui Y Furue M Sanada H Tachibana T Nakayama T Sugama J Furuta KTachi M Tokunaga K Miyachi Y Development of the DESIGNR with anobservational study an absolute evaluation tool for monitoring pressureulcer wound healing Wound Repair Regen “Sachdev AH Pimentel M Gastrointestinal bacterial overgrowth pathogenesisand clinical significance Ther Adv Chronic Dis “Groah SL Schladen M Pineda CG Hsieh CH Prevention of pressure ulcersamong people with spinal cord injury a systematic review PM R “Vantrappen G Janssens J Hellemans J Ghoos Y The interdigestive motorcomplex of normal subjects and patients with bacterial overgrowth of thesmall intestine J Clin Invest “Zoetendal EG Raes J van den Bogert B Arumugam M Booijink CC TroostFJ Bork P Wels M de Vos WM Kleerebezem M The human small intestinalmicrobiota is driven by rapid uptake and conversion of simplecarbohydrates ISME J “ Miazga A Osinski M Cichy W Zaba R Current views on theetiopathogenesis clinical manifestation diagnostics treatment andcorrelation with other nosological entities of SIBO Adv Med Sci “ 0cKubota BMC Gastroenterology Page of Khoshini R Dai SC Lezcano S Pimentel M A systematic review ofdiagnostic tests for small intestinal bacterial overgrowth Dig Dis Sci “ Heintschel M Heuberger R The potential role of zinc supplementation onpressure injury healing in older adults a review of the literature Wounds“Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations Han TR Kim JH Kwon BS Chronic gastrointestinal problems and boweldysfunction in patients with spinal cord injury Spinal Cord “Ebert E Gastrointestinal involvement in spinal cord injury a clinicalperspective J Gastrointestin Liver Dis “ Gungor B Adiguzel E Gursel I Yilmaz B Gursel M Intestinal microbiota inpatients with spinal cord injury PLoS One 2016111e0145878 Harari D Sarkarati M Gurwitz JH McGlincheyBerroth G Minaker KLConstipationrelated symptoms and bowel program concerning individualswith spinal cord injury Spinal Cord “ Menter R Weitzenkamp D Cooper D Bingley J Charlifue S Whiteneck GBowel management outcomes in individuals with longterm spinal cordinjuries Spinal Cord “ De Looze D Van Laere M De Muynck M Beke R Elewaut A Constipationand other chronic gastrointestinal problems in spinal cord injury patientsSpinal Cord “Lynch AC Wong C Anthony A Dobbs BR Frizelle FA Bowel dysfunctionfollowing spinal cord injury a description of bowel function in a spinalcordinjured population and comparison with age and gender matchedcontrols Spinal Cord “Krogh K Nielsen J Djurhuus JC Mosdal C Sabroe S Laurberg S Colorectalfunction in patients with spinal cord lesions Dis Colon Rectum “ Chen CY Chuang TY Tsai YA Tai HC Lu CL Kang LJ Lu RH Chang FY LeeSD Loss of sympathetic coordination appears to delay gastrointestinaltransit in patients with spinal cord injury Dig Dis Sci “ Cheng X Zhang L Xie NC Xu HL Lian YJ Association between smallintestinal bacterial overgrowth and deep vein thrombosis in patients withspinal cord injuries J Thromb Haemost “Kirsch M Bozdech J Gardner DA Hepatic portal venous gas an unusualpresentation of Crohn's disease Am J Gastroenterol “Jones RM Neish AS Recognition of bacterial pathogens and mucosalimmunity Cell Microbiol “ Hoog CM Lindberg G Sjoqvist U Findings in patients with chronicintestinal dysmotility investigated by capsule endoscopy BMCGastroenterol Singh VV Toskes PP Small bowel bacterial overgrowth presentationdiagnosis and treatment Curr Treat Options Gastroenterol “ Quigley EM AbuShanab A Small intestinal bacterial overgrowth Infect DisClin N Am “ viiiix Melchior C Gourcerol G Bridoux V Ducrotte P Quinton JF Leroi AMEfficacy of antibiotherapy for treating flatus incontinence associated withsmall intestinal bacterial overgrowth a pilot randomized trial PLoS One2017128e0180835 VandenbrouckeGrauls CM Vandenbroucke JP Effect of selectivedecontamination of the digestive tract on respiratory tract infections andmortality in the intensive care unit Lancet “Silvestri L van Saene HK Casarin A Berlot G Gullo A Impact of selectivedecontamination of the digestive tract on carriage and infection due togramnegative and grampositive bacteria a systematic review ofrandomised controlled trials Anaesth Intensive Care “ Camus C Salomon S Bouchigny C Gacouin A Lavoue S Donnio PYJavaudin L Chapplain JM Uhel F Le Tulzo Y Shortterm decline in allcause acquired infections with the routine use of a decontaminationregimen combining topical polymyxin tobramycin and amphotericin Bwith mupirocin and chlorhexidine in the ICU a singlecenter experienceCrit Care Med “ Olaitan AO Morand S Rolain JM Mechanisms of polymyxin resistanceacquired and intrinsic resistance in bacteria Front Microbiol Lauritano EC Gabrielli M Scarpellini E Lupascu A Novi

Thyroid_Cancer

RUFYs a Family of EffectorProteins Involved in IntracellularTrafficking and CytoskeletonDynamicsRmy Char1 and Philippe Pierre123 Aix Marseille Universit Centre National de la Recherche Scientifique Institut National de la Sant et de la RechercheMdicale Centre d™Immunologie de MarseilleLuminy Marseille France Institute for Research in Biomedicine and IlidioPinho Foundation Department of Medical Sciences University of Aveiro Aveiro Portugal Shanghai Instituteof Immunology School of Medicine Shanghai Jiao Tong University Shanghai ChinaIntracellular trafficking is essential for cell structure and function In order to performkey tasks such as phagocytosis secretion or migration cells must coordinate theirintracellular trafficking and cytoskeleton dynamics This relies on certain classes ofproteins endowed with specialized and conserved domains that bridge membraneswith effector proteins Of particular interest are proteins capable of interacting withmembrane subdomains enriched in specific phosphatidylinositol lipids tightly regulatedby various kinases and phosphatases Here we focus on the poorly studied RUFY familyof adaptor proteins characterized by a RUN domain which interacts with small GTPbinding proteins and a FYVE domain involved in the recognition of phosphatidylinositol3phosphate We report recent findings on this protein family that regulates endosomaltrafficking cell migration and upon dysfunction can lead to severe pathology at theanismal levelKeywords RUFY cancer neurodegenerative diseases immunity RUN FYVE phosphatidylinositol 3phosphatecytoskeletonINTRODUCTIONThe anization of cells into multiple membranous compartments with specific biochemicalfunctions requires complex intracellular traffic and sorting of lipids and proteins to transport themfrom their sites of synthesis to their functional destination Intracellular transport involves lipidvesicles or tubules with the capacity to fuse with one another or to be secreted They collectivelyparticipate in the dynamic exchanges necessary for cell homeostasis Rothman S¸reng Membrane traffic is tightly coordinated with protein synthesis signal transduction ofenvironmental stimuli and cytoskeleton anization allowing the implementation of key cellularfunctions such as endocytosis exocytosis or migration McMahon and Gallop Habtezion VegaCabrera and PardoLpez MacGillavry and Hoogenraad Margaria Tapia Buratta Stalder and Gershlick Several families of molecular components required for orchestrating membrane vesicle exchangeand transport during this process are conserved They include adaptor and coat proteins smallGTPbinding proteins GTPases as well as Synaptosome Associated Protein SNAP ReceptorEdited byRoberto BotelhoRyerson University CanadaReviewed byDaniel G S CapellutoVirginia Tech United StatesBrian Paul CeresaUniversity of Louisville United StatesCorrespondenceRmy CharcharcimlunivmrsfrPhilippe PierrepierrecimlunivmrsfrSpecialty sectionThis was submitted toMembrane Traffica section of the journalFrontiers in Cell and DevelopmentalBiologyReceived June Accepted July Published August CitationChar R and Pierre P TheRUFYs a Family of Effector ProteinsInvolved in Intracellular Traffickingand Cytoskeleton DynamicsFront Cell Dev Biol 103389fcell202000779Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingSNARE proteins and SNARE binding proteins Juliano The vast superfamily of GTPases is involved in the establishmentor regulation of virtually every step of intracellular membranetrafficking They behave as molecular switches that can alternatebetween active and inactive states through GTP binding andhydrolysis into GDP Takai Stenmark Thelargest group of GTPases involved in intracellular membranetraffic is the Rab proteins family Lamb Rab GTPasesspecifically localize to diï¬erentintracellular compartmentsregulating vesicle formation and sorting as well as transportalong the cytoskeletal network Each Rab protein can be recruitedto specific membrane subdomains of a defined anelle andis associated to multiple eï¬ectors controlling membrane fusionand trafficking Rab interaction with the membrane fusioncomplexes and cytoskeleton regulators is therefore crucial forcellular functions including endocytosis and autophagy Chenand WandingerNess Bruce Geng Thomas and Fromme Yuan and Song Here we review the literature concerning a lesswell knownfamily of proteins involved in the complex biochemical crosstalkestablished between the cytoskeleton and intracellular vesiclesThis small group of proteins was named RUFY for œRUN andFYVE domaincontaining RUFYs share a common structuraldomain anization including an Nterminal RUN domainone or several coiledcoil CC repeats and a Cterminal FYVEdomain A The molecular structures of the diï¬erentRUFY proteins has been described Dunkelberg and GutierrezHartmann Mari KukimotoNiino Kitagishi and Matsuda but their function in endocyticregulation and their physiological relevance at the anismallevel are still poorly characterized Kitagishi and Matsuda Terawaki We revisit here how the rufy genefamily was annotated and propose the addition of a novelmember the fyco1 FYVE and Coiledcoil containing domain gene given its sequence and functional similarities withthe other rufy genes Pankiv Terawaki We also highlight recent findings on the implication ofRUFY proteins in the regulation of cytoskeleton and endosomedynamics and their contribution to immunity cancer andneurodegenerative diseasesEndocytosis and AutophagyEndocytosis and autophagy are membrane traffic pathwaysrequired for degradation and recycling ofextracellularand intracellular components respectively Birgisdottir andJohansen These pathways have a common endpoint at thelysosome where their cargo is degraded These both pathwaysintersect at several stages throughout vesicle formation transportand fusion and share some of the components of their molecularmachineries BThere are numerous coexisting endocytic pathways whichinitiate by the formation of nascent endocytic vesicles formedfrom plasma membrane invaginations and scissions Theseendocytic vesicles undergo homotypic fusion and are rapidlytargeted to sorting endosomes SE Sorting events initiated inSE determine the fate of internalized cargo molecules such asrecycling to plasma membrane degradation in lysosomes orother trafficking events Naslavsky and Caplan BOn their way to degradation sorted cargo accumulate inearly endosomes EE that further mature into late endosomesLEthrough multiple events of cargo and lipid sortingLate endosomes adopt a membrane anization termedmultivesicular bodies that are enriched in lysobisphosphatidicacid and contain intraluminal vesicles Gruenberg NextLE potentiate their hydrolytic competence by fusing withlysosomes Pillay resulting in the degradationoftheir contents providing nutrients and key factors tothe cell Doherty and McMahon Kaksonen and Roux Notably endosomes play a role in signal transductionby serving as signaling platforms either for surface activatedreceptors like Tolllike receptors and epidermal growth factorreceptor or metabolic sensors such as mechanistic targetof rapamycin complex mTORC1 Arg¼ello Often they promote the degradation of their targets leadingto signaltermination Chung The endocyticpathway has also specialized functions in diï¬erentiated cellssuch as neurotransmitter release and recycling in neurons orantigen processing and presentation in professional antigenpresenting cellsArg¼ello SolDom¨nech Hinze and Boucrot Endocytosis events and endosomes positioning ishighly dependent on the dynamic and spatial reanizationofinclude actinintermediate filaments or microtubules Fletcher and Mullins Pegoraro the diï¬erent cytoskeleton networks thatlike B cells or dendritic cellsComplementary to endocytosis autophagy is an intracellularprocess by which cells degrade and recycle their own cytoplasmicmaterials Mizushima and Komatsu Autophagy plays acentral role in many physiological processes including stressmanagement development immunity and aging Puleston andSimon Zhong F®lfan Moretti Doherty and Baehrecke Autophagy ispartially controlled though mTORC1 activity and is responsiblefor degradation and recycling of misfolded proteins as wellas obsolete anelles Galluzzi The endpoint ofautophagy is to deliver cytoplasmic material to lysosomes wherelike for endocytosed cargo it is degraded Several autophagyprocesses can be distinguished based on the entry mode ofthe cytosolic components destined for degradation BMacroautophagy involves engulfment of cytoplasmic contentsinto a double membrane vesicle termed the autophagosomeThe autophagosome fuses then with lysosomes becomingin which its cargo is degraded Galluzzian autolysosome The presence ofspecific phosphoinositideslipidstogether with Rab GTPases at a given membranecompartment is often directly correlated with compartmentfunction One of the common mechanism regulating endocytosisand autophagy is an accumulation of phosphatidylinositol phosphate PtdIns3P at surface of EE and on intraluminalvesicles of multivesicular endosomes and on autophagosomesNascimbeni B PtdIns3P is also observedat sites of LC3ˆ’associated phagocytosis another pathway ofinternalization used by the cells to ingest large particulatematerial or microbes PtdIns3P is therefore a beacon used by theFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingFIGURE RUN and FYVE domain containingproteins in the endolysosomal pathway A Schematic representation of the RUFY proteins family B Description ofthe endolysosomal and autophagy pathways and presumed functional locations of RUFY proteins Extracellular material is ingested by endocytosis orphagocytosis The action of different endosomes allows cargo to be sorted recycled or degraded in a complex and regulated process involving fusion maturationand transport along the cytoskeleton Alternatively during autophagy obsolete components present in cytosol are captured in autophagosomes prior fusion withlysosomes and degradation macroautophagy or directly internalized through endosomal invagination microautophagy SE sorting endosome EE earlyendosome TGN trans golgi network LE late endosome MVBs multi vesicular bodies RE recycling endosome MT microtubule CT centrioles ER endoplasmicreticulum The location of PI3P and RUFY proteins known activity is shown Created with BIoRendercomFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and Traffickingcellular machinery to regulate endosomal sorting and autophagyBirgisdottir and Johansen RUN DomainsThe presence of a single copy of a RUN and a FYVE domainat their extremities is the key characteristic defining the RUFYfamily members RUN domains were named after three proteinsbearing similar peptide motifs RPIP8 UNC14 and NESCAnew molecule containing SH3 at the carboxyˆ’terminus Ogura Matsuda RUN domains are present inmultiple proteins RUN proteins in a large panel of anismsFigure and principally allow direct interactions with smallGTPases of the Rap and Rab families Callebaut Yoshida RUN domains adopt a hydrophobicglobular structure bearing six conserved blocks named A to FFigure 3A These blocks correspond to eight αhelices andsome 310helices The first helix is crucial to limit hydrophobicexposure and maintain protein solubility of RUNcontainingproteins Callebaut KukimotoNiino In spite of strong conservation among the domains presentin RUNcontaining proteins the proteins they interact withtheir eï¬ectors are highly variable Mari and thestructural features of the RUN domain alone are not sufficientto define binding specificity for one or several members of theGTPase superfamily Fukuda Most RUN domainbearing proteins bind small GTPases but interactions with othermolecules like kinesin have also been described Boucrot A direct physical link between RUN proteins withactin filaments and microtubules has been also demonstratedTorti reinforcing the idea that these molecules arealso critical for cellular functions requiring actin remodelingsuch as migration or phagocytosis Price and Bos Bos Miertzschke Xu Figure 4AAdditional functions for RUN domains have been describedfor example for the RUN domain present in NESCA whichblocks TRAF6mediated polyubiquitination of the NFkappaBessential modulator and consequently induces NFkB activationThis is just one of the ways in which RUN proteins canact in signal transduction and the coordination of membranetraffic with actin dynamics upon external stimulation Yoshida As well as promoting endosomal fusion throughtheir binding to Rab or Rap GTPases Callebaut FIGURE Evolution of RUN and FYVE domain or rufy genes among living anisms Diagram illustrating the evolution of the rufy genes Species representative ofvarious taxonomic groups are listed data were extracted from the Differential Expression Atlas Genes database EMBLEBI Next to each species studied thenumber corresponds to the number of genes having in its sequence a FYVE green RUN blue or both red domain The œX corresponds to the appearance of acommon rufy ancestor geneFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingFIGURE Molecular anization of RUN and FYVE domains from the RUFY proteins family Alignment of the protein sequences of the RUN A and FYVE Bdomains of the RUFY proteins family in human and mouse A RUN consensus blocks are represented by segments A“F Rpip8 sequence is used as RUN domainreference B FYVE conserved motives and zinc fingers are represented by segments In the alignment œx is any amino acid and œ represents positively chargedamino acid Eea1 sequence is used as FYVE domain reference For all alignment amino acids are colored according to their properties Cyan for hydrophobicpositions A V I L M turquoise for aromatic positions F Y W H red for basic residues K R purple for acidic residues D E green for polar uncharged N Q ST salmon for cysteine C orange for glycine G and yellow for proline P Gray numbers below alignment means the amino acids position after alignment Blacknumbers surrounding the alignments represent the start left and end right positions of the domains in the peptide sequence of each protein Alignment wererealized with Seaviewer analyzer software Gouy Accession numbers for protein are following human Rpip8 NP_0011382971 mouse Rpip8NP_0580391 human Eea1 NP_0035573 mouse Eea1 NP_0010019321 human RUFY1 NP_0794343 mouse RUFY1 NP_7661451 human RUFY2NP_0604574 mouse RUFY2 NP_0817012 human RUFY3 NP_0557761 mouse RUFY3 NP_0818061 human RUFY3XL NP_0010325191 mouseRUFY3XL NP_0012767031 human RUFY4 NP_9408852 mouse RUFY4 NP_0011641121 human FYCO1 NP_0787892 mouse FYCO1NP_0011037232Yoshida their interaction with motor proteins likekinesin or myosin suggests a role for RUN domains in regulatingvesicular and anelle transport Callebaut Yoshida Via these diï¬erent mechanisms RUN proteins havebeen implicated in neuronal development Honda 2017bsignaling Sun migration Yoshida and regulation of various cellular function like endocytosis orexocytosis Kitagishi and Matsuda FYVE DomainsFYVEdomainbearing proteins for Fab1 YOTBZK63212Vac1 and EEA1 are specifically found in association withmembranous anelles enriched in PtdIns3P and highlyconserved among eukaryotes including yeast Hayakawa Figure FYVE domains adopt a zinc finger conformationMisra and Hurley Kutateladze and Overduin Inaddition to FYVE ten types of zinc finger folds have beenFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingFIGURE RUFY proteins are important for intracellular trafficking signaling and cytoskeleton dynamics A Schematic representation of the RUN and FYVEdomains activity of RUFY proteins RUN domains act on signaling endosomal protein trafficking and cytoskeletal network dynamics via small GTPase proteins FYVEdomains bind PtdIns3P and regulates autophagy and endosome trafficking B Function of RUFY proteins in homeostatic conditions C Consequences ofalterations in RUFY proteins functions at the cellular and anismal levelcharacterizedincluding conventional Gal4 GATA1 TFIISMetRS LIM RING domain PKC CRD and PHD domainsZinc fingers are structural conformations adopted by peptidechains upon coordination of two Zn2 cations within a cysteinerich region Schwabe and Klug Stenmark Unlike most molecules bearing zinc fingers FYVE proteinsdisplay only one copy of the domain located at any positionalong the peptide chain highlighting its autonomy as a structuralunit FYVE zinc fingers can stabilize proteinprotein or proteinDNARNA interactions Dunkelberg and GutierrezHartmann A œclassical FYVE domain has eight potential zinccoordinating tandem cysteine positions and is characterized byhaving basic amino acids around the cysteines Many membersof this family also include two histidine residues in a sequencemotif including WxxD CxxC RHHCxCG and RVC whereœx means any amino acid and œ a positively charged aminoFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and Traffickingacid Figure 3B Most deviations from this sequence canreduce the domain affinity for zinc and destabilize it Stenmark Misra and Hurley Stenmark and Aasland Kutateladze and Overduin Within this structuralframework specific modifications in the nonconserved residuesof the domain can radically aï¬ect FYVE protein subcellularlocalization and function by forming a œturret loop and adimerization interface Hayakawa With regard to their affinity for PtdIns3P FYVE domaincontaining proteins are mostly found associated to EE orphagosomes Stenmark Gaullier Stenmark and Aasland Figure 4A The presence ofFYVE domains is therefore correlated to the regulation ofmembrane traffic through specific recognition of PtdIns3Pdomains by œRHHCxCG motifs Gaullier and modulation by associated phosphatidylinositol kinasesPtdIns3P is generated from phosphatidylinositol by Class IIIPtdIns 3kinases PI3Klike Vps34 on target membranessuch as nascent autophagosome omegasomes Melia or EE Di Paolo and De Camilli Raib Scott B In turn accumulation ofPtdIns3P recruits and activates eï¬ector proteins containingFYVE domains favoring transport or fusion of target anellesStenmark and Gillooly Axe Burman andKtistakis Schink Affinity for PtdIns3P isdetermined by the pair of histidine residues present in theœRHHCXCG motif of the FYVE domain Stahelin Diraviyam Lee He This affinity can also be harnessed by FYVE proteins to linkendosomes with mRNA ribonucleoprotein ps mRNPand associated ribosomes playing a role in their longdistancetransport in the cell Pohlmann Importantlymany FYVE proteins homodimerize Dimerization multiplies theconserved residues displayed by the diï¬erent signature motifspresent in the FYVE domain and contributes to a network ofhydrogen bonding and electrostatic interactions that providespositive selection for binding several PtdIns3P head groupsPHdependent insertion of FYVE domain into cell membranesHe Pankiv is reinforced by additionalhydrophobic membrane interactions with the turret loop andortandem lysine residues These nonspecific interactions promoteFYVE domain access to phosphate head groupsthat arehindered by the close packing of lipid molecules This bivalentmechanism increases therefore greatly FYVE domains specificityfor PtdIns3Penriched domains and discrimination againstother mono or polyphosphorylated PtdIns species Misra andHurley Stenmark and Aasland Dumas Kutateladze and Overduin FYVE proteins are therefore key players in endocytosis andautophagy and mutations in FYVE domains can alter profoundlythese functions as well as cellular homeostasis Kamentseva For example EEA1 protein early endosome antigen isknown to be crucial for endosome dynamics and any mutation inits conserved residues or the oligomerization site can drasticallyreduce the affinity between its FYVE domain and PtdIns3PStenmark Gaullier In this contextRUFYs proteins by bearing a Nterminal RUN domain oneor several copies of a coiledcoil domain next to a CterminalFYVE domain A have all the features required tocarryout specific adaptor functions to regulate endocytosis orautophagy by impacting on anelle fusion and mobility alongthe cytoskeletonThe RUFY Proteins FamilyThe RUFY family encompass four genes named rufy1 to sharing homologies and displaying specific tissue expressionand alternative splicing Rufy genes are relatively conservedgenes absent from prokaryotes and fungi Upon evolution theemergence of the common ancestor appeared in vertebrates andarthropods which possess one ortholog CG31064 Figure No RUFY protein could be detected In Caenorhabditis elegansand only a FYVEbearing protein T10G35 considered as anortholog of human EEA1 shows some sequence similaritieswith the RUFY family T10G35 exhibits PtdIns3P bindingactivity and is involved in endocytosis being mostly expressedin epidermis and intestine of C elegans Hayakawa In chordates Rubicon RUN domain and cysteinerichdomain containing Beclin 1interacting protein and FYVE AndCoiledCoil Domain Autophagy Adaptor FYCO1 displaystructural and functional features potentially categorizing themas RUFY proteins Rubicon was identified as a componentthe Class III PI3K complex and a negative regulatorofof autophagy and endosomaltrafficking Matsunaga Zhong Like RUFYs Rubicon containsmultiple functional domains that interact with other proteinsincluding a RUN a CC and a FYVElike domains Wong However despite these similaritiesthe poordegree of sequence homology and the lack of conservationof its FYVElike domain which was found not to bind toPI3P Burman and Ktistakis prevented Rubicon™sintegration within the RUFY proteins family conversely toFYCO1 which we propose here to name RUFY5 and detail thecharacteristics belowRUFY1RUFY1 previously named Rabip4 is an kDa proteinmainly expressed in the brain kidneylung placenta andtestis There are two RUFY1 isoforms Rabip4 and Rabip4™that has an additional amino acid upstream oftheNterminal RUN domain A They were both shownto interact with the small endosomal GTPases Rab4 Rab5and Rab14 Fouraux Vukmirica Table RUFY1 inactivation inhibits efficient recycling ofendocytosed transferrin implicating RUFY1 in the regulationof EE functions through cooperative interactions with Rab4and Rab14 Cormont Yamamoto Nag This was further demonstrated by the alteration ofepidermal growth factor receptor endocytic trafficking kineticsin cells depleted of RUFY1 Gosney and thehijacking of RUFY1 by the bacteria P gingivalis to escapelysosomal degradation Takeuchi In melanocytesRUFY1 was found to form a complex with rabenosyn5Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and TraffickingTABLE Summary of RUFY proteins functional interactionsProtein AliasesBinding partnerFunctionsRUFY1Rabip4 Rabip4™ZFYVE12RUFY2LZFYVE Rabip4rKIAA1537 FYVE13RUFY3Singar1 RIPXZFYVE30 KIAA087RUFY4ZFYVE31FYCO1RUFY5ZFYVE7 RUFY3CTRCT18 CATC2Rab4EtkRab14AP3Rabenosyn5KIF3ABRab4AAP3 complexPODXL1Rab33ARab4ARab6ARETRap2FascinRab5Rab33AGPM6aRap2STEFYial2complexPAK1FOXK1HOXD9Rab7Recycling endosomal traffickingRegulation of endocytosis through its interaction with RUFY1RUFY1™s recruitment endosome tethering and fusionRegulates spatial distribution of lysosomeSorting endosome pathway in endosomal membrane inmelanocytes and segregates tyrosinaserelated protein1Increases cell proliferation migration and invasionEndosome dynamic Golgi complexassociated Rab33 andautophagosome formation on omegasomesLead to a fusion of the RET tyrosine kinase domain to a RUNdomain and a coiledcoil domain appear to be critical fortumorigenesisControl neuronal polarityControl the growth of axons and neuronal growth coneActs on endosomal traffickingFacilitates cell polarityInduce cell migration and invasion in gastric cancerIncreases cells migration RUFY3mediated with metastasis invasionin colorectal cancerHOXD9 transactivate RUFY3 and it overexpression induce gastriccancer progression proliferation and lung metastasisAutophagosome formation and lysosome clusteringMAP1LC3ABAutophagosome formation and elongationRab7Kinesin1Endosomal transport by acting with microtubule plus enddirectiontransportAllows translocation from the late endosome lysosome andautophagosome to the plasma membrane through plusendmicrotubule transportStudyCormont Yang Yamamoto Ivan Nag Zhi Fukuda Kitagishiand Matsuda Staubitz JanoueixLerosey Wei Yoshida Fukuda Honda 2017aWang Xie 2017aZhu Terawaki Cheng Olsvik Wang KrauŸ and Haucke Raib KIF3AB Rab4A and adaptor protein3 AP3 to diï¬erentiallyregulate tyrosinaserelated protein1 and tyrosinase sorting inendosomes contributing to melanosome maturation Nag Table Moreover silencing the Rabip4™isoform ofRUFY1 was shown to promote outgrowth of plasma membraneprotrusions and to regulate the spatial distribution of lysosomesat their tips through an interaction with AP3 Ivan Figures 1B 4B RUFY1 is also capable of controllingcell migration by regulating integrin trafficking Vukmirica presumably via endocytosis In full agreementwith a role of RUFY1 in regulating endosomal dynamics asingle nucleotide polymorphism S705A in the rufy1 gene wasassociated with high blood glucose levels and type diabetesmellitus susceptibility in an exomewide association studyEWAS Yamada This result is consistent with theearly finding that Rabip4 expression leads to Glucose transporter Glut1intracellular retention Cormont Interestingly RUFY1 display a SH3binding motif œPxxPxPembedded in the FYVE domain and is able to interactingwith the epithelial and endothelialtyrosine kinase ETKand possibly regulates endocytosis through this interactionYang Another EWAS aiming to find earlyonsetAlzheimer™s Disease AD susceptibility genes identified RUFY1among genes involved in endolysosomal transport and knownto be important for the development of AD Kunkle Figure 4CRUFY2lungRUFY2 or Leucine zipper FYVEfinger protein LZFYVE is a kDa protein originally identified as an activating transcriptionfactor2 interactor embryogenesis Dunkelberg and GutierrezHartmann preferentially located in the nucleus andexpressed during After development RUFY2 expression remainshigh in the brainliver and the gastrointestinal tractYang RUFY2 displays two Nterminalleucinezipper domains as well as a Cterminal FYVEfinger domainAlthough it is likely to have a nuclear function at early stagesof embryonic development the presence of a FYVE domainsuggests a cytoplasmic role for RUFY2 in regulating membranetraffic in fully diï¬erentiated cells Importantly the RUN domainof RUFY2 was shown to associate specifically with the Golgicomplexassociated Rab33A Fukuda Table GivenFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cChar and PierreRUFYs Proteins and Traffickingthe reported interaction of Rab33A and Rab33B with Atg16Land its putative role in regulating autophagy Fukuda and Itoh RUFY2 could contribute to autophagosome formationthrough a dual interaction with Rab33A and PtdIns3P onomegasomes Figures 1B 4B Irrespective of its function rufy2expression is subject to modulation by the micro RNA miR155BofillDe Ros which is an important regulator ofimmune cells development and inflammatory responses Ceppi The rufy2 gene is also frequently found mutatedin cancer cells with the most frequent mutations convertingit into a strong target for nonsense mediated mRNA decaythereby decreasing considerably its expression Shin Figure 4CRUFY3RUFY3 also known as Rap2interacting protein X RIPXKukimotoNiino or Single AxonRelated Singar1Mori is the best characterized member of theRUFY family RUFY3 the smallest of the RUFY proteins witha molecular weight of kDa A is mostly expressedin neurons Kitagishi and Matsuda Neuronal RUFY3 isatypical since it lacks a FYVE domain and is considered as partof the RUFY family based on strong sequence similarities withthe other members notably in the RUN and coiledcoil domainsFigure 2A RUFY3 is distributed between the cytosol and at theplasma membrane but not in intracellular vesicles presumablybecause it lacks a FYVE domain In artificial conditions likefollowing expression of the dominant gain of function mutantform of Rab5 Q79L in U937 cells RUFY3 was found associatedin large vesicle structures and to coimmunoprecipitate withRab5 via an interaction with its carboxyl terminal domain andsurprisingly not its RUN domain Yoshida LikeRUFY2 RUFY3 was also shown in a 2hybrid screen and bycoimmunoprecipitation to bind Rab33 through its coiledcoildomain CC1 Fukuda In 293T and 3Y1 celllines however RUFY3 was shown not to interact with severalsmall GTPasesincluding Rab2 Rab5 Rab7 Rho and RasThis suggests that either RUFY3 requires cell specific partnerproteins or posttranslation modifications to be able to bind tosmall GTPases RUFY3 was first described as interacting withRap2 a small Raslike GTPase via a residue fragment “ located in the RUN domain JanoueixLerosey KukimotoNiino Table Together with Rap1 Rap2interacts with Ras eï¬ectors such as Raf PI3K and Ral guaninenucleotide dissociation stimulator inhibiting activation of theirdownstream targets and thus suppressing Ras oncogenic activityKukimotoNiino Nussinov In the adultnervous system Rap1 and Rap2 also regulate the maturationand plasticity of dendritic spine and synapses By forminga complex together with Rap2 and Fascin RUFY3 interactswith the filamentous actin network and controls the growth ofaxons and neuronal growth cone Wei Table Recent mechanistic studies indicate that RUFY3 accumulates inlipid rafts by forming a Glycoprotein M6A GPM6aRUFY3Rap2STEFYial2 complex Honda 2017a Table This complex activates the Rac guanine nucleotide exchangefactor Honda 2017b impacting actin anization andpromoting neuronal polarity and growth Figure 4B RUFY3seems therefore to have diï¬erent axogenic functions in brainMori Honda 2017b and not surprisingly rolesfor RUFY3 in amyotrophic lateral sclerosis Arosio major depressive disorder Aberg and AD Zelaya have been reported Olfactory dysfunction occurs in of AD cases and is correlated with elevated rufy3 expressionin glomerular and mitral layers of the olfactory bulb Zelaya RUFY3 is cleaved by caspase and critically required forcaspasemediated degeneration of tropomyosin receptor kinaseA positive sensory axons in vitro and in vivo Hertz Figure 4C Removal of neuronally enriched RUFY3 is able toblock caspase 3dependent apoptosis while dephosphorylation ofRUFY3 at residue S34 appears required for its degradation Hertz Analysis of rufy3deficient mice supports a seconddistinct function for RUFY3 in neuronal growth and polaritysince mutant embryos show defects in axonal projection patternsThese occur in addition to the prevention of CASP3dependentapoptosis in dorsal root ganglions RUFY3 appears therefore to bekey for nervous system development remodeling and functionexplaining the embryonic lethality displayed upon rufy3 geneticinactivation in mouse Hertz With the current advance in genomics and single cell RNAsequencing specific gene expression patterns can be revised andmore accurately defined Analysis of several genomic databasesBioGPS NCBI Human Atlas Protein ImmGen Ensembl revealthat in addition to neurons RUFY3 expression can be detectedin other tissues and cell types Moreover the rufy3 gene appearsto have many transcriptional variants leading to the expressionof diï¬erent protein isoforms Two of these isoforms display aCterminal region extended by amino

Thyroid_Cancer

inhibitor with Temozolomide results in significant apoptosis in glioblastoma via the p65 and actin cytoskeleton regulatory pathwaysNaze G Avci1 Sadaf Ebrahimzadeh‘Pustchi1 Yasemin M Akay1 Yoshua Esquenazi2 Nitin Tandon Jay‘Jiguang Zhu Metin Akay1Glioblastoma GBM is the most malignant brain tumor characterized by intrinsic or acquired resistance to chemotherapy GBM tumors show nuclear factor‘κB activity that has been associated with tumor formation growth and increased resistance to therapy We investigated the effect of inhibitor BAY ‘ with Temozolomide TMZ on the signaling pathways in GBM pathogenesis GBM cells and patient‘derived GBM cells cultured in 3D microwells were co‘treated with BAY ‘ and TMZ or BAY ‘ and TMZ alone and combined experiments of cell proliferation apoptosis wound healing assay as well as reverse‘phase protein arrays western blot and immunofluorescence staining were used to evaluate the effects of drugs on GBM cells The results revealed that the co‘treatment significantly altered cell proliferation by decreasing GBM viability suppressed pathway and enhanced apoptosis Moreover it was found that the co‘treatment of BAY ‘ and TMZ significantly contributed to a decrease in the migration pattern of patient‘derived GBM cells by modulating actin cytoskeleton pathway These findings suggest that in addition to TMZ treatment can be used as a potential target to increase the treatment™s outcomes The drug combination strategy which is significantly improved by inhibitor could be used to better understand the underlying mechanism of GBM pathways in vivo and as a potential therapeutic tool for GBM treatmentGlioblastoma multiforme GBM is the most malignant primary brain tumor in the central nervous system Current standard of care therapy includes surgery followed by radiotherapy and concomitant and adjuvant chemotherapy with the alkylating agent Temozolomide TMZ which provides survival benefits for patients with GBM1 However even with the advances in surgical resection combined with TMZ therapy and irradiation the prognosis for newly diagnosed GBM patients remains poor In fact due to its rapid proliferation increased invasion and migration capacity and chemoresistance to the alkylating agents a0the median survival is only a0months with the ˜Stupp™ regimen radiation with daily TMZ — “ a0weeks followed by cyclic TMZ2 and 5year survival rate is less than which is the lowest longterm survival rate of malignant brain tumors3“ TMZ methylates DNA at the O6 positions of guanine and DNA repair enzyme O6methylguanine methyltransferase MGMT removes alkyl groups from O6 position of guanine in DNA making cells resistant to TMZ6 Therefore new therapies are necessary to prevent cell proliferation and induce apoptosis for GBM patientsNuclear factorkappa B NFκB is a regulatory transcription factor of the Rel gene family including p50 cRel RelB or p65 subunits It is involved in the control of tumor cell proliferation migration immune response and apoptosis7“ Studies have shown that NFκB gene was involved in the regulation pathways of different cancer types such as thyroid cancer head and neck squamous cell carcinoma and colorectal cancer711“ Increased 1Department of Biomedical Engineering University of Houston Cullen Blvd Houston TX USA 2UTHealth Neurosurgery McGovern Medical School Memorial Hermann at Texas Medical Center The University of Texas Health Science Center at Houston Houston TX USA email makayuheduScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cactivation of NFκB has also been identified in GBM tumors where the expression of NFκB was much higher in GBM tissue compared with nonGBM tissue1415 NFκB also promotes chemoresistance to TMZ and regulates MGMT activity in GBM by promoting MGMT gene expression through NFκB binding sites within the MGMT promoter16 NFκB inhibitors such as parthenolide do not completely eradicate tumors therefore they are mostly used in combination with other drugs17 When used in combination with TMZ NFκB inhibitor parthenolide has been shown to activate mitochondrial apoptosis signaling in U87MG and U373 GBM cells which lead to cell death18 and had a combined effect on cell cytotoxicity in LN18 and T98G glioma cells19 NFκB inhibitor CBL0137 has been shown to bind DNA leading the functional inactivation of the Facilitates Chromatin Transcription FACT complex a chromatin remodeling complex regulating transcription replication and DNA repair2021 In a0vitro evaluation of the CBL0137 on FACT p53 and NFκB has been done using U87MG and A1207 GBM cells It was shown that CBL0137 induced loss of chromatinunbound FACT activated p53 and inhibited NFκB dependent transcription21 In a0vivo studies showed that CBL0137 was effective in increasing survival rates in TMZresistant orthotopic mouse models21 Moreover Wang et a0al indicated that NFκB inhibitor BAY suppresses the expression of MGMT and enhances the TMZinduced apoptosis in TMZ resistant U251 cells22 However there is still a lack of characterization of the precise pattern of NFκB activation in combination with TMZ in GBM cell populations that have been a0surgically resected from patientsIn vitro and in a0vivo identifications and validations of molecular targets of GBM are important as they can progress into clinical studies Studies reported that combining multiple gene targets may prevent tumor growth and improve the treatment strategy for GBM23“ Both Bay and TMZ exert antitumoral activities individually in different tumor types28“ Therefore in this study we aimed to analyze functionally the combined effect of Bay and TMZ in different GBM cells For this purpose first we used our 3D PEGDAbased hydrogel microwell platform31“ to provide reliable preclinical models that can recapitulate in a0vivo features of the GBM tumors We cultured GBM cells U87 and LN229 and patientderived GBM cells in 3D microwells for a more precise and personalized treatment approach We then treated GBM cells with Bay and TMZ in combination or alone Our results indicated that the cotreatment of Bay and TMZ significantly reduced cell viability in all three cell lines in correlation with a significant decrease in the spheroid size The levels of NFκB protein and its subunits p65 and p50 were also significantly decreased compared with the control and single drug applications Similar a0decreases in the cell viability and protein levels were observed in all three GBM cells Tumor biopsy samples could give more realistic information about how tumors respond to drugs when they are used for in a0vitro or in a0vivo studies35“ Therefore we decided to continue our experiments with only using our patientderived GBM cells We treated patientderived GBM cells with Bay and TMZ or alone and analyzed specific cellular proteins along with their posttranslational modifications via reversephase protein arrays RPPA to elucidate the mechanism of action of the proteins3839 We observed that several cell signaling pathways including cell metabolism proliferation apoptosis were significantly affected by the combination of the drugs which were consistent with the literature4041 Furthermore our RPPA data revealed that there was a significant change in the modulation of actin cytoskeleton and following experiments including western blot analysis for the expression of FAK protein and wound healing assay for cell migration patterns confirmed the RPPA results We observed a significant decrease in both actin fluorescence intensity and migration pattern in the a0cotreated patientderived GBM cells To the best of our knowledge the effect of cotreatment of Bay and TMZ has never been studied previously on the actin modulation of patientderived GBM cells These results suggested that Bay and TMZ induced alteration in the a0actin filament anization by reducing the level of focal adhesion protein which might implicate in cell apoptosis The effect of Bay with TMZ necessitates further exploration to better understand its mechanism of action in GBM and potential therapeutic tools for GBM treatmentResultsCo‘treatment of Bay ‘ and TMZ reduced viability of GBM cells We used our previously a0published data to select the most effective drug concentrations for this study42 We cultured LN229 U87 and patientderived cells in the microwells for a0days where they formed 3D spheroids and we added a0µM of Bay and a0µM of TMZ in combination or alone Then we cultured the spheroids for more days with or without drug Control group did not receive any treatment The cell viability assay was performed on day after drug administration The results showed that the a0cotreatment significantly reduced cell viability of GBM cells LN229 and U87 and patientderived GBM cells cultured in 3D PEGDA microwells respectively as shown in Fig a01ac When they were used alone TMZ reduced cell viability to and p and Bay reduced cell viability to and in LN229 U87 and patientderived GBM cells respectively compared to control groups Fig a01d However when they were used in combination the viability of the cells significantly decreased to and in LN229 U87 and patientderived GBM cells respectively compared to control groups p Fig a01d Tumor cells are generally less sensitive to drug treatments in 3D cultures than in 2D cultures4344 This could reflect reduced compound access or differences in the response to cell death To confirm that cotreatment was more effective compared to single drug use we quantified the size of the spheroids using ImageJ45 Our data showed that after a0days of drug treatment the spheroids™ sizes were significantly reduced in the cotreatment by and in LN229 Fig a01e U87 Fig a01f and patientderived GBM cells p Fig a01g respectively compared to control group p When we compared the spheroids™ sizes of the cotreatment with TMZ alone there was a reduction of and in LN229 U87 and patientderived GBM cells respectively p Finally the spheroids™ sizes of the cotreatment compared with Bay alone showed a decrease of and in LN229 U87 and patientderived GBM cells respectivelyScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Representative images of the GBM tumor cells cultured in the PEGDA microwells a“c LN229 U87 and patientderived GBM cells were cultured in the microwells for a0days respectively After day Bay and TMZ were applied either alone or in combination onto the cell spheroids Control group did not receive any treatment The cells were cultured with or without drugs additional more days The images were taken on Day Day and Day after the drug application to observe the disruption in the spheroids Dotted black lines represent the edge of the tumor spheroid Scale bars a0µm d Bar graph showing trypan blue staining for cell viability of LN229 U87 and patientderived GBM cells e“g Spheroid size quantification was done using ImageJ for LN229 U87 and Patientderived GBM cells respectively Twotailed ttest followed by Wilcoxon test were done GraphPad Prism v5 Data represent the mean ± SD of three biological replicates p and p Suppression of activity in GBM cells by co‘treatment of Bay ‘ and TMZ As a readout of NFkB activity after drug treatment we first quantitatively assessed the cytoplasmic activation of phosphorylated NFκB p65 subunit in both treated and untreated groups in all GBM cells NFκB pp65 subunit activity was observed in the control groups of all three GBM cells Fig a02a NFκB pp65 subunit activity decreased to and when TMZ applied alone and and when Bay was applied alone in LN229 U87 and patientderived cells respectively However the decrease in NFκB pp65 subunit was reduced to when LN229 U87 and patientderived cells respectively were cotreated p Fig a02a Bay specifically inhibits NFκB activation by blocking phosphorylation of IκBα46 In independent experiments we analyzed the abundance of phosphorylated NFκB p65 NFκB p50 and IκBα in all three GBM cells Qualitative and quantitative western blot analysis revealed that the exposure to Bay with TMZ significantly downregulated the abundance of NFκB p65 NFκB p50 and IκBα compared with control and Bay or TMZ alone Fig a02b Please note that loading controls were used for each experiment but only the representative loading control for p and tP65 and p and tP50 was presented Fig a02b The cell viability assay cells™ size and protein expressions in all three GBM cells revealed similar results without any dramatic change Therefore considering the importance of using patientderived tumor cells to elucidate the mechanism of drugs and respective signaling pathways35“ we further continued our experiments using patientderived GBM cellsApoptosis was promoted by co‘treatment of Bay ‘ and TMZ RPPA technology is designed for multiplexed antibodybased relative quantification where each array is tested with a validated antibody specific to a particular protein along with their particular posttranslational modifications47 In the attempt to elucidate the mechanism of action of Bay with TMZ by which NFκB subunits were modulated and to identify downstream signaling molecules we employed RPPA platform using our drug treated or untreated patientderived GBM cells RPPA results showed that many oncogenic pathways were altered by the drug treatments but more specifically by the cotreatment Fig a03a Decreased expression of NFκB was not only associScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 NF“kB activity in LN229 U87 and patientderived GBM cell lines a NF“kB p65 subunit activity in LN229 U87 and patientderived GBM cell lines respectively The cells cultured with or without drugs for a0days were collected from the microwells and subjected to ELISA Data represent the mean ± SD of three biological replicates p and p b Representative immunoblots LN229 U87 and patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels bottom panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p ated with changes in the a0NFκB pathway but also with apoptosis cell metabolism and proliferation which were confirmed by the analysis of downregulated RPPA proteins in Enrichr KEGG libraries4849 Fig a03c p One of the specific pathways given by RPPA was apoptosis Apoptosis is one of the important mechanisms that regulates cell death and suppress tumorigenesis Studies have demonstrated that Bcl2 family proteins can positively and negatively regulate apoptosis by regulating antiapoptotic protein Bcl2 and proapoptotic protein Bax4050 Our RPPA data using patientderived GBM cells showed that the fold change of Bcl2 relative to control was times higher in cotreated group TMZ alone Bay alone respectively Fig a03b In order to further confirm whether the expression of a0these proteins were downregulated by the cotreatment we performed western blot analysis Our results showed a similar decrease in Bcl2 protein expression in the cotreatment compared with the control and single drug a0treatment Fig a03d In contrast Bax protein fold change relative to control was times higher in cotreated group TMZ alone Bay respectively where we observed a significant increase after the cotreatment of Bay with TMZ compared with the control p Fig a03b Bcl2Bax ratio is a key indicator in susceptibility of the cells to apoptosis Western blot results confirmed the change in Bcl2Bax ratio in the cotreatment compared with the control group and single a0drug treatment Fig a03d Our RPPA data also showed a significant increase in the cleavedcaspase protein Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 The effect of Bay and TMZ on signaling pathways in patientderived GBM cells a Heat map presentation of RPPA analysis showing the changes in the protein expression RPPA was performed on lysates treated with Bay and TMZ alone or in combination All relative protein level data points were normalized to the a0control group Red and green indicate up and down regulations respectively in the heat map The samples were run in duplicate n b Fold change of the a0selected proteins relative to the a0control group via RPPA Data represent the mean ± SD of two biological replicates p p Wilcoxon rank sum test c Analysis of downregulated RPPA proteins shows a a0significant activation in numerous Enrichr KEGG pathways The pathways were a0sorted by p value ranking d Representative immunoblot validation of significantly altered proteins involved in different KEGG pathways Patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p fold change relative to control times higher in the cotreatment compared with times higher in TMZ alone and times higher in Bay alone p Fig a03b To confirm if cotreatment triggered apoptosis correlated with caspase activation we performed western blot analysis with procaspase3 cas3 and cleavedcaspase3 Ccas3 We observed that Bay and TMZ induced apoptosis was associated with cas3 Fig a03d Please note that loading controls were used for each experiment but only the representative loading control for Bax cas3 and Ccas3 was presented Fig a03d Moreover another important mechanism of NFκB activation in GBM regulates through AKT phosphorylation of IκB Our RPPA data showed relative fold changes of in the cotreated group TMZ alone and Bay alone respectively p Fig a03b The western blot results also confirmed a significant decrease in the abundance of AKT pT308 Fig a03dTo further investigate whether cotreatment of Bay with TMZ can lead to glioma cell apoptosis and to confirm our RPPA and western blot results we performed apoptosis assay TUNEL The patientderived GBM cells were cotreated with Bay with TMZ or single drug treated and subjected to TUNEL assay to detect DNA damage Fig a04a The results indicated that TUNEL cells in the cotreatment were increased tenfold compared with control and and 24folds compared with TMZ alone and Bay alone respectively p Fig a04b Additionally in some TUNEL cells we observed a typical ring type chromatin aggregation underneath the nuclear membrane which suggested an early stage apoptosis51 Fig a04a red arrows There were also a few TUNEL cells that lacked the typical apoptotic ringlike nuclear structure indicating that they were either at a different stage of apoptosis or alternatively undergoing necrosis52 that we have not investigated furtherCo‘treatment of Bay ‘ with TMZ changed actin anization by inhibiting FAK phosphorylation and cell migration Actin filaments Factin are one of the main components of the cellular cytoskeleton which regulates actin dynamics and migration process in the cells The disruption of the actin cytoskeleton inhibits cell migration and adhesion53 Depolymerization or cleavage of actin lamins and other cytoskeletal proteins have been also found to be involved in cell apoptosis54“ To confirm the RPPA results showing changes in the actin modulation pathway and to understand the mechanism that regulates cytoskeletal Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Apoptosis assay TUNEL a Fluorescent images of TUNEL cells in patientderived GBM cells TUNEL assay was performed on cells treated with Bay and TMZ in combination or alone in the microwells Cells were collected from the microwells trypsinized and replated into 8well chamber slides TUNEL cells green with ringlike nuclear stain are indicated with red arrows Nuclei were counterstained with DAPI blue b Numbers of TUNEL cells are presented as mean ± SD of three biological replicates p and p X20 objective Scale bars a0µmanization we treated patientderived GBM cells co treated with Bay with TMZ or single drug treated 3D spheroids collected from the microwells were stained with phalloidin green and DAPI blue Staining cells with fluorescently conjugated phalloidin is considered the most reliable method of accurately labeling Factin in fixed cells57 In the control group intact cells formed finemeshed networks with a distinct Factin anization on both day Fig a05a upper panel and day Fig a05a bottom panel In single drug treated cells actin was still found to be polymerized to filaments as it can be seen by its interaction with phalloidin at both days and However the cells which were cotreated with Bay and TMZ lost their Factin anization and their shape compared with the control and the single drug treated groups at day Fig a05a bottom panel Changes in the a0actin distribution within the cells were quantified by measuring the staining intensity using Fiji Macro ImageJ as described previously5859 At day we observed a a0significant decrease in the fluorescence intensity of phalloidin when the cells were cotreated with Bay and TMZ compared with the a0control and single drug treated groups p Fig a05b To investigate the drug related Factin mechanism we examined the levels of FAK protein following cotreatment or single drug treatment As shown in Fig a05c cotreatment significantly decreased the level of phosphorylated FAK compared with both control and single drug applications p Furthermore we investigated cell migration patterns of the patientderived cells that were cotreated with Bay and TMZ or single drug treated We collected 3D spheroids from microwells after drug treatment and replated them in 24well plate to perform scratch wound healing assay We noted a significant increase in cell density in the scratch area in both control and Bay alone after and a0h of scratch formation p Fig a06a Although compared with the a0control cells both cotreatment and TMZ alone groups showed a decrease in the cell migration into the scratch area after a0h we observed that after a0h the migration rate of the cotreated cells was significantly slower than the cells that were treated with TMZ alone p Fig a06b These results indicated that the disanization of actin microfilaments was concomitant with the cell apoptosis after the a0cotreatment of Bay with TMZDiscussionDespite the increase in the median survival of GBM patients from to months4 the clinical efficacy of standard of care therapy including TMZ chemotherapy combined with surgery and radiotherapy is still limited Due to challenges in treating GBM significant attempts have been made to develop single or combined drug treatments60“ However given the cost long time frame and risks of failure associated with developing a new drug repurposing available drugs may be the most effective alternative therapeutic strategy Therefore it is important to evaluate potential drug combinations for GBM treatmentScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Changes in the actin cytoskeleton and migration pattern in patientderived GBM cells cotreated with Bay and TMZ or single drug treated in the microwells a Upper panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin green and DAPI blue Bottom panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin and DAPI Scale bars a0µm b Intensity of staining obtained with phalloidin was measured in each cell using ImageJ and displayed as boxplots with to confidence intervals A twoway ANOVA with Dunnett™s multiple comparisons test was performed to determine statistical relevance Three biological replicates n p p c Representative immunoblots show the levels of FAK pTyr397 and total FAK in patientderived GBM cell lysates cotreated with Bay and TMZ or single drug treated for a0days in the microwells The levels of the proteins were quantified using ImageJ right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p Due to the cell repellent property of PEGDA hydrogel tumor cells can form aggregates at the bottom of the microwells and selfassemble into spheroids in each well within a0days following cell seeding313363 Compared with 2D monolayer cell culture 3D spheroids have an important advantage their larger size Thus often drug effects can easily be monitored over time by measuring the size and shape of spheroids4344 Additionally using 3D in a0vitro tumor models can better recapitulate in a0vivo features of the tumors We used PEGDA hydrogelbased microwell platform313363 in order to culture different types of a0GBM cells commercially available GBM cell lines LN229 U87 and a0patientderived GBM cells However we investigated the effect of the drugs on the patientderived GBM cells more in detail since growing tumors from tumor biopsy samples could give very detailed information about how tumors respond to drugs35“ Considering the precious nature of the patient samples this platform which requires fewer cells compared with 2D monolayer cultures provides us with a robust tool to recapitulate in vivo features of GBM tumors and to test our drug combinationsNFκB is one of the major transcription factors associated with GBM and responsible for activating a series of cellular responses including cell proliferation survival invasion and apoptosis6465 Previous studies have shown that NFκB can activate Akt and promote cell survival and proliferation by downregulating the expression of phosphatase and tensin homolog deleted on chromosome ten1866 NFκB pathway can inhibit cell apoptosis by inhibiting a stressactivated protein kinase and a mitogenactivated protein kinase signaling pathway67 It can also be activated in response to treatment with cytotoxic drugs such as vinca alkaloids and topoisomerase inhibitors Several studies have demonstrated the activation of NFκB in GBM patientderived stemlike cells cultures96869 Moreover alkylating agents TMZ can activate NFκB through DNA damage pathway activation7071 The combination effect of Bay and TMZ have been showed in our previous study where we determined the most effective drug concentrations on GBM cells using our microfluidics platform42 Another study that investigated the combined effect of NFκB inhibitor BAY with TMZ showed that combined drug application induced TMZ resistant in U251 GBM cells22 However the characterization of the precise pattern of NFκB activation in different GBM cell populations from surgically resected tissues still remains elusive Therefore in this study we investigated the interaction of Bay with TMZ and their effects on the LN299 and U87 GBM cell lines as well as patientderived GBM cells in order to recapitulate NFκB activation as in a0vivo features of the GBM and its signaling pathways We applied a0µM of Bay and a0µM of TMZ3442 in combination or alone for all three GBM cell types First we observed a significant decrease in both cell viability and size of the spheroids in the cotreatment compared with control and single drug application Then we showed quantitatively and Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Cell migration of patientderived GBM cells by wound healing assay a Patientderived cells were cotreated with Bay and TMZ or single drug treated in the microwells trypsinized and replated in 24well plates After they reached to their confluency a scratch wound was formed with a 200μl tip and cells were incubated for the next a0h Images were taken 4x at a0h a0hr and a0hr Scale bars a0µm b The wound width was measured with ImageJ and the average wound width was shown Data represent the mean ± SD of three biological replicates p and p oneway ANOVA with Tukey™s post hoc testqualitatively the expression of NFκB in all three GBM cell types a0We noted a significant decrease in the cotreated group compared with control and single drug application Our western blot data also confirmed the decrease in the abundance of pP65 pP50 and pIKBa that Bay has been shown to inhibit its phosphorylation46 However in the cotreated group the decrease was significantly higher compared to both control and single drug application This data showed that cotreatment of Bay and TMZ has more effect on the inhibition of NFκB pathway than Bay or TMZ alone and suggests a a0decreased downstream transcription of oncogenic proteins72 Although there were slight differences in the NFκB expression patterns in three different GBM cell types a0we focused on the patientderived cells in the rest of the study due to their ability to better recapitulate the genomic similarities to primary disease7374Proteins that interact with each other activate multiple pathways which can result in apoptosis according to tissue type and pathological condition Glioblastoma tumors express high levels of antiapoptotic BCL2 family proteins such as Bcl2 and BclxL which may cause glioblastoma cells to resist apoptosis75 The proapoptotic members of Bcl2 family such as Bax and Bak are necessary for their proapoptotic effect Interactions and the ratio between antiapoptotic Bcl2 and proapoptotic Bax are decisive factors in the induction of apoptosis7677 Active NFκB can prevent cells from apoptosis by stimulating the expression of genes and promoting cell proliferation Although patientderived GBM samples have been shown to be highly resistant to apoptosis77 our data revealed changes in the expression of various members of Bcl2 family and NFκB signaling pathway after cotreatment of Bay and TMZ Our RPPA results outlined distinct molecular profiles in which apoptotic P53 signaling and NFκB signaling pathways were significantly affected after the a0cotreatment These results supported that the inhibition of NFκB expression could inhibit the expression of Bcl2 and promote the expression of Bax thus promote apoptosis Our data also suggested the possible interaction between Bcl2 and p53 in Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Proposed schematic of the a0signaling pathways involved in Bay and TMZmediated inhibition in GBM patientderived cells The effect of combined therapy of Bay and TMZ was achieved through the inhibition of SrcFAKVinculin which regulate the cytoskeleton anization through MAPKs JNK and PI3KAKT signaling pathways Exposure to both Bay and TMZ also leads to receptormediated activation of Bax but not Bcl2 in the subsequent inhibition of the downstream NFκB transcription factor Inhibition of NFκB in turn causes cell deathregulating cell survival and death7778 The activation of extrinsic and intrinsic molecular pathways can lead to the proteolytic activation caspases The extrinsic pathway is triggered by proapoptotic ligands that activate cell surface death receptors and procaspase8 which in turn leads to the cleavage of caspase3 and apoptosis79 Our results determined that the a0cotreatment significantly inhibited the expression of caspase3 while the expression of cleaved caspase3 was increased Additionally TUNEL assay which detects DNA strand breaks which could occur as an event in the apoptosis showed a dramatic increase in the TUNEL cells after the cotreatment compared with the a0control and single drug application Altogether these results suggested that the inhibition of cell proliferation Bcl2 and caspase3 by a0the cotreatment of Bay and TMZ may occur through the NFκB mediated apoptosis and they might be tightly coupled8081The literature provides evidence that supports crosstalk between PI3KAktmTOR signaling pathway and NFκB which is downstream of Akt NFκB activation in GBM regulates through AKT phosphorylation of IκB resulting in an activated NFκB that translocates to nucleus8283 Our data showed that when Bay was used with TMZ there was a decrease in the abundance of PI3Kp110 AktpS473 AktpT308 and mTORpS2448 This preliminary data is important to suppo

Thyroid_Cancer

"Long non‘coding RNA FOXD2‘AS1 regulates the tumorigenesis and progression of breast cancer via the S100 calcium binding protein A1Hippo signaling pathwayPEI HUANG1 and JINHUI XUE21Department of Pathology The First Affiliated Hospital of Zhengzhou University Zhengzhou Henan 2Department of Pathology Zhengzhou Central Hospital Affiliated to Zhengzhou University Zhengzhou Henan PR ChinaReceived November Accepted April 103892ijmm20204699Abstract Breast cancer is one of the most prevalent cancer types and is accompanied by a high incidence and mortality rate severely threatening women's health globally Long noncoding RNA forkhead box d2 adjacent apposite strand RNA lncRNA FOXD2‘AS1 has been identified to function as an oncogene in human cancers however it has rarely been investigated in breast cancer The aim of the present study was to investigate the role of FOXd2AS1 in breast cancer and to clarify the underlying mechanisms The expression of FOXD2‘AS1 in breast cancer cell lines was first quantified by reverse transcription‘quantitative PCR and the biological function of FOXD2‘AS1 was then determined Cellular proliferative ability was determined by Cell Counting kit‘ assay and wound healing and Transwell assays were conducted to assess the cell migratory and invasive ability corresponding protein expression levels were determined by western blot analysis In addition experimental animal models were established by the subcutaneous injection of MDA‘MB‘ cells into the right axillary lymph nodes of BALBc nude mice and the effects of FOXD2‘AS1 on tumor growth were observed The results indicated that FOXD2‘AS1 expression was upregulated in breast cancer cell lines and that FOXD2‘AS1 downregulation significantly inhibited the proliferation migration and invasiveness of MCF‘ and MDA‘MB‘ cells S100 calcium binding protein A1 S100A1 was also upregulated in breast cancer cell lines and was positively regulated by FOXD2‘AS1 Furthermore the inhibition of S100A1 and the overexpression of the serinethreonine‘protein kinase large tumor suppressor homolog LATS1 inhibited the FOXD2‘AS1‘induced Correspondence to Dr Pei Huang Department of Pathology The First Affiliated Hospital of Zhengzhou University East Jianshe Road Zhengzhou Henan PR ChinaE‘mail huangpei193163comKey words FOXD2‘AS1 S100 calcium binding protein A1 breast cancer large tumor suppressor homolog Hippocellular proliferation migration and invasiveness in breast cancer Experimental mouse models revealed that FOXD2‘AS1 downregulation significantly inhibited tumor growth and that the levels of phosphorylated p‘YAP and p‘LATS1 were upregulated by FOXD2‘AS1 knockdown indicating that the inhibition of FOXd2AS1 activated Hippoyesassociated protein signaling On the whole the findings of the present study suggest that the FOXD2‘AS1S100A1Hippo axis is involved in the tumorigenesis and progression of breast cancer In the future these may contribution to the identification of more effective breast cancer treatmentsIntroductionAs one of the most prevalent malignancies breast cancer is a primary cause of mortality among gynecological cancer cases and with increasing morbidity and mortality rates it poses a considerable threat to women's health worldwide In statistics from the American cancer Society estimated newly diagnosed cases and deaths from breast cancer in the United States The leading causes of the high death rate are distal metastasis and resistance to the existing treatments despite improvements in early diagnosis and systemic treatment the incidence of breast cancer and metastasisrelated mortality is steadily increasing Therefore there is an urgent need to elucidate the mechanisms responsible for the disordered cellular metastasis and to enhance our understanding of the tumorigenesis and development processes hence facilitating the identification of more efficient breast cancer treatmentsLong noncoding RNAs lncRNAs are a group of RNAs nucleotides in length which lack protein‘coding capacity Numerous studies have revealed that lncRNAs have versatile biological functions in pathological and physiological processes including tumorigenesis ‘ lncRNAs are considered to regulate the development of various types of cancer including breast cancer For instance LINC01089 is downregulated in breast cancer tissues and cell lines and LINC01089 overexpression increases tumor cell proliferation migration and invasiveness As an oncogene that regulates breast cancer cell proliferation and apoptosis hepatocellular 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERcarcinoma upregulated EZH2‘associated lncRNA is closely associated with the clinical progression of breast cancer These results indicate the indispensability of research on lncRNAs and breast cancerlncRNA forkhead box d2 adjacent apposite strand RNA FOXD2‘AS1 is a novel non‘coding RNA identified to be an oncogene in human cancers FOXD2‘AS1 has been shown to be upregulated in various types of cancer including glioma osteosarcoma and papillary thyroid cancer as well as breast cancer ‘ A previous study indicated that FOXD2‘AS1 participates in regulating the development of breast cancer via the miR‘‘5pPFN2 axis and that it may be a potential biomarker for the diagnosis and prognosis of breast cancer However to the best of our knowledge there are no additional data regarding the investigation of FOXd2AS1 in breast cancer and its effects and the underlying mechanisms on the regulation of breast cancer cell invasion and metastasis Thus the aim of the present study was to determine the role and potential mechanisms of action of FOXd2AS1 in breast cancer and to provide further support for its use in clinical diagnosis and treatmentMaterials and methodsDatasets The present study evaluated the expression level of FOXD2‘AS1 in breast cancer samples using The Cancer Genome Atlas TCGA dataset which was downloaded from the TCGA data portal tcga‘data‘nci‘nih‘govezxjtlueducn The TCGA data subset for breast cancer included normal samples and tumor samples The Mann‘Whitney test was used to determine statistically significant differences between normal and tumor samples P005 was considered to indicate a statistically significant differenceCell culture A human normal breast epithelial cell line MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ were purchased from the American Type Culture Collection ATCC The cells were incubated in RPMI‘ medium supplemented with fetal bovine serum FBS in a humidified incubator at ˚C CO2 Transfection To overexpress FOXd2AS1 an overexpression vector pcdNA FOXd2AS1 and its corresponding negative control vector pcDNA‘NC were synthesized by Shanghai GenePharma Co Ltd Short hairpin shRNAs targeting FOXD2‘AS1 nM shRNA‘FOXD2‘AS1‘ and shRNAFOXd2AS12 and a negative scramble control shRNA shRNA also purchased from Shanghai GenePharma Co Ltd were used to knock down FOXD2‘AS1 expression In addition pcdNALATS1 shRNAS100A11 and shRNAS100A12 were obtained from Shanghai GenePharma Co Ltd to overexpress LATS1 or to knock down S100A1 respectively The shRNA sequences were as follows shRNA‘FOXD2‘AS1‘ targets GGA CTC CAC TCT TCG CTT A shRNA‘FOXD2‘AS1‘ targets GCT TCC AGG TAT GTG GGA A shRNA‘S100A1‘ targets GAT CCG GAG ACC CTC ATC AAC GTG TTC TTC CTG TCA GAA ACA CGT TGA TGA GGG TCT CCT TTT TG shRNA‘S100A1‘ targets GAT CCG TGG ACT TCC AGG AGT ATG TGC TTC CTG TCA GAC ACA TAC TCC TGG AAG TCC ACT TTT TG Cells were transfected with pcDNA FOXD2‘AS1 nM pcdNALATS1 nM pcdNANc nM shRNAFOXd2AS11 ngµl shRNAFOXd2AS12 ngµl shRNAS100A11 ngµl shRNAS100A12 ngµl shRNA ngµl or co transfected with pcdNA FOXd2AS1 and pcdNALATS1 or cotransfected with pcDNA FOXD2‘AS1 and shRNA‘S100A1 using Lipofectamine® transfection reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's instructions Lipofectamine reagent was first mixed with vectors to form a reagent‘vector complex followed by incubation with cells at ˚C for h The transfection efficacy was assessed by reverse transcription‘quantitative PCR RT‘qPCR after h of transfectionRNA extraction and RT‘qPCR Total RNA was extracted from all cell lines using TRIzol® reagent Takara Bio Inc according to the manufacturer's instructions Total RNA was then reverse transcribed into cDNA using the PrimeScript„¢ RT Master kit and the mRNA expression levels were quantified using the SYBR Premix Ex Taq„¢ kit Both Takara Bio Inc with a Fast Real‘Time PCR System Applied Biosystems Thermo Fisher Scientific Inc The sequences of specific primers used for RT‘qPCR were as follows FOXDA‘AS1 forward '‘TGG ACC TAG CTG CAG CTC CA‘' and reverse '‘AGT TGA AGG TGC ACA CAC TG‘' S100A1 forward '‘GAG TAT GTG GTG CTT GTG GC‘' and reverse '‘CTT GGA CCG CTA CTC TTG CG‘' large tumor suppressor homolog LATS1 forward '‘ACC GCT TCA AAT GTG ACT GTG ATG CCA C CT‘' and reverse '‘CTT CCT TGG GCA AGC TTG GCT GAT CCT CT‘' and GAPDH forward '‘GCG AGA TCG CAC TCA TCA TCT ‘' and reverse '‘TCA GTG GTG GAC CTG ACC ‘' The data were displayed as ‘ΔΔCq values with GAPDH as the constitutive marker The PCR conditions were as follows ˚C for min cycles of ˚C for sec and ˚C for sec followed by ˚C for minCell Counting Kit‘ CCK‘ assay cell proliferation was determined using the Cell Counting Kit‘ assay CCK‘ Dojindo Molecular Technologies Inc Briefly cells were seeded into a ‘well plate and incubated for h at ˚C Following culture for the indicated periods of time and h µl of the CCK‘ reagent were added to each well and the plate was incubated at ˚C for a further h The optical density values at nm were then measured using a microplate spectrophotometer Thermo Fisher Scientific IncWestern blot analysis The total protein was extracted from the cells using RIPA lysis buffer Beyotime Institute of Biotechnology and quantified using a BCA protein assay kit Thermo Fisher Scientific Inc The same amount of each protein sample µg was subjected to SDS‘PAGE the proteins were then transferred onto PVDF membranes EMd Millipore and was blocked in nonfat milk for h at room temperature The membranes were then incubated with primary antibodies against cyclinE1 cat no ab33911 Abcam cyclin‘dependent kinase‘ CDK2 cat no ab32147 Abcam p21 cat no ab109520 Abcam matrix metalloproteinases MMP2 cat no ab92536 Abcam MMP9 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE was added to the lower 24well chamber After h cells on the upper surface were removed and cells attached to the lower surface were stained with crystal violet Beijing Solarbio Science Technology Co Ltd at room temperature for min The cells were viewed under a light microscope magnification x100 CKX41 Olympus Corporation and the invasive ability of the cells was determined using ImageJ software version by counting the number of cells attached to the lower surfaceCell cycle analysis The cell cycle distribution was determined by flow cytometry After being subjected to the indicated treatments the cells were collected and fixed in ethanol at ‘ËšC overnight The cells were then washed twice with PBS and incubated in the dark with RNase A and PI staining solution Roche Diagnostics at ˚C for min Finally the cell samples were analyzed using a FACSCalibur flow cytometer and CellQuest software version both from BD BiosciencesIn vivo experiments The present study was approved by the First Affiliated Hospital of Zhengzhou University and the animal experiments were performed according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals A total of 5weekold male BALBc nude mice were purchased from the Experimental Animal Center of Shanghai Institute for Biological Sciences and housed in a standard environment ˚C humidity h lightdark cycle with free access to food and water Each mouse was subcutaneously injected at the right axillary lymph nodes with 10x107 MdAMB468 cells which were stably transfected with either the shRNA negative control shRNA or shRNA‘FOXD2‘AS1 The weights and tumor volumes tumor volume x length x width2 of the mice were monitored every days until the mice were sacrificed At days after the injection all the mice were sacrificed by cervical dislocation that caused a sharp section of the spinal cord followed by an instantaneous cardiac arrest After the cessation of the heartbeat and respiratory arrest of the mice was confirmed the tumors were excised photographed and stored for the further investigationStatistical analysis data are presented as the means ± standard deviation SD from ‰¥ independent experiments and each experiment was conducted in triplicate The data were analyzed using SPSS statistical software version SPSS Inc and the differences among groups were analyzed using one‘way analysis of variance followed by Tukey's post hoc test P005 was considered to indicate a statistically significant differenceResultsFOXD2‘AS1 expression is upregulated in breast cancer cells The TCGA database cancergovtcga was used to identify the association between FOXD2‘AS1 and breast cancer by evaluating the expression profiles of FOXD2‘AS1 in breast cancer tissues and normal tissues The results of TCGA analysis revealed a significantly higher FOXd2AS1 expression in breast cancer tissues than in normal tissues Fig 1A Human Figure FOXD2‘AS1 is upregulated in breast cancer cells A The Cancer Genome Atlas TCGA database cancergovtcga was used to identify the association of FOXD2‘AS1 with breast cancer by collecting the profiles of FOXD2‘AS1 in breast cancer tissues and normal tissues P0001 vs normal samples B mRNA level of FOXD2‘AS1 in human normal breast epithelial cell line MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ was determined by RT‘qPCR P001 and P0001 vs MCF‘10A cells cat no ab38898 Abcam S100 calcium binding protein A1 S100A1 cat no Cell Signaling Technology Inc phosphorylated p‘yes‘associated protein YAP cat no Cell Signaling Technology Inc YAP cat no Cell Signaling Technology Inc serinethreonine‘protein kinase LATS1 cat no Cell Signaling Technology Inc p‘LATS1 cat no Cell Signaling Technology Inc mammalian STE20‘like protein kinase MST1 cat no Cell Signaling Technology Inc MST2 cat no Cell Signaling Technology Inc and GAPDH cat no ab8245 Abcam at ˚C overnight The membranes were washed with Tris‘buffered saline with Tween TBST and incubated with horseradish peroxidase HRP‘conjugated goat anti‘mouse IgG secondary antibody cat no sc‘ Santa Cruz Biotechnology Inc at room temperature for h The protein bands were visualized using an enhanced chemiluminescence kit Amersham Pharmacia Biotech and quantified using ImageJ software version National Institutes of HealthWound healing assay The cellular migration rate was determined using a wound healing assay The cells were seeded into a ‘well plate and cultured to confluence A wound was produced in each monolayer using a ‘µl pipette tip and the plate was washed times with PBS to remove detached cells The cells were then cultured in the fresh medium without FBS Following incubation for h the wound‘healing ability was assessed under a light microscope magnification x100 CKX41 Olympus Corporation and the widths of the wounds were measured at and hTranswell assay The cell invasive rate was determined with a Transwell assay cells 4x104well in serum‘free medium were placed in the upper chamber of each insert [which had been precoated with µl of Matrigel Bd biosciences at ˚C for h] and complete medium containing FBS 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 suppresses cell proliferation migration and invasion in breast cancer A and B Following transfection with shRNA‘FOXD2‘AS the mRNA level of FOXD2‘AS1 was measured by RT‘qPCR in MCF‘ and MDA‘MB‘ cells C and D CCK‘ assay was performed to determine cell proliferation following transfection E‘G Cell cycle distribution was determined and analyzed by FACS H and I The protein expression of cyclin E1 CDK2 and p21 was determined by western blot analysisnormal breast epithelial cell line MCF‘10A and human breast cancer cell lines McF7 MdAMB468 MdAMB453 and BT549 were also obtained to detect the mRNA levels of FOXD2‘AS1 The results revealed that FOXD2‘AS1 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure Continued Knockdown of FOXD2‘AS1 suppresses cell proliferation migration and invasion in breast cancer J Wound healing assay was performed to detect the migration of both MCF‘ and MDA‘MB‘ cells K and L Relative migration rate of MCF‘ and MDA‘MB‘ cells was quantified respectively M Transwell assay was performed to detect the invasion of both McF7 and MdAMB468 cells N and O Relative cell invasive rate of McF7 and MDA‘MB‘ cells was quantified respectively P MMP‘ and MMP‘ protein expression in MCF‘ transfected with shRNA‘FOXD2‘AS1 was detected and quantified Q MMP‘ and MMP‘ protein expression in MDA‘MB‘ transfected with shRNA‘FOXD2‘AS1 was detected and quantified P005 P001 and P0001 vs shRNA‘NC CDK2 cyclin‘dependent kinase‘ MMP matrix metalloproteinase expression was markedly upregulated in all breast cancer cells particularly in the MCF‘ ER‘positive breast cancer cell line and MdAMB468 cells triplenegative breast cancer cell line Fig 1B which were used for further experiments even 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 regulates the HippoYAP signaling pathway A The protein expression of HippoYAP signaling pathway‘related genes p‘YAP YAP p‘LATS1 LATS1 MST1 and MST2 was determined by western blot analysis in MCF‘ cells B The protein expression of HippoYAP signaling pathway‘related genes p‘YAP YAP p‘LATS1 LATS1 MST1 and MST2 was determined by western blot analysis in MDA‘MB‘ cells P005 P001 and P0001 vs shRNA‘NC LATS1 large tumor suppressor homolog MST mammalian STE20‘like protein kinase YAP yes‘associated protein though there may be some variability in the results between the cell lines These findings indicate that FOXD2‘AS1 is upregulated in breast cancerFOXD2‘AS1 knockdown suppresses breast cancer cell proliferation migration and invasiveness To further elucidate the role of FOXd2AS1 in breast cancer FOXd2AS1 was knocked down in both the McF7 and MdAMB468 cells Due to a higher transfection efficacy shRNA‘FOXD2‘AS1‘ referred to as shRNA‘FOXD2‘AS1 was subsequently used for breast cancer cell experimentation Fig 2A and B The results of CCK‘ assay indicated that FOXD2‘AS1 knockdown significantly inhibited the proliferative ability of the MCF‘ and MdAMB468 cells Fig 2c and d The cell cycle was then analyzed by flow cytometry which revealed that FOXd2AS1 knockdown increased the percentage of cells in the G1 phase whereas it decreased that in the S phase for both the MCF‘ and MDA‘MB‘ cells Fig 2E‘G Furthermore FOXd2AS1 knockdown decreased the protein expression levels of cyclin E1 and CDK2 and increased the expression of 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure S100A1 is upregulated in breast cancer cells A The mRNA level of S100A1 in MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ was determined by RT‘qPCR P005 P001 and P0001 vs MCF‘10A cells B and C In shRNA‘FOXD2‘AS1‘transfected McF7 and MdAMB468 cells the protein expression of Sl00A1 was detected by western blot analysis P005 and P0001 vs shRNA‘NC D and E Following transfection with pcDNA‘FOXD2‘AS1 the mRNA level of FOXD2‘AS1 in MCF‘ and MDA‘MB‘ cells was detected by RT‘qPCR P001 and P0001 vs pcDNA‘NC MCF‘ and MDA‘MB‘ cells were transfected with shRNA‘S100A1 and the protein expression and mRNA level of S100A1 in F and G MCF‘ and H and I MDA‘MB‘ cells were determined by western blot analysis and RT‘qPCR respectively P001 and P0001 vs shRNA‘NC S100A1 S100 calcium binding protein A1 p21 Fig 2H and I These findings indicate that FOXD2‘AS1 knockdown suppresses cellular proliferation by regulating the cell cycle specifically by preventing G1 to S phase progression Moreover FOXD2‘AS1 knockdown significantly decreased the migration rate Fig 2JL and the invasiveness Fig 2MO of the McF7 and MdAMB468 cells The 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure S100A1 mediates FOXD2‘AS1‘induced cell proliferation migration and invasion in breast cancer Following transfection with pcDNA‘FOXD2‘AS1 cells were transfected with shRNA‘S100A1 A and B CCK‘ assay was performed to determine the proliferation of the differently treated cells C Migratory ability of MCF‘ cells in the different treatment groups was determined by wound healing assay D Invasive ability of MCF‘ cells in the different treatment groups was determined by Transwell assay E Migratory ability of MDA‘MB‘ cells in the different treatment groups was determined by wound healing assay F Invasive ability of MDA‘MB‘ cells in the different treatment groups was determined by Transwell assay P0001 vs pcDNA‘NC P001 and P0001 vs FOXD2‘AS1 shRNA‘NC S100A1 S100 calcium binding protein A1 expression levels of MMP‘ and ‘ which are critical to the migration invasion and metastasis of breast cancer cells were both downregulated following FOXD2‘AS1 knockdown Fig 2P and Q indicating that FOXD2‘AS1 may enhance cellular migration and invasiveness by regulating MMP‘ and ‘FOXD2‘AS1 knockdown regulates the HippoYAP signaling pathway The HippoYAP signaling pathway is reportedly involved in the progression of breast cancer In the present study the levels of specific proteins involved in the YAPHippo signaling pathway were assessed in the MCF‘ and MdAMB468 cells following FOXd2AS1 knockdown Western blot analysis revealed that the levels of p‘YAP and p‘LATS1 were significantly upregulated while those of MST1 and were significantly downregulated by FOXD2‘AS1 knockdown Fig 3A and B Thus the results confirmed that FOXD2‘AS1 regulates the HippoYAP signaling pathway in breast cancer cellsS100A1 mediates FOXD2‘AS1‘induced breast cancer cell proliferation migration and invasiveness S100A1 is a 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure Overexpression of LATS1 inhibits FOXD2‘AS‘induced cell proliferation migration and invasion A and B mRNA level of LATS1 in MCF‘ and MDA‘MB‘ cells was detected by RT‘qPCR in LATS1‘overexpressing cells P0001 vs overexpression‘NC oe‘NC Following transfection with pcDNA‘FOXD2‘AS1 cells were transfected with pcDNA‘LATS1 to overexpress LATS1 C and D CCK‘ assay was performed to determine the proliferation of the differently treated cells E Migratory ability of MCF‘ cells in the different treatment groups was determined by wound healing assay F Invasive ability of MCF‘ cells in the different treatment groups was determined by Transwell assay G Migratory ability of MDA‘MB‘ cells in the different treatment groups was determined by wound healing assay H Invasive ability of MDA‘MB‘ cells in the different treatment groups was determined by Transwell assay P0001 vs pcDNA‘NC P0001 vs FOXD2‘AS1 shRNA‘NC LATS1 large tumor suppressor homolog calcium‘binding protein of the S100 protein family which is not only upregulated in but is also involved in the progression of ovarian cancer In the present study the expression of S100A1 was evaluated in the breast cancer cell lines indicating that S100A1 was significantly upregulated in breast cancer cells particularly in the MCF‘ and MDA‘MB‘ cells compared with the McF10A cells Fig 4A In McF7 and MdAMB468 cells transfected with shRNAFOXd2AS1 it was found that the protein expression level of S100A1 was downregulated Fig 4B and C To further investigate the role of S100A1 in FOXD2‘AS1‘mediated cellular proliferation migration and invasiveness breast cancer cells were transfected with an expression vector pcdNAFOXd2AS1 Fig 4d and E and shRNAS100A1 to inhibit S100A1 protein and mRNA expression Fig 4FI As shown in Fig 5A and B the overexpression of FOXd2AS1 significantly promoted the proliferation of McF7 and MdAMB468 cells which was subsequently reversed by the downregulation of S100A1 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 suppresses tumor progression of breast cancer in vivo A Each mouse was injected with 10x107 MdAMB468 cells which were stably transfected with either shRNA negative control or shRNA‘FOXD2‘AS1 subcutaneously at the right axillary lymph node Following sacrifice the tumors were excised and photographed B and C During the experiment the mouse weight and tumor volume tumor volume x length x width2 was monitored every days until the mice were sacrificed D Protein expression levels of p‘YAP YAP p‘LATS1 and LATS1 in the extracted tumors were determined by western blot analysis and were then quantified P0001 vs shRNA‘NC LATS1 large tumor suppressor homolog YAP yes‘associated protein Wound healing and Transwell assays demonstrated that FOXD2‘AS1 overexpression significantly increased MCF‘ cell migration and invasiveness respectively which were also reversed by the downregulation of S100A1 Fig 5C and D A similar result was observed in the MDA‘MB‘ cells Fig 5E and F These results suggest that S100A1 regulates the FOXd2AS1mediated proliferation migration and invasiveness of breast cancer cellsOverexpression of LATS1 inhibits FOXD2‘AS‘induced cell proliferation migration and invasiveness Since the HippoYAP signaling pathway is involved in the FOXd2AS1mediated characteristics of breast cancer cells HippoYAP signaling was further investigated for its regulatory role in breast cancer cell proliferation migration and invasiveness For this purpose LATS1 was overexpressed in the McF7 and MdAMB468 cells Fig 6A and B and the results of CCK‘ assay revealed that LATS1 overexpression significantly inhibited FOXD2‘AS1‘induced cellular proliferation Fig 6C and D Furthermore the results of wound healing and Transwell assays revealed that LATS1 overexpression significantly inhibited the FOXD2‘AS1‘induced migration and invasiveness of both the McF7 Fig 6E and F and MDA‘MB‘ cells Fig 6G and HFOXD2‘AS1 knockdown suppresses breast cancer tumor progression in vivo From the aforementioned results the role of FOXd2AS1 in both the McF7 and MdAMB468 cells was confirmed To explore the role of FOXD2‘AS1 in breast cancer in vivo mice were injected with MdAMB468 cells which were stably transfected with either an shRNA negative control or shRNA‘FOXD2‘AS1 Following sacrifice 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE the tumors were excised and photographed Fig 7A Tumors in the shRNA‘FOXD2‘AS1 group were the smallest in size directly reflecting the suppressive effect of FOXD2‘AS1 knockdown on tumor growth During the experiment body weights and tumor volumes were recorded every days Body weight increased at a slower rate in the shRNAFOXd2AS1 group and the tumor volumes of this group also increased at a slower rate than those in the other groups Fig 7B and C Additionally western blot analysis of the extracted tumor tissues indicated a significant increase in p‘YAP and p‘LATS1 expression in the shRNA‘FOXD2‘AS1 group Fig 7D which was consistent with the in vitro results These findings thus suggest that FOXD2‘AS1 knockdown suppresses the progression of breast cancer and regulates the HippoYAP signaling pathway in vivoDiscussionBreast cancer is one of the most common types of human tumors particularly among females The high rates of metastasis and recurrence typically result in the deterioration and death of patients with breast cancer Increasing evidence suggests that lncRNAs function as oncogenic or antitumor genes in various tumor types cells types and the microenvironment and that they may be used as effective and specific biomarkers for clinical diagnosis and prognosis Due to its oncogenic properties lncRNA FOXd2AS1 has been investigated in several malignant tumors In the present study FOXD2‘AS1 expression was found to be upregulated in breast cancer cell lines thus it was knocked down in MCF‘ and MdAMB468 cell to investigate its role in breast cancer FOXd2AS1 knockdown inhibited the proliferation migration and invasiveness of McF7 and MdAMB468 cells and inhibited tumor growth in vivo Notably S100A1 expression was also found to be upregulated in breast cancer cells and further investigation revealed that S100A1 was inhibited following FOXd2AS1 knockdown indicating that the expression of FOXd2AS1 and S100A1 was positively associated in breast cancer cells Subsequent experiments revealed that the overexpression of FOXD2‘AS1 significantly accelerated tumorigenesis by promoting cellular proliferation migration and invasiveness However the effects of FOXd2AS1 were reversed by the downregulation of S100A1 These results suggest that both FOXD2‘AS1 and S100A1 knockdown exert antitumor effects on the progression of breast cancer and that FOXD2‘AS1 may exert its oncogenic functions by regulating S100A1S100A1 is a calcium‘binding protein belonging to the S100 protein family which exhibit a range of biological properties surrounding cellular proliferation metastasis immune evasion and angiogenesis and are also involved in tumorigenesis For example S100A4 enhances p53dependent apoptosis and facilitates more aggressive tumor progression S100A6 has been reported to be upregulated in human osteosarcoma colorectal carcinoma and hepatocellular carcinoma which was mostly associated with its suppressive properties towards cancer cell migration and tumor metastasis Therefore S100 proteins play an important role in the development and progression of tumors highlighting the necessity to further understand their roles and potential underlying mechanisms In the present study S100A1 expression was found to be upregulated in breast cancer cell lines FOXd2AS1 overexpression was shown to accelerate breast cancer progression by promoting cellular proliferation migration and invasiveness and functional experiments demonstrated that the knockdown of S100A1 reversed the effects induced by FOXD2‘AS1 Furthermore S100A1 knockdown suppressed breast cancer progression by inhibiting the proliferation migration and invasiveness of McF7 and MDA‘MB‘ cells In agreement with these findings S100A1 has been reported to be overexpressed in ovarian cancer and to be associated with lymph mode metastasis the overexpression of S100A1 was shown to enhance cellular proliferation and migration whilst its inhibition exerted an opposite effect on ovarian cancer cells Moreover high tumor expression levels of S100A1 have been shown to be positively associated with decreased relapse‘free survival time in an endometrioid subtype of ovarian and endometrial cancers It was thus hypothesized that S100A1 functions as an important regulator in breast cancer and may therefore be a promising therapeutic target for this as well as other types of gynecological cancerThe Hippo pathway is an important signaling pathway that regulates cellular proliferation and apoptosis the activation of which is triggered by the phosphorylation of the large tumor suppressor kinases LATS1 and LATS2 The Hippo pathway is very complex as a number of kinases relay upstream signals to LATS to regulate this pathway The STE20 protein kinases MST12 as the core components of the Hippo pathway are considered responsible for the phosphorylation and activation of LATS12 YAP a downstream effector of the Hippo pathway is highly activated in various types of cancer and targeting YAP may effectively suppress tumorigenesis Both dysregulated Hippo signaling and aberrant YAP activation contribute to cancer progression In the present study FOXD2‘AS1 knockdown significantly increased the phosphorylation of YAP and LATS1 indicating that the Hippo signaling pathway was activated by FOXD2‘AS1 downregulation Notably it was found that MST12 expression levels were downregulated by

Thyroid_Cancer

performance demonstration of a hybrid compton camera with an active pinhole for wide‘band X‘ray and gamma‘ray imagingAkihisa omata1 Jun Kataoka1 Kazuya fujieda1 Shogo Sato1 eri Kuriyama1 Hiroki Kato2 Atsushi toyoshima3 takahiro teramoto3 Kazuhiro ooe2 Yuwei Liu2 Keiko Matsunaga2 takashi Kamiya2 tadashi Watabe2 eku Shimosegawa2 Jun Hatazawa2X‘ray and gamma‘ray imaging are technologies with several applications in nuclear medicine homeland security and highenergy astrophysics However it is generally difficult to realize simultaneous wideband imaging ranging from a few tens of keV to MeV because different interactions between photons and the detector material occur depending on the photon energies for instance photoabsorption occurs below keV whereas Compton scattering dominates above a few hundreds of keV Moreover radioactive sources generally emit both X‘ray and gamma‘ray photons in this study we develop a œhybrid compton camera that can simultaneously achieve X‘ray and gamma‘ray imaging by combining features of œcompton and œpinhole cameras in a single detector system Similar to conventional compton cameras the detector consists of two layers of scintillator arrays with the forward layer acting as a scatterer for highenergy photons keV and an active pinhole for lowenergy photons keV The experimental results on the performance of the hybrid camera were consistent with those from the Geant4 simulation We simultaneously imaged Am keV and Cs keV in the same field of view achieving an angular resolution of —¦ FWHM for both sources in addition imaging of At was conducted for the application in future nuclear medicine particularly radionuclide therapy the initial demonstrative images of the At phantom were reconstructed using the pinhole mode using keV and Compton mode using keV exhibiting significant similarities in sourceposition localization We also verified that a mouse injected with MBq of At can be imaged via pinhole‘mode measurement in an hourIn the field of nuclear medicine it is essential to visualize the distribution of radioisotopes in a patient™s body Particularly a radiological diagnosis that enables noninvasive visualization of the pathology from outside the body is important The general approach is to visualize nuclear gamma rays emitted from radioactive tracers Two common techniques”single photon emission computed tomography SPECT and positron emission tomography PET”play important roles in the diagnosis However they image a specific energy range of either Xrays or gamma rays SPECT can image gamma rays with energies less than a0keV with the use of the collimator whereas PET can image positron emitters that emit 511keV gamma rays These lead to a limited number of radioactive tracers that can be imaged only with current SPECT and PET scanners In this context a Compton camera12 that can conduct imaging in a wide energy band is reckoned to cause a breakthrough in nuclear medicine34 Attempts are being made to develop Compton cameras aiming the image of prompt gamma rays emitted from the patient™s body during proton therapy5“ In addition several works have aimed to realize tracer visualization using the Compton camera For example Fontana et a0al describe the Compton camera as an 1Graduate School of Advanced Science and Engineering Waseda University Tokyo Japan 2Graduate School of Medicine Osaka University Osaka Japan 3Institute for Radiation Science Osaka University Osaka Japan email omt22fujiwasedajpScientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Comparison of the simulated energy response of the intrinsic efficiency left and angular resolution right with the results of actual measurementselectronically collimated SPECT device which is however optimized for higher energies above a few hundreds of keV9 Yoshida et a0al proposed whole gamma imaging WGI wherein a conventional PET scanner is converted by inserting an additional scatterer to create a Compton camera10 They succeeded in realizing the triple gamma ie PETCompton imaging of 44Sc which emits keV and a0keV gamma rays however the application to imaging SPECT tracer remains to be researched in the future In this context the simultaneous capture of SPECT and PET images has also been reported using a Compton camera consisting of SiCdTe semiconductors which is however limited both in terms of detection efficiency and angular resolution11“ We argue that such difficulties can be easily overcome with the hybrid camera proposed in this paper This increases the variety of radioactive tracers available for nuclear medicine14“ The emergence of new tracers may solve current problems such as manufacturing costs of tracers in addition to achieving improvements in the diagnosis quality A Compton camera also enables the simultaneous imaging of multiple tracers this significantly increases the information obtained from living anisms in a single diagnosisMoreover nuclear medicines involving radioactive sources are applied not only in the diagnosis but also in the treatment of diseases such as cancer Particularly radionuclide therapy RNT1920 which uses the targeted radionuclide by administering radioisotopes to patients is widely used because it damages cancer cells while limiting the exposure of healthy tissues to radiation For instance RNT with α ps is receiving significant attention because of its high therapeutic potential owing to the higher ionization power for damaging cancer cells2122 Nevertheless once administered into the body it is difficult to determine the distribution and pharmacokinetics of a radionuclide For the safety and effectiveness of RNT it is conceivable to visualize the characteristic Xrays and nuclear gammarays emitted simultaneously with the alpha decay Some of these characteristic X rays and nuclear gamma rays can be visualized by SPECT Furthermore the use of a Compton camera for the in a0vivo visualization of 223Ra an αp emitter used for RNT was suggested in our previous study23 However these radionuclides emit several Xrays and gamma rays with different branching ratios Some emit strong characteristic Xrays that can be imaged with a SPECT whereas others emit highenergy gamma rays that can be imaged only with a Compton camera see Table a0 Imperatively a simple and costeffective imaging system that is sensitive to both Xrays and gamma rays is highly desiredThis paper proposes a œhybrid Compton camera that realizes simultaneous wideband imaging from a few tens of keV to approximately an MeV combining some features of œCompton cameras and œpinhole cameras in a single detector system Although the hybrid camera consists of two layers of positionsensitive detectors similar to a Compton camera1323 the front detector has a hole in the center Compton and pinhole imaging are enabled using the front detector as a scatterer for highenergy photons a0 keV and an active pinhole for lowenergy photons a0 a0keV We developed a prototype of the hybrid camera Simulation and experimental results depicted resolutions better than —¦ full width at half maximum FWHM in the range of “ a0keV In addition the imaging of At is receiving attention as a source applicable to RNT with αps24“ We initially investigated the capability of our hybrid camera system with a simple phantom of At and thus conducted mouse imagingResultsperformance evaluation The imaging performance of the hybrid camera was initially evaluated by simulations using Geant428 The simulation configuration includes the scintillator arrays the MPPC arrays and the metal case which are detailed in the œMethods section A monochromatic point source placed a0cm from the camera and at the center of the field of view FOV was imaged The detector performance was investigated between and a0keV for both the pinhole and Compton modes Considering the energy and spatial resolution of the actual detectors we utilized a reconstruction flow as described in the œMethods section The simulation showed that pinhole imaging is difficult over a0keV whereas Compton imaging is active at approximately a0keV or higher Figure a0 left details the intrinsic detection efficiency that indicates the proportion of events detected either as the pinhole or the Compton mode to all radiation emitted towards the detector The intrinsic detection efficiency Ç«int is expressed by the following equation using the absolute detection efficiency Ç«absScientific RepoRtS 101038s41598020710195Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Upper The experimental configuration of the simultaneous measurement of Am and 137Cs Lower The MLEM reconstructed images of the Am a0keV source analyzed by the pinhole mode left and the Cs a0keV source analyzed by the Compton mode rightÇ«int 4Ï�ǫabswhere 01 denotes the solid angle of the detector viewed from the source Figure a0 right shows the angular resolution of the pinhole and Compton images as a function of energy For Compton imaging we use the angular resolution measure ARM that is commonly used to measure the angular resolution of Compton cameras For measuring the angular resolution of pinhole imaging the �θ value is obtained by geometrically converting the position resolution of the image to angular resolution Note that for the source placed at the center of the FOV the angular resolution �θ can be calculated as follows�θ arctan�x2lwhere 01x and l denote the position resolution FWHM of the pinhole image and the distance between the camera and the source respectively The angular resolution was better than —¦ in the range of “ keV exhibiting the higher efficiency of the proposed hybrid camera than those of the conventional camerasSubsequently the fundamental imaging performance of the hybrid camera was evaluated by performing experiments under the same geometry as in the simulation The measurements were recorded at energies of a0keV and a0keV for pinhole imaging and a0keV and a0keV for Compton imaging The obtained angular resolutions were consistent with the values predicted by the simulation as represented by the red plots in Fig a0 rightExperimental demonstration of wideband imaging To examine the performance of the hybrid camera we conducted simultaneous imaging of Am and Cs sources As shown in Fig a0 upper these sources were placed cm away from the camera and at —¦ and ˆ’ —¦ respectively from the center of the FOV The Am source was reconstructed in the pinhole mode a0keV whereas the Cs source was reconstructed in the Compton mode a0keV From Fig a0 lower each convergence indicated the correct positions depicting the potential of broadband imaging using the hybrid cameraAs a next step extended sources were measured to examine the validity of the camera system including the image reconstruction technique The extended a0mm × a0mm œLshaped sources were reproduced by moving on a stage that automatically moved at a constant speed a0mmmin The image of an œLshaped Am source Scientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0cFigure a0 The MLEM reconstructed images of œLshaped sources Pinhole reconstruction of the Am source left and Compton reconstruction of the Cs source right that were measured separatelyFigure a0 Energy spectrum of At obtained by a LaBr3 scintillator coupled to a PMTwas reconstructed by the pinhole mode using events with energies around a0keV Subsequently the image of an œLshaped Cs source a0keV was also reconstructed by the Compton mode MLEM reconstruction images of each measurement are presented in Fig a0 At imaging of a small bottle Furthermore we investigated the performance of the hybrid camera with the imaging of a At source which is an αps source for RNT Initially the energy spectrum of At was obtained by a LaBr3 scintillator coupled to a photomultiplier tube PMT Figure a0 shows peaks of characteristic Xrays mainly a0keV and gamma rays with energies of and a0keV Furthermore the intensity of Xrays is approximately three orders of magnitude higher than that of the gamma raysNext placing a0cm away from the hybrid camera at —¦ the center of the FOV and —¦ to the right a small bottle was imaged with μL of At a0MBq The measurement times were and a0min at —¦ and —¦ respectively The images were reconstructed using a0keV Xrays in the pinhole mode and a0keV gamma rays in the Compton mode The measurements resulted in pinhole events and Compton events at —¦ and pinhole events and Compton events at —¦ The pinhole images of the a0keV Xrays and the Compton images of the a0keV gamma rays are presented in Fig a0 At imaging of a mouse To investigate the capability of the hybrid camera for animal imaging imaging of a mouse [8weekold male ICR mouse SLC Japan Hamamatsu Japan] with a At tracer was conducted The mouse length a0mm weight a0g was injected with At a0kBq a0h before the measurement The mouse was euthanized by an overdose of isoflurane a0h after the injection The hybrid camera was positioned on the right side of the mouse a0mm from the body axis as shown in Fig a0 left The measurement time was a0h resulting in pinhole events and Compton events From Fig a0 center the pinhole image depicted that the distribution of the At converged on the thyroid stomach and bladder from events obtained after a0h of measurement Moreover although the Compton image confirms the concentration near the Scientific RepoRtS 101038s41598020710195Vol1234567890wwwnaturecomscientificreports 0cFigure a0 The pinhole MLEM reconstruction image left and the Compton MLEM reconstruction image right of a bottle with At at the center of the FOV upper and —¦ to the right lowerFigure a0 Left Experimental configuration of the measurement of the mouse administered with 211At Center The pinhole MLEM reconstructed image obtained by a0h of measurement Right The Compton MLEM reconstructed image obtained by a0h of measurementcenter accumulation is splitting possibly owing to the lack of event statistics as shown in Fig a0 right All the animal experiments were approved by the animal ethics committees of Osaka University and were performed according to the institutional guidelinesScientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0cRadionuclide211At213Bi225Ac223Ra212BiX a0ray keV Gamma ray keV absolute intensity Pinhole Comptoncid31cid31cid31cid31cid31cid31Table Examples of αp emitters for RNT and their main Xrays and gamma rays and their absolute intensities in percentage accompanied by the adaptability of the pinhole mode andor the Compton modeFigure a0 The configuration of the hybrid camera left Schematic view of the pinhole event center for the lower energy range and the Compton event right for the higher energy range that are used for the pinholeCompton reconstruction in the hybrid cameraDiscussionIn the imaging experiment with the At inside a small bottle the source position was obtained by pinhole reconstruction using the events accumulated in the first s The time taken to localize the convergence through the pinhole mode was of that thorough the Compton mode This is due to the intense Xray emission from the 211At as shown in Fig a0 a0keV Xrays are statistically advantageous over a0keV gamma rays for imaging 211At In this regard it is comprehensible that Compton events are not enough to reconstruct the distribution of the At tracer although it can be imaged through the pinhole mode There are several αemitting nuclides that can be used for RNT in the future2122 The distribution of these nuclides should be comprehensible and controlled by monitoring characteristic Xrays and nuclear gamma rays from outside the body Table a0 summarizes several properties of αemitting nuclides that are planned for clinical use Moreover Xray or gammaray energy suitable for a specific imaging application may use different nuclides and consequently different energies as the a0keV for At investigated here Although some nuclides are easy to image with lowenergy Xrays others are suitable for gammaray imaging for example Ac and 212Bi The wide scope of the hybrid cameras allows us to select the appropriate energy from lowenergy Xrays to highenergy gamma rays and thus potentially cover a wide energy range including the conventional SPECT a0keV PET a0keV and Compton camera subMeV to MultiMeV for various applications This leads to a reduction in the measuring time thus reducing the burden on the patient In addition wideband imaging can image multiple tracers simultaneously which significantly increases a patient™s complete medical information obtained from a single diagnosisTo improve the performance for further applications the hole size in the front detector should be adjusted as a tradeoff between the efficiency and the resolution of pinhole imaging Additionally the application of the depthofinteraction technique29“ to the backward detector may improve the resolution Therefore the energy range of the pinhole and Compton modes can be adjusted based on the density andor thickness of the detectors By revising the structure of the detector imaging in a wider band can be realized Particularly Compton cameras developed for gammaray astronomy have realized imaging in high energy bands such as “ a0MeV32 By applying the configuration of the hybrid camera as proposed in this paper these Compton cameras can perform pinhole imaging without compromising on the performance of the original Compton camera Currently we are developing a new hybrid camera that covers a0keV to a0MeV by adopting the configuration of the scintillator as described in Kishimoto et a0al and Hosokoshi et a0al MethodsConfiguration of the hybrid camera The configuration of the hybrid camera is detailed in Fig a0 left The hybrid camera consists of a pair of positionsensitive detectors capable of receiving the reaction position and the energy deposit of each event The detectors are composed of Cedoped Gd3Al2Ga3O12 GAGG scintilScientific RepoRtS 101038s41598020710195Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Left Energy spectrum of all the events detected in either detector black the events accumulated by with the front detector green the events obtained only with the rear detector blue and the events reacted with both detectors red accumulated from placing Am and Cs sources simultaneously in front of the detector Right 2D energy spectrum of coincidence events from the front detector scatterer and the rear detector absorber The area painted red corresponds to the energy cut range for a0keV Compton events The brightest area Efront ˆ¼ a0 a0keV corresponds to backscattering eventsModePinholeComptonCoincidence selectionAnticoincidence rearCoincidence front and rearEnergy cutRearSum and frontTable Factors of event selection for each reconstruction modelator arrays3334 coupled with multipixel photon counter MPPC arrays29 A × array of × × a0mm3 GAGG pixels is used as the front detector and a × array of × × a0mm3 GAGG pixels is used as the rear detector The front detector has an active × mm2 pinhole in its center The distance between the detectors is a0cm The energy resolutions of the front and rear detectors are and FWHM at a0keV respectively The position resolution for gammaray interaction is equal to the pixel size both for the pinhole and Compton mode The time range of the coincidence is set to be ± a0 a0μs and the count rate capability is ‰¤ a0 a0kHz The camera is enclosed in a mmthick metal mainly tungsten density a0gcm3 case except the front surfaceThe proposed hybrid camera enables imaging similar to that of a pinhole camera in addition to the conventional Compton camera Commonly the Compton camera which consists of a scatterer and an absorber uses events that undergo Compton scattering in the scatterer and photoabsorption in the absorber as shown in Fig a0 right For each event the scattering angle of Compton scattering is calculated using Compton kinematics restricting the arrival direction in the conical area called the Compton cone The position of the radiation source is identified by superimposing the Compton cones However photons with energy lower than several hundreds of keV cannot be imaged by the Compton camera owing to the increased probability of photoabsorption in the front scatterer The hybrid camera can also make use of such photoabsorption events We have devised a method to operate the camera as a Compton and a pinhole camera by utilizing the front scatterer with a hole in the center As shown in Fig a0 center the arrival directions of lowenergy photons are limited by analyzing the scatterer as an active pinhole Among lowenergy events detected in the rear detector the events that are not detected in the front detector can be considered to have passed through the hole in the front detector Note that Compton andor pinhole reconstruction can be selected analytically after the measurementReconstruction flow for each mode The selection of valid events for Compton and pinhole modes are parameterized with two factors coincidence selection and energy cut Coincidence is an event pattern hitting one or both of the detectors Specifically only anticoincidence events wherein only the rear detector is triggered are used for pinhole mode imaging whereas coincidences between the front and rear detectors are used for Compton mode reconstruction An energy cut is used to restrict the energy range of photons deposited in each detector The event selection criteria for each mode are summarized in Table a0 From Fig a0 left blue the spectrum from the rear detector in the anticoincidence mode simultaneously irradiated with Am and Cs sources shows that a0keV Xray is accurately masked in the front detector Therefore the pinhole events Scientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0cRadionuclide241Am137Cs211At211AtTarget keV ModePinholeComptonPinholeComptonEnergy cut keV Erear Efront Efront Erear Erear Efront Efront Erear Table The numbers for applied energy cutsare selected by the energy cuts with the corresponding energy range On the other hand Compton events are chosen from candidates of coincident events Figure a0 left red shows the spectrum from coincidence events obtained with mixed Am and Cs measurement Therefore Compton events are chosen from the region of interest reddiamond restricted from the total energy deposit a0keV a0 Efront Erear a0keV and energy deposit of front detector to reject the backscattering events Efront a0keV Furthermore the events whose scattering angles cannot be determined geometrically are deleted A red diamond in the 2D spectrum of the coincidence events shown in Fig a0 right corresponds to valid events for Compton image reconstruction The quantitative numbers applied for energy cut are summarized in Table a0 In image reconstruction maximum likelihoodexpectation maximization MLEM a statistical approximation method153536 is used to improve the image quality This method uses statistical iterations to locate sources with greater accuracy and better signaltonoise ratio alternate to the simple back projection method The number of iterations is for each mode except for the œLshaped Compton image which is In the pinhole mode the signaltonoise ratio is also improved by subtracting the background image that was reconstructed from the events next to the source energy rangeReceived May Accepted August References Schönfelder V et al The imaging Compton telescope COMPTEL on the gamma ray observatory IEEE Trans Nucl Sci “ 101109TNS198443333 Schöenfelder V et al Instrument description and performance of the imaging gammaray telescope COMPTEL aboard the Compton gammaray observatory Astrophys J Suppl Ser 10108619179 Todd R Nightingale J Everett D A proposed γ camera Nature “ 10103825113 2a0 Kataoka J et al Ultracompact Compton camera for innovative gammaray imaging Nucl Instrum Methods Phys Res Sec A Acceler Spectrom Detect Assoc Equip “ 101016jnima201709048 Koide A et al Precision imaging of MeV gamma rays using a 3D position sensitive Compton camera Sci Rep “ 101038s4159 Mochizuki S et al Highprecision Compton imaging of MeV prompt gammaray toward an online monitor for proton therapy Nucl Instrum Methods Phys Res Sect A Detect Assoc Equip “ 101016jnima201811032 Golnik C et al Tests of a Compton imaging prototype in a monoenergetic MeV photon field”a benchmark setup for prompt gammaray imaging devices J Instrum P06009 101088174802211106P0600 Hilaire E Sarrut D Peyrin F Maxim V Proton therapy monitoring by Compton imaging influence of the large energy spectrum of the promptγ radiation Phys Med Biol 101088003191556183127 Fontana M Dauvergne D Létang J M Ley JL Testa E Compton camera study for high efficiency SPECT and benchmark with Anger system Phys Med Biol 10108813616560aa926 a Yoshida E et al Whole gamma imaging a new concept of PET combined with Compton imaging Phys Med Biol Nakano T et al Imaging of 99mTcDMSA and 18FFDG in humans using a SiCdTe Compton camera Phys Med Biol 05LT01 10108813616560ab8e8 10108813616560ab33d 10108813616560aae1d Sakai M et al In a0vivo simultaneous imaging with 99m Tc and F using a Compton camera Phys Med Biol Sakai M et al Compton imaging with 99m Tc for human imaging Sci Rep “ 101038s4159 z Kishimoto A et al Development of a compact scintillatorbased highresolution Compton camera for molecular imaging Nucl Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip “ 101016jnima201606056 Kishimoto A et al First demonstration of multicolor 3D in a0vivo imaging using ultracompact Compton camera Sci Rep “ 101038s4159 w Motomura S et al Gammaray Compton imaging of multitracer in biological samples using strip germanium telescope IEEE Trans Nucl Sci “ 101109TNS200789420 Motomura S Kanayama Y Haba H Watanabe Y Enomoto S Multiple molecular simultaneous imaging in a live mouse using semiconductor Compton camera J Anal At Spectrom “ 101039B8029 64D Motomura S et al Improved imaging performance of a semiconductor Compton camera GREI makes for a new methodology to integrate biometal analysis and molecular imaging technology in living anisms J Anal At Spectrom “ 101039C3JA3 0185K Hamilton J G Soley M H Studies in iodine metabolism by the use of a new radioactive isotope of iodine Am J Physiol Legacy Content “ 101152ajple gacy19391273557 Odonoghue J Bardies M Wheldon T Relationships between tumor size and curability for uniformly targeted therapy with betaemitting radionuclides J Nucl Med “ Poty S Francesconi L C McDevitt M R Morris M J Lewis J S Alpha emitters for radiotherapy from basic radiochemistry to clinical studies”part J Nucl Med “ 102967jnume d11720465 Sgouros G et al MIRD Pamphlet No Abridged radiobiology and dosimetry of αp emitters for targeted radionuclide therapy J Nucl Med “ 102967jnume d10805865 Scientific RepoRtS 101038s41598020710195Vol1234567890wwwnaturecomscientificreports 0c Fujieda K et al First demonstration of portable Compton camera to visualize 223Ra concentration for radionuclide therapy Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip 101016jnima201916280 Brown I Astatine211 its possible applications in cancer therapy Int J Radiat Appl Instrum Part A Appl Radiat Isot “ 101016088328898690273 X Willhauck M J et al The potential of 211Astatine for NISmediated radionuclide therapy in prostate cancer Eur J Nucl Med Mol Imaging 101007s0025 Watanabe S et al Difference in thyroid uptake between Astatine211 and Iodine123 in normal rats a comparative study between oral and intravenous administration J Nucl Med “ Watabe T et al Enhancement of At uptake via the sodium iodide symporter by the addition of ascorbic acid in targeted αtherapy of thyroid cancer J Nucl Med “ 102967jnume d11822263 Agostinelli S et al GEANT4”a simulation toolkit Nucl Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip “ 101016S0168 Kataoka J et al Recent progress of MPPCbased scintillation detectors in high precision Xray and gammaray imaging Nucl Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip “ 101016jnima201411004 Kishimoto A et al Development of a dualsided readout PET module using largearea monolithic MPPCarrays IEEE Trans Nucl Sci “ 101109TNS201222332 Kataoka J et al Handy Compton camera using 3D positionsensitive scintillators coupled with largearea monolithic MPPC arrays Nucl Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip “ 101016jnima201307018 Hosokoshi H et al Development and performance verification of a 3D positionsensitive Compton camera for imaging MeV gamma rays Sci Rep “ 101038s4159 z Kamada K et al Composition engineering in ceriumdoped Lu Gd3Ga Al5O12 singlecrystal scintillators Cryst Growth Des Kamada K et al inch diameter single crystal growth and scintillation properties of CeGd3Al2Ga3O12 J Cryst Growth “ 101021cg200 694a “ 101016jjcrys gro201111085 Dimmock M R Nikulin D A Gillam J E Nguyen C V An CL implementation of pinhole image reconstruction IEEE Trans Nucl Sci “ 101109TNS201221977 Wilderman S a0J Clinthorne N a0H Fessler J a0A Rogers W a0L Listmode maximum likelihood reconstruction of Compton scatter camera images in nuclear medicine In IEEE Nuclear Science Symposium Conference Record IEEE Nuclear Science Symposium and Medical Imaging Conference Cat No 98CH36255 vol a0 “ 101109NSSMI C199877387 IEEE AcknowledgementsThis research was supported by JSPS KAKENHI Grant Number JP15H05720 20H00669 and JPMJER1905 ERATOFS The At was supplied through the Supply Platform of Shortlived Radioisotopes supported by JSPS GrantinAid for Scientific Research on Innovative Areas Grant Number 16H06278Author contributionsJK conceived the concept of this research AO developed the hybrid camera AO JK KF SS and EK conducted the experiments HK AT TT KO YL KM TK TW ES and JH conducted the experiments using 211At AO wrote the manuscriptcompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to AOReprints and permissions information is available at wwwnaturecomreprintsPublisher™s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Authors Scientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0c'

Thyroid_Cancer

Signal Transduction and Targeted Therapywwwnaturecomsigtrans REVIEW The potent roles of saltinducible kinases SIKs in metabolichomeostasis and tumorigenesisZicheng Sun12 Qiwei Jiang1 Jie Li2 and Jianping Guo1Saltinducible kinases SIKs belong to AMPactivated protein kinase AMPK family and functions mainly involve in regulatingenergy responserelated physiological processes such as gluconeogenesis and lipid metabolism However compared with anotherwellestablished energyresponse kinase AMPK SIK roles in human diseases especially in diabetes and tumorigenesis are rarelyinvestigated Recently the pilot roles of SIKs in tumorigenesis have begun to attract more attention due to the finding that thetumor suppressor role of LKB1 in nonsmallcell lung cancers NSCLCs is unexpectedly mediated by the SIK but not AMPK kinasesThus here we tend to comprehensively summarize the emerging upstream regulators downstream substrates mouse modelsclinical relevance and candidate inhibitors for SIKs and shed light on SIKs as the potential therapeutic targets for cancer therapiesSignal Transduction and Targeted Therapy 101038s4139202000265wINTRODUCTIONSaltinducible kinase SIK was first identified in the adrenalglands of high salt dietfed rats in Further the SIK familyincluding SIK1“SIK3 are characterized as serinemembersthreonine kinases that belong to AMPactivated protein kinaseAMPK family23 Later SIKs have shown selfphosphorylationand play an important role in regulating adrenocortical functionunder the stimulation of high salt or adrenocorticotropichormone ACTH1 Of note the SIK1 is abundantly expressedin the adrenal cortex as well as in the adipose and neuraltissues3“ while both SIK2 and SIK3 are ubiquitous in humansand mainly expressed in adipose and neural tissues respectively3 In addition these SIK family members are dysregulatedin various cancers including ovarian breast prostate and lungcancers indicating that SIKs may execute crucial roles in tumoroccurrence or progression36In recent years although the roles of SIKs in tumorigenesis havedrawn much attention due to their association with TGFSmadAKT Hippo NFκb and other signaling pathways involved incancer progression6“ similar to the AMPK kinases the potentialroles of SIKs in tumorigenesis are still controversial as oncogene ortumor suppressor in a tissue context dependent manner Therefore the purpose of this review is to comprehensively summarizethe upstream regulators downstream effectors clinical relevanceas well as candidate inhibitors of SIKs to highlight the potentialstrategy to target SIKs for cancer therapiesTHE UPSTREAM REGULATORS AND DOWNSTREAMSUBSTRATES OF SIKSSIK1 gene is located in human chromosome while SIK2 andSIK3 genes are both located on chromosome SIKs sharesimilar structures to AMPKrelated kinases including AMPKα1α2in SIKsspecificallySADAB MARK1“ NUAK12 and SNRK all of which can bephosphorylated and activated by liver kinase B1 LKB1 GenerallyAMPKrelated kinases consist of two common domains possessing an Nterminal serinethreonine kinase domain KD followedby a ubiquitinassociated UBA domain18“ Beyond that SIKs arealso composed of a central sucrose non fermenting SNF1homology SNH domain and a long Cterminal domainFig 1a2022 The Nterminal KD contains a LKB1 phosphorylationsite and is relatively conserved across SIK family However theSNH domain is distinctthe similaritypercentage of SIK2 and SIK3 compared that of SIK1 is and respectively The Cterminal domain contains multipleprotein kinase A PKA phosphorylation sites and is highlyconserved between SIK1 and SIK222 Like other AMPK familymembers an activation loop Tloop exists in the KD of SIKswhich near the substratebinding pocket and is phosphorylatedand activated by LKB1 Fig 1a b1922 In addition there is also anautophosphorylation residue in the Tloop which is essential forthe kinase activity of SIK1 and SIK223 On the other hand a UBAdomain has also been defined within the SNH domain24 andmutations derived from the UBA domain notably decreased LKB1mediated SIK phosphorylation and kinase activation24 partially viapreventing SIK interacting with adapter protein to promoteSIK nuclear transport2425 Similar to AMPK kinases the Thr322residue in SIK1 SNH domain could also be activated by calciumdependent protein kinase CaMKmediated phosphorylation2026similar results were observed in SIK2 kinase and resulted in SIK2degradation27 SIKs are considered rapid turnover proteins due tothe phosphorylation by PKA PKC and tyrosine kinase in theirCterminal region Fig 1a1720 Thus SIK family members share asimilar structure and play redundant and distinctroles inregulating biological processes especially in metabolic homeostasis which will be further summarized in the following sections1Institute of Precision Medicine the First Affiliated Hospital Sun YatSen University Guangzhou Guangdong China and 2Department of Breast and Thyroid Surgery theFirst Affiliated Hospital Sun YatSen University Guangzhou Guangdong ChinaCorrespondence Jie Li Lijie78mailsysueducn or Jianping Guo guojp6mailsysueducnReceived May Accepted July The Authors 0cThe potent roles of saltinducible kinases SIKs in metabolic Sun et alFig The diagram structure of SIKs and related kinases a The structure and phosphorylation residues are illustrated SIKs are composed ofKD kinase domain containing an LKB1 phosphorylation site SNH domain containing UBA ubiquitinassociated domain and Cterminaldomain containing multiple PKA phosphorylation sites b The structure of AMPKrelated family kinases are illustrated These kinases share asimilar structure with SIKs AMPKα subunits are composed of KD AID autoinhibitory domain αlinker containing two αRIM regulatorysubunitinteracting motif and αCTD Cterminal domain SADs are composed of KD UBA domain and KA1 kinaseassociated domain it isNterminal next to the AIS sequence autoinhibitory sequence MARKs are composed of KD UBA domain spacer and tail domain includingthe KA1 domain NUAKs are composed of KD and UBA domain SNRK is composed of KD and UBA domain The phosphorylation sites on theTloop of AMPKrelated family kinases are illustrated The AMPKrelated family kinases can be directly phosphorylated and activated by LKB1on their Tloop right panel c SIK and AMPK downstream substrate phosphorylations are illustrated SIKs phosphorylated LXRKHSTXSXXXL motif underlined phosphorylated residue X any residue and the identified AMPK substrates phosphorylation sites reside in theknown AMPK phosphorylation consensus sequence LMIXRKHXXSXXXLVIF are illustratedsuch as energy deprivationSIK upstream regulatorsActing as AMPKrelated kinases SIKs exhibit a similar activationproperty with AMPK1922 in an LKB1mediated phosphorylationdependent manner Fig 1a b18“ Importantly physiologicalchangesinsulin or glucagonperturbation all manipulate SIK kinase activity29 For exampleinsulin stimulation or chronic hyperglycemia could increase SIKprotein level and kinase activity30“ By contrast Patel et al33reported that insulin did not regulate SIK2 phosphorylation andactivity Different from other AMPKrelated family members SIKscould be specifically activated by the sodium homeostasis2 As aresult sodium intakeinduced calcium ‚ux affected by NaCa2exchange system NCE1 could cause CaMKmediated SIK1phosphorylation and activation263435 which was argued byanother study36Liver kinase B1LKB1 protein kinase was initially identified inPeutz Jeghers syndrome PJS37 and later it has been considered amaster serinethreonine kinase involved in diverse physiologicalsuppressorfunctionsin LBK1 tumorprocesses38 Accumulating evidence has demonstrated that LKB1can phosphorylate and activate many AMPKrelated kinases ontheir Tloop Fig 1b18“ Genetically deletions of LBK1 arefrequently occurred in NSCLCs especially in KRASG12DbearingNSCLCs3940 indicating that LBK1 is a potent tumor suppressene Although previous efforts mainly devoted to the studies ofAMPK rolesrecentlydepletion of AMPKα1 or AMPKα2 could not markedly impairLKB1 tumor suppressive roles in KRASG12Ddriven NSCLC models41indicating that other substrates will play more important roles inmediating LBK1 tumor suppressor functions As such SIK1 andSIK3 have been revealed as the predominant downstream targetsof LKB1 in mediating antitumorigenesis effect in NSCLC4243While some studies provided that SIK2 underwent autophosphorylation and activation in vitro independent on the presenceof LKB17 Therefore whether other members of AMPK subfamilymediating LBK1 functions in metabolic homeostasis and tumorigenesis need more investigations especially in combination withtheir conditional KO mouse modelsSignal Transduction and Targeted Therapy 0ctriphosphate IP3Ca2“CaMK Ca2“CaMK is another important upstream regulator of SIKs in an LKB1 independent manner744 In the absence ofLKB1 there is still a residual activation of SIK1 which may be dueto the activation by CaMK4243 Phospholipase C PLC can boostCa2 ‚ux from endoplasmic reticulum ER to the cytoplasmvia inositolthus activating theCaMK which leads to the phosphorylation and activation ofSIK2 at Ser358745 PKA can also phosphorylate SIK2 at Ser358720But PKA is not involved in PLCmediated SIK2 phosphorylation atSer358 and activation7 As discussed earlier sodium mediatedSIK1 activation is also through CaMK263435 Interestingly a studyfound that CaMK IIV phosphorylated SIK2 at Thr484 leading toSIK2 degradation and promoting CREBmediated transcriptionFig 1a27receptorProtein kinase A PKA one of the members of AGC kinase is atetrameric holoenzyme composed of homodimer including twokinds of regulatory subunits RIα and RI RIIα and RII and threecatalytic subunits Cα C or Cγ4647 PKA activity depends onthe binding of cAMP with the regulatory subunits leading to therelease of active catalytic subunits and then phosphorylatingdiverse substrates46 Pathologically mutations in PKA catalyticsubunit promoted adrenal cortical tumorigenesis and Cushing™ssyndrome4849 Although PKA is not considered as an oncogenePKA has an active role in several cancers50“ and induce thetransformation of human mammary stromal cells into epithelialcells MET53 Until now all three SIK family members have beendiscovered to undergo PKAmediated phosphorylation andinhibition Bioinformatic analyses imply that SIKs contain multiplemotifs harboring PKA phosphorylation and binding sitesRSXSXP underlined phosphorylated residue X any residue202954 When these potential phosphorylation residues areabolished the binding of SIKs with is largely eliminatedwhich markedly antagonizes PKA inhibitory roles on SIKs2954Notably changes in these residues do not affect LKB1mediatedSIKs activation Biologically PKA can phosphorylate SIK1 topromote its nucleus translocation55“ which could be efficientlyblocked by mutating these two arginine residues within thephosphorylation motif56 Similarly PKA directly phosphorylatesSIK2 to regulate its stabilization and relocation by modulating itsinteraction with Meanwhile the deletion of PKA notonly promotes SIK1 protein stability but also transcriptionallyaccelerates SIK1 expression59 Hence PKA would be a criticalnegative upstream regulator of SIKs to compete with LKB1 ingoverning SIK physiological or pathological functionsOther upstream regulators Aside from phosphorylation otherposttranslational modifications PTMs such as acetylation alsoplay important roles in governing SIK activity Of note p300mediated acetylation inhibited ATP binding with and activating ofSIK2 by disturbing its phosphorylation at Thr175 conversely SIK2can also directly phosphorylate and regulate p300 acetyltransferase60 In addition HDAC6 has been identified to activate SIK2 byremoving its acetylation modification9 In addition RNF2 an E3ligase has been revealed to ubiquitinate and in turn degrade SIK1in hepatoma cells61 Consequentlythe specific regulatorymechanisms of other PTMs to SIKs need to be further exploredfor fully understanding the upstream regulation for SIKsSIK downstream substratesSimilar to AMPK in recognizing the substrate motif LMIXRKHXXSXXXLVIF6263 SIKs phosphorylates substrates containing LXFig 1c5455 A variety of metabolicRKHSTXSXXXL motifregulatorsincluding CRTC and class IIa HDACs are commonsubstrates of both AMPK and SIKs29 Importantly SIKs but AMPKscan directly phosphorylate some specific substratesincludingCRTCCREB and PPase methylesterase1 PME1 to involve inmetabolic homeostasis34Signal Transduction and Targeted Therapy The potent roles of saltinducible kinases SIKs in metabolic Sun et alHDAC Histone deacetylases functionally remove the acetylationmodification from both histone and nonhistone proteins64Among the histone deacetylases class IIa HDACs HDAC4 and are inhibitors of different transcription factors especially forMEF2 family64 All three SIK family members have emerged as newkinases for class IIa HDACs5765 SIKmediated HDAC phosphorylation promotes its binding with and facilitates its transportfrom nucleus to cytoplasm and then represses MEF2dependenttranscription5765 Therefore SIKs can regulate the development ofskeletal muscle skeleton regulatory T cells as well as leukemiaand other pathological processes via manipulating class IIa HDACsas discussed above2957596466CREBregulated transcription coactivatorcAMP response elementbinding proteincAMP response elementbinding protein CREBand CREBregulated transcription coactivator CRTC affect cellproliferation differentiation metabolism and other biologicalprocesses67 Increased CREB activity confers to tumor progressionchemotherapy resistance and reduced survival68 CREB is anotherwellestablished SIK downstream effector Although SIKs could notdirectly phosphorylate CREB they can inhibit CREB in a kinasedependent manner69 CRTC is a coactivator of CREB includingCRTC1“ and favors to stabilize CREB or directly contacts withCREB promoters70 CRTC is also helpful for the recruitment ofhistone acetyltransferase p300 for CREB transcriptional activitySIKs can directly phosphorylate CRTC block its association with and inhibit its nuclear transport where CRTC binds andenhances CREB driven gene transcription820313271 SIKs alsopromote COP1mediated CRTC1 ubiquitination and degradationby phosphorylating its multiple residues67 In keeping with thesefindings SIK2 could abrogate CRTC2 acetylation by phosphorylating p300 and integrate with the phosphorylation of CRTC2 tofacilitate COP1mediated CRTC2 ubiquitination and subsequentdegradation3272 Of note CREB could transcriptionally boost theexpression of Sik1 by binding its enhancer in mouse skeletalmuscle cells57 Thereforeit is possible that there is a negativefeedback loop between SIKs and CRTC“CREB signaling pathway to‚uence cellular malignanciesPME1Na KATPaseThe NaKATPase is widely distributedon the cell membrane and functions to transport sodium andpotassium ions and maintain the balance of osmotic pressure73The activated SIK1 phosphorylates PME1 causing its dissociationfrom the complex of PP2APME1 NaKATPase34 as a resultPP2A dephosphorylates NaKATPase and attains its catalyticactivity34 On the other hand SIKs also transcriptionally regulatethe NaKATPase either by directly inhibiting the entry of CRTCinto the nucleus to transcript ATP1B1 gene which encodes a NaKATPase subunit2 or by indirectly repressing the hormonesinduced NaKATPase expression74 via an increased CYP11Aand StAR mRNA levels to promote the adrenocorticotropichormone production5575“Other downstream signaling pathwaysTGFSmad pathwayIn normal epithelial cells TGFSmadsignaling pathway is considered to play an anticancer role byinducing cell cycle arrest and apoptosis78 However during thelate stage of tumorigenesis TGFSmad promotes cancer cell EMTand plays a pilot role in promoting cancer79 SIK1 is considered asa transcriptional substrate of TGFSmad pathway80 meanwhileactivated SIK1 may regulate the contraction phenotype of vascularsmooth muscle cells by inhibiting TGF1 signaling to prevent highsalt intakecaused hypertension81 Recent studies also indicatethat SIKs function as a negative feedback in the TGF signal byformatting the SIK1“Smad7“SMURF2 SMADspecific E3 ubiquitinprotein ligase complex to ubiquitinate ALK5 to repress TGFsignaling pathway8082 Notably high glucosemediated downregulation of SIKs results in the stabilization of ALK5 in mesangial 0cThe potent roles of saltinducible kinases SIKs in metabolic Sun et alFig The roles of SIKs in the regulation of glucose lipid metabolism and ‚ammation are illustrated SIKs™ regulatory effect is mainlythrough phosphorylating CRTC and class IIa HDACs to retain them in the cytoplasm Therefore SIKs repress various gene expression and theninhibit gluconeogenesis lipogenesis steroidogenesis and the production of IL10 Besides SIK2 upregulates GLUT4 expression by inhibitingtranscriptional repressor ATF3 leading to glucose uptake SIKs promote NFκb signaling pathway and production of ‚ammatory factors suchas IL1 IL6 IL12 TNFα and iNOS through downstream substrates CRTC However SIK1 and SIK3 inhibit the interaction of TRAF6 and TAB2and then repress NFκb signaling pathway Energy deprivation and hormone insulin glucagon and ACTH presence all control the activity ofSIKs kinase and regulate their effect on metabolismcells83 Furthermore one study suggested that SIK1 phosphorylated polarity protein partitioningdefective Par3 to promote itsdegradation via both proteasome and lysosome manners84 Arecent research also revealed that SIK inhibitors could repress theTGFmediated transcriptional capability of plasminogen activator inhibitor PAI1 and cellular apoptosis without affecting thephosphorylation or nuclear translocation of R“Smads complex85Of note this might be via SIK1 but not SIK2 or SIK3 to controlSmadassociated transcriptional cofactors via phosphorylatingCRTC85Hippo pathwayThe Hippo signaling pathway was conservativeand initially identified in drosophila which plays a major role incontrolling an size86 SIK2 and SIK3 have been proved to beupstream regulators of the Hippo signaling pathway in drosophilaMechanisticallythey can directly phosphorylate the scaffoldprotein Salvador Sav a core component of Hippo complex toprevent the oncogene driven inhibition of Yki an ortholog of Yesassociated protein YAP87 As an important hub of Hipposignaling pathway YAP activation leads to the inhibition of cellcontact and facilitates tumor cell metastasis8688 Specifically SIK2can directly trigger YkiYAP transcription activity to increase theYkiYAP target gene expression and promote tissue overgrowth87indicating the potential oncogenic role of SIK2 in tumorigenesisNFκb signaling pathway NFκb signaling pathway is one of thewellestablished ‚ammatory pathways by which SIKs couldmanipulate the production of‚ammatory factors Fig Meanwhile CRTC and class IIa HDACs two important downstreamregulate NFκb signalingsubstratespathway89“ However it is noteworthy that the effects of SIK1and SIK3 on NFκb signaling pathway seem to be distinct theyprefer to inhibit the binding of TAB2TRAF6 to repress the NFκbsignal92negativelyofSIKsPI3KAKT signaling pathwayTo date the correlation betweenSIKs and AKT signaling pathway is focused on SIK2 Fig SIK2 leads to a decrease of AKT phosphorylationwhich may be due to the SIK2mediated IRS1 phosphorylationthus inhibiting the insulin signaling pathway30 However in tumorcells the effect of SIK2 on the AKT signaling pathway seems to bechanged to elevating PI3KAKT activity7101193 MechanisticallySIK2 can directly phosphorylate p85 a regulatory subunit of PI3Kcomplex to activate the AKT kinase activity7 As a result SIKinhibitors such as ARN3236 can efficiently reduce AKT phosphorylation and activation in ovarian cancer cells10 Howeverthere is no compelling evidence proven that SIK2 could directlybind and regulate AKT kinase activity7101193 therefore the directconnection between SIKs especially the SIK1 and SIK3 and AKTneed to be further exploredBIOLOGICAL FUNCTIONS OF SIKS IN METABOLICHOMEOSTASISSIK functions in gluconeogenesisGluconeogenesis is a biological process in which noncarbohydrateprecursors including lactic acid glycerin amino acids etc aretransformed into carbohydrates including glucose or glycogenwhich can be manipulated by insulin and glucagon controlled theexpression of glucose6phosphatase G6Pase and phosphoenolpyruvate carboxykinase PEPCK293194 Specifically PEPCK andG6Pase control the initial and final steps of gluconeogenesisrespectively95 Proliferatoractivated receptor gamma coactivatorPGC1α a directtarget of CREB can largely elevate theexpression of PEPCK and G6Pase96 Meanwhile PGC1α is alsoassociated with histone acetyltransferase HAT p30097 and servesas a key regulator in liver gluconeogenesis and the focal target ofcAMPPKACREB axis98 On the other hand insulin can block theeffect of PGC1α and interfere with the activation of gluconeogenesis through AKTmediated forkhead box FoxO1 phosphorylation99 Furthermore insulin can also regulate the activity of PGC1α by governing its acetylation and phosphorylation100101SIKs and their substrates such as CRTC and class IIa HDAC arelargely involved in gluconeogenesis Fig SIK1 was firstfound to inhibit gluconeogenesis in the hepatocytes and itsmRNA and protein levels under fasting conditions increasedSignal Transduction and Targeted Therapy 0cfourfold relative to feeding conditions31 Moreover overexpression of SIK1 in primary hepatocytes suppressed forskolin or cAMPinduced an increase in Pck1 gene expression via phosphorylationof CRTC231 Subsequent studies have proven that SIK2 and SIK3have a similar effect3233102 Dentin et al reported that SIK2 is adownstream substrate of PI3KAKT signaling pathway response toinsulin subsequently followed by CRTC2 phosphorylation32 Itohet al102 showed that SIK3 knockout in hepatocytes was associatedwith elevated mRNA of Pgc1a Pepck and G6pc gene Collectivelyall three SIK isoforms can inhibit gluconeogenesis possibly via SIKmediated CRTC phosphorylation and restriction in the cytoplasm2054 CRTC played a key role in gluconeogenic by bindingCREB to transcriptionally promote gluconeogenic genes expression such as G6PC PEPCK1 and PGC1α gene29103 By contrast inthe case of starvation glucagon can also inhibit the catalyticactivity of SIKs via PKAmediated phosphorylation and facilitategluconeogenesis29 On the other hand SIKs directly phosphorylated class IIa HDACs to block their nuclear translocation2029 andinteraction with FoxO1 on PEPCK and G6Pase promoter regionsthereby stimulating gluconeogenesis104 Conceivably SIK inhibitors could compromise the phosphorylation of CRTC23 andHDAC45leading to gluconeogenic gene expression andglucose production203354 As such lossoffunction mutations ofSIKs or deficiency ofantagonizegluconeogenesis202933could efficientlyLKB1While SIKs have markedly involved in diverse signaling pathways to regulate gluconeogenesis several studies demonstratedthat SIK1 and SIK2 did not impact gluconeogenesis alone inmouse model33105 Of note in liver specific Sik1 and Sik2 doubleKO mice CRTC phosphorylation and gluconeogenesis were not‚uenced33105 instead SIK3 plays a key role in regulatinggluconeogenesis rather than SIK1 and SIK2102 Under the conditions of lactateinduced gluconeogenesis the blood glucose levelof Sik3 but not Sik1 and Sik2 KO mice were rapidly increased thanthat of WT mice indicating that Sik3 plays an important role inmouse gluconeogenesis66102 Though SIKs display a controversialrole in gluconeogenesisit is generally accepted that SIKs canreduce insulin sensitivity and promote energy storage byinhibiting gluconeogenesisareThese proteinsSIK functions in glucose uptakeThe process of glucose uptake mainly depends on the expressionof sodiumdependent glucose transporter and glucose transporterGLUT106 The majority of peripheral glucose uptake in adiposetissue and skeletal muscle are achieved by insulinresponsiveglucose transporter GLUT4106 Importantly GLUT4 expressionhas been negatively regulated by various upstream regulatorsincluding but notlimited to HDAC4 CRTC23 and proteinphosphatase 2A PP2A107all wellestablished SIK2 downstream substrates indicating that SIK2 is apositive regulator of glucose intake by upregulating GLUT4expression Fig “ Meanwhile CREB upregulated theexpression of transcriptional repressor activating transcriptionfactor ATF3 and thereby downregulated the GLUT4 resultingin promoting insulin resistance107110 Consistently inactivating SIKpharmacologically or genetically could reduce GLUT4 expressionand glucose uptake107“ However SIK1 promotes insulinresistance and inhibits glucose uptake in skeletal muscle possiblyvia directly phosphorylating insulin receptor substrate IRS Sik1 KO did not lead to hyperglycemia and gluconeogenesissignificantly improved glucose toleranceperipheral insulin sensitivity and skeletal muscle glucose uptakeupon highfat diet due to elevated expression of GLUT4 GLUT1and GLUT12105in vivo butSIK functions in lipid metabolismIn addition to its role in glucose metabolism SIKs also seems tofunction as a negative regulator of lipid metabolism Fig LipidSignal Transduction and Targeted Therapy The potent roles of saltinducible kinases SIKs in metabolic Sun et alis an important source of energy and substance for cell homeostasis and its metabolic process is tightly regulated by a complexnetwork112 The fatty acid a vital and raw materialfortriglycerides112 is synthesized mainly by two key enzymesacetylCoA carboxylase ACC and fatty acid synthase FAS112SIK1 represses lipogenic gene expression such as Acaca acetylCoA carboxylase Fasn FAS Srebf1 sterol regulatory elementbinding transcription factor and Scd1 stearoylCoA desaturase possibly via an SREBPs sterol regulatory elementbindingproteinmediated transcriptional regulation113 Overexpression ofSIK1 in hepatocytes induced high mRNA levels of the lipogenicgene Srebf1 Fasn and Scd1 and high protein levels of ACC andFAS113 SREBP1c is directly phosphorylated by SIK1 at Ser329which is proposed to be required for SIK1 in repressing lipogenicgene expression113 Steroids are another kind of lipid includingestrogen progesterone and adrenocorticotropic hormone77114Steroidogenic acute regulatory protein StAR and cytochromeP450 cholesterol side chain cleavage P450scc are two keyenzymes in steroidogenesis77114 StAR regulates the transport ofcholesterol from the outer membrane to the inner membrane inmitochondria which is the key ratelimiting step of steroidsynthesis114 In addition CYP11A gene encodes P450scc acholesterol side chain cleavage enzyme that catalyzes theconversion of cholesterolto pregnenolone a precursor ofsteroid77 SIK1 plays a key role in steroidogenesis and adipogenesis mediated by governing ACTH signaling pathway2255115 ThemRNA levels of SIK1 in mouse adrenal cortex cells Y1 cellsstimulated by ACTH peaked rapidly within h then decreasedgradually and returned to the basic level after h However themRNA levels of P450scc and StAR began to rise after a few hoursreaching the highest levels after h115 The transcription of SIK1occurred before the ACTH stimulated StAR and P450scc transcription so it can be speculated that SIK1 is associated withsteroidogenic gene expression115 On the other hand SIK1overexpression significantlyrepressed the ACTHdependentexpression of P450scc and StAR in Y1 cells115 Subsequent studiesdemonstrated that SIK1 repressed the efficient operation of theCREB transcription activation complex thereby inhibiting the CREdriven transcription of the CYP11A gene and the StAR gene inY1 cells5575“Du et al116 found that similar to SIK1 SIK2 can also repress theexpression of lipogenic genes FAS ACC2 and SCD1 and thiseffect can be reversed by depleting SREBP1In addition SIK2promotes fatty acid synthesis by upregulating SREBP1c expression thus promoting the transcription of Fasn in ovarian cancercells93 Meantime SIK2 also promotes cholesterol synthesis byupregulating SREBP2 expressionto transcriptionally elevatecholesterol synthetase 3hydroxy3methylglutarylcoenzyme Areductase HMGCR93Importantly SIK2 phosphorylated andinhibited p300 activity leading to the decreased acetylation ofcarbohydrate response elementbinding protein ChREBP whichplays a positive role in lipogenic and gluconeogenesis60 SIK2 alsophosphorylated IRS1 to attenuate insulin driven lipogenesis inhuman adipocytes55 Another study also showed depletion of SIK2promoted increased adipogenic potential and insulin resistance inpreadipocytes in a CRTC2dependent manner110 SIK2 controlledFAO in liver and skeletal muscle as such Sik2 KO mice displayedthe decreased key enzymes in the process of FAO such ascarnitine carnitine palmitoyltransferase CPT1 mitochondrialmedium chain acyl COA dehydrogenase MCAD and peroxisomalacylCoA oxidase ACOX1110 Inconsistently SIK2 promotes FAOby phosphorylating ACC1 and inhibiting CPT1A in ovarian cancercells resulting in promoting abdominal metastasis7 In additionsome studies showed that Sik2 KO mice do not impact lipidmetabolism in vivo33SIK3 has been reported as a new energy regulator by promotinglipid storage in Drosophila through compromising the activity ofHDAC4 and CRTC20117 SIK3 also regulated cholesterol and bile 0cThe potent roles of saltinducible kinases SIKs in metabolic Sun et alTable The summary of SIK mouse modelsSIK members Mouse model typesFunctional characteristicsSIK1SIK1SIK1SIK1SIK1SIK2SIK2SIK2SIK2SIK3SIK3SIK3SIK3SIK3SIK3Sik1flflGDF9Cre miceSik1flflTBGCre miceSik1flflMyf5Cre miceSik1ˆ’ˆ’ mice generated from Sik1 KO ES cellsKSik1 KrasLSLG12DR26LSLluclucSik1flfl miceKTH11LSLCas9 KrasLSLG12DR26LSLTomatoH11LSLCas9 miceSik1ˆ’ˆ’ miceSik2ˆ’ˆ’ mice Sik2lacZSik2flflCreˆ’ miceSik2ˆ’ˆ’ mice generated from Sik2 KO ES cellsSik2ˆ’ˆ’ mice generated from Sik2 KO ES cellsSik3ˆ’ˆ’ mice generated from Sik3 KO ES cellsSik3ˆ’ˆ’ mice generated from Sik3 KO ES cellsSik3flflCol11α211EnhCre and Col11α2ERCre miceSik3ˆ’ˆ’ mice generated from Sik3 KO ES cellsSik3ˆ’ˆ’ mice generated from Sik3 KO ES cellsSIK1T182A SIK2T175A and SIK3T163A single anddouble KI mice created by ES cells gene targetingtechnologiesAbnormal glucose metabolismElevated insulin secretion and more osteogenic potentialIncreased tumor size and burdenIncreased tumor sizeHigh blood pressureHyperglycemia and hypertriglyceridemianormal glycemiaEnhanced neuronal survivalPreventing left ventricular hypertrophyDystrophic including lipodystrophy hypolipidemia hypoglycemiaand hyperinsulinemia with cholestasis and cholelithiasisphenotypeDwarfism and skeletal deformitiesAchondroplasia and resistance to the osteoarthritisAbnormal circadian rhythmsPro‚ammatory phenotypeAnti‚ammatory phenotypeReferencesacid metabolism by combining with retinoic acid metabolism andmight alter energy storage in mice118 Inhibition of fatty acidsynthesis was observed in Sik3 KO mice118 however the roles ofSIK3 in regulating lipid metabolism are not good evaluated inmammal animals20 Based on these observations although SIKshave been considered to play important roles in lipid metabolismthe mechanism of SIKs regulating lipid metabolism has not beenwell elucidated yet Thus more efforts are desired in the future toexplore the diverse and distinct roles of SIK family members inlipid metabolic homeostasisSIK functions in ‚ammationInflammation is an important pathological change tightly relatedto tumorigenesis Inflammation predominantly changes the tumormicroenvironment and accelerates tumor occurrence growth andmetastasis119 An important aspect of controlling ‚ammation isreprogramming macrophages to promote transformation fromclassic activated macrophage M1 macrophage to regulatorymacrophage M2 macrophage120 Of note SIKs act as molecularin regulating M1“M2 macrophage transformationswitchesFig The observation that SIK inhibitors compromisedCRTC3 phosphorylation in TLRstimulated macrophagesled toincreased CREBdependent gene expression including IL10 andreduced pro‚ammatory cytokine expression such as TNFα andIL6871 Importantly IL10 drives an anti‚ammatory function bypromoting the expression ofregulatory M2b macrophagemarkers such as SPHK1 LIGHT and Arg18120 Similar results werealso observed in dendritic cells DCs71 On the other hand SIKinhibitors decreased the production of pro‚ammatory cytokines but not IL10 in IL1mediated macrophages possibly dueto the insufficient CRTC3 phosphorylation71 Moreover otherupstream regulators for example prostaglandin E2 PGE2 alsoinduced IL10 production via the PKA“SIK“CRTC signaling pathway in the quiescent

Thyroid_Cancer

Lasting and SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaMarissa R Keever1 Pan Zhang2 Courtni R Bolt1 Adrienne M Antonson1Haley E Rymut1 Megan P Caputo1 Alexandra K Houser1 Alvaro G Hernandez3Bruce R Southey1 Laurie A Rund1 Rodney W Johnson14 andSandra L RodriguezZas12456 Department of Animal Sciences University of Illinois at UrbanaChampaign Urbana IL United States Illinois InformaticsInstitute University of Illinois at UrbanaChampaign Urbana IL United States Highthroughput Sequencingand Genotyping Unit Roy J Carver Biotechnology Center University of Illinois at UrbanaChampaign Urbana ILUnited States Neuroscience Program University of Illinois at UrbanaChampaign Urbana IL United States Departmentof Statistics University of Illinois at UrbanaChampaign Urbana IL United States Carl R Woese Institute for GenomicBiology University of Illinois at UrbanaChampaign Urbana IL United StatesThe prolonged and sexdependent impact of maternal immune activation MIA duringgestation on the molecular pathways of the amygdala a brain region that ‚uencessocial emotional and other behaviors is only partially understood To address thisgap we investigated the effects of viralelicited MIA during gestation on the amygdalatranscriptome of pigs a species of high molecular and developmental homology tohumans Gene expression levels were measured using RNASeq on the amygdalafor 3weekold female and male offspring from MIA and control groups Amongthe genes that exhibited significant MIA effect a prevalence of differentiallyexpressed genes annotated to the neuroactive ligand“receptor pathway glutamatergicfunctions neuropeptide systems and cilium morphogenesis were uncovered Genesin these categories included corticotropinreleasing hormone receptor glutamatemetabotropic receptor glycoprotein hormones alpha polypeptide parathyroidhormone receptor vasointestinal peptide receptor neurotensin proenkephalinand gastrinreleasing peptide These categories and genes have been associatedwith the MIArelated human neurodevelopmental disorders including schizophreniaand autism spectrum disorders Gene network reconstruction highlighted differentialvulnerability to MIA effects between sexes Our results advance the understandingnecessary for the development of multifactorial therapies targeting immune modulationand neurochemical dysfunction that can ameliorate the effects of MIA on offspringbehavior later in lifeKeywords immune activation pigs RNAseq neuropeptides glutamatergic pathway GABAergic pathwayINTRODUCTIONThe maternal immune response triggered by pathogens and other environmental stressors duringgestation can also elicit an indirect response by the fetal immune cells Kroismayr Odorizzi and Feeney Prins Viral infection during gestation for exampleactivates a cytokinerelated signaling cascade and molecules from this process can cross theEdited byNo¨lia Fern ndezCastilloCentre for Biomedical NetworkResearch CIBER SpainReviewed bySilvia PellegriniUniversity of Pisa ItalyTewarit SarachanaChulalongkorn University ThailandCorrespondenceSandra L RodriguezZasrodrgzzsillinoiseduSpecialty sectionThis was submitted toNeurogenomicsa section of the journalFrontiers in NeuroscienceReceived May Accepted July Published August CitationKeever MR Zhang P Bolt CRAntonson AM Rymut HE Caputo MPHouser AK Hernandez AGSouthey BR Rund LA Johnson RWand RodriguezZas SL Lastingand SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaFront Neurosci 103389fnins202000774Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaplacenta and reach the fetal brain The resulting maternalimmune activation MIA can impactfetal developmentalprocesses and exert longterm postnatal eï¬ects in the oï¬springRutherford The relationship between MIAand neurodevelopmental disordersincluding schizophreniaspectrum disorders SSD and autism spectrum disorders ASDand neurodegenerative disorders such as Alzheimer™s diseaseAD in oï¬spring has been established Knuesel Canetta Mattei These diseases sharesome behavior symptoms comorbidities such as eating disordersand genetic and environmental ie MIA agents Canitanoand Pallagrosi The previous neurological disorders havebeen associated with abnormal structure and dysregulationof the amygdala Schumann FernandezIrigoyen and share genes and molecular mechanismsincluding histocompatibility complex MHC genes Andersand Kinney glutamatergic and GABAergicassociatedgenes Bourgeron Marin Li andmitochondrial activity processes Pieczenik and Neustadt Sragovich socialinteractionThe fetal amygdala is susceptible to ‚ammatory signals andthe plasticity of this brain structure to MIA can lead to alterationsof the developmental trajectory These disruptions may havelonglasting and maladaptive consequences for the oï¬springdue to the significant role that the amygdala plays in manyneurological pathways Located in the forebrain the amygdala‚uencescognition neuroendocrinebehavior learning memory emotion and autonomic systemsThe amygdala also modulates the response of these processesto stressors including pathogenic infections and those resultingfrom management practices such as weaning Tian The amygdala experiences high uptake of gonadalhormones and is anatomically connected to other sexuallydimorphic nuclei Therefore this brain region is involved inregulation of several dimorphic functions such as aggressionsexual behavior gonadotropin secretion and integration ofolfactory information Hines Evidence supports thediï¬erential activation of the amygdala to stimuli between malesand females Killgore and YurgelunTodd includingdiï¬erences in the sexual responses and emotional memoryHamann and diï¬erential vulnerability to insult Baird Due to the interconnected and multiregulatorynature of this brain structureinsults to the amygdala canimpact the individual™s social locomotor and feeding behaviorPetrovich and Gallagher growth and reproductivephysiology health status and immunological response tosecondary stressorsRecent studies lend support to the link between MIA andaltered amygdala function Carlezon In miceMIA elicited by polyinosinicpolycytidylic acid [PolyIC]increased the synaptic strength of glutamatergic projectionsfrom the prefrontal cortex to the amygdala Li In field tests mice exposed to MIA spentless timein the center and traveled a higher distanceindicativeof a higher anxiety behavior incidence than the controlcounterparts These findings suggest that the change in thebalance between excitation glutamatergic and inhibitiontherefore aï¬ecting brain circuitsspike output offeedforward GABAergic modified theamygdala neuronsthatcould regulate behavior in SSD and ASD A candidate genestudy of the eï¬ects of social stress during gestation reportedthatthe expression of a corticotropinreleasing hormonereceptor in the amygdala of 10weekold pigs was higherin females than in males Rutherford Thisstudy concluded that prenatal stress substantially increasedanxietyrelated behaviorstheimpact of maternal stressors during gestation on specificamygdala molecular profiles and associated neurological orbehavioral disorders in the oï¬spring later in life highlightthe complexity of the molecular mechanisms underlying thepathophysiology of MIAin female pigs Studies ofetalvirusadvantages ofrodents when consideringstudying a pig modelto pigsResearch on the lasting eï¬ects of MIA in pigs complementsAntonson et althe insights oï¬ered by rodent modelsstem Theratherfrom the greater homology of humansan physiologythan toin particular brain growth andsize development anddevelopment processesMurphy A pigmodel that has oï¬ered insights into MIA employs porcinereproductivePRRSVtothein the brain andmicroglia ie macrophagelike cellsisin neonatal pigsAntonson associated with behavioralelicit MIA Thisand respiratorysyndromechallengeactivatesimmunechangesThe study of MIA elicited by PRRSV allows for thecharacterization of the impact of a live viral pathogen thatselfreplicates in the host evoking extended activation ofimmune pathways PRRSV challenge during gestation is a wellcharacterized replicable and eï¬ective method for inducingMIA in pigs Antonson In additionPRRSV outbreaks impose a major economic burden to thelivestock industry PRRSV is an enveloped singlestranded RNAvirus thatinfects alveolar macrophages causing interstitialpneumonia and increased serum levels ofthe cytokinesinterleukin betainterleukin and tumor necrosis factoralpha Antonson The persistent repercussionsof MIA on the molecular pathways ofthe pig amygdalaare yet to be investigated Moreover the potentially distinctvulnerability to the prolonged eï¬ects of MIA between sexesremains unknownThe overarching goal of the present study is to advancethe understanding of the impact of MIA on the molecularmechanisms ofthe amygdala Three supporting objectivesare explored a characterization of prolonged transcriptomechanges elicited by viral MIA in pigs a species that hashigh neurodevelopmental homology with humansandfood production valueidentification of molecularpathwaysthat present diï¬erential vulnerability to MIAbetween sexes and c understanding the eï¬ect of MIA onmolecular interactions assisted by gene network inferenceThe findings from these complementary analyses supportthe use of multipleto amelioratethe potential detrimental eï¬ect of MIA on the oï¬springphysiology and behaviortherapeutictargetsbFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaMATERIALS AND METHODSAnimal ExperimentsAll experimental procedures used published protocols Antonson The animal studies were approved by theIllinois Institutional Animal Care and Use Committee IACUCat the University of Illinois and are in compliance with the USDAAnimal Welfare Act and the NIH Public Health Service Policy onthe Humane Care and Use of AnimalsCamborough gilts born and raised at the University of Illinoisat UrbanaChampaign herd were inseminated at days ofage using PIC boar sperm Antonson All gilts were PRRSV negative and were moved at gestationday GD into diseasecontainment chambers maintainedat —¦C and a h lightdark cycle with lights on at AM The gilts were fed daily kg of a gestational diet andhad ad libitum water access One week after acclimation fourgilts were intranasally inoculated with live PRRSV strain P129BV School of Veterinary Medicine at Purdue University WestLafayette IN United States using mL of — median tissueculture infectious dose TCID50 diluted in sterile Dulbecco™smodified Eagle medium DMEM mL total volume Thefour gilts in the Control group were intranasally inoculatedwith an equal volume of sterile DMEM PRRSV inoculationcorresponded to the last third of gestation in pigs and humansduring initiation of rapid fetal brain growth Antonson PRRSV and Control groups were housed in separatecontainment chambersThe rectal temperatures and diet consumption of the giltswere recorded daily until farrowing Antonson The PRRSVinoculated gilts were oï¬ered the maximumfed daily and feed refusal was measured The Control giltswere fed the same amount consumed by the PRRSVinoculatedgilts on the previous day The daily body temperature andfeed intake levels were compared using a mixedeï¬ects modelanalyzed with PROC MIXED SAS Institute Inc Cary NCUnited States The model included the eï¬ects of gilt treatmentand replicate while accommodating for heterogeneity of variancebetween MIA groupsFarrowing was induced with an intramuscular injection of mg of Lutalyse dinoprost tromethamine Pfizer New YorkNY United States on GD in consideration that the averagegestation length is approximately days Antonson Gilts farrowed in individual farrowing crates ofstandard dimensions — m After farrowing thegilts were fed twice a day up to kg of a nutritionallycomplete diet for the lactating period and water remainedavailable ad libitum Pigs received intramuscular injections ofiron dextran mgpig Butler Schein Animal Health DublinOH United States and Excede for Swine mgpig ZoetisParsippany NJ United States to control for respiratory diseasesThe pigs remained with their mothers until PD The bodyweight of pigs was measured daily and analyzed using the mixedeï¬ects model in SAS PROC MIXED SAS Institute Inc CaryNC United States The model included the eï¬ect of MIA andthe random eï¬ect of gilt accommodating for heteroscedasticitybetween pig treatment and sex groups The impact of MIA wasstudied at PD because this is a common age to wean pigsThe study of transcriptome profiles from older pigs could beconfounded with changes in diet and environment associatedwith weaning while profiles from younger pigs would hinder theassessment of the prolonged eï¬ects of MIARNA Extraction and SequencingA balanced experimental design was studiedincluding pigs evenly distributed between maternal PPRSV activatedMPA group of pigs and Control gilts CON group of pigseach group encompassing males and females denoted Maand Fe respectively At PD pigs were removed fromthe farrowing crate and anesthetized intramuscularly using atelazolketaminexylazine drug cocktail mg of tiletamine mg of zolazepam reconstituted with mL ketamine gL and mL xylazine gL Fort Dodge AnimalHealth Fort Dodge IA United States at a dose of mLkgbody weight following protocols Antonson Following anesthetization pigs were euthanized using anintracardiac injection of sodium pentobarbital mgkg bodyweight Fata Plus Vortech Pharmaceuticals Dearborn MIUnited States Pig brains were extracted the amygdalae wererecognized using the stereotaxic atlas of the pig brain Felix dissected out flash frozen on dry ice and stored at ˆ’—¦Cfollowing published protocols Antonson RNA wasisolated using EZNA isolation kit following the manufacturer™sinstructions Omega Biotek Norcross GA United States TheRNA integrity numbers of the samples were above indicatinglow RNA degradation The RNASeq libraries were preparedwith TruSeq Stranded mRNAseq Sample Prep kit Illumina IncSan Diego CA United States The libraries were quantitatedby qPCR and sequenced on one lane on a NovaSeq for cycles from each end of the fragments using NovaSeqS4 reagent kit FASTQ files were generated and demultiplexedwith the bcl2fastq v220 conversion software Pairedend reads nt long were obtained and the FASTQ files are availablein the National Center for Biotechnology Information GeneExpression Omnibus GEO database experiment accessionnumber GSE149695RNA Sequence Mapping and DifferentialExpression AnalysisThe average Phred quality score of the reads assessed usingFastQC Andrews was across all read positions andtherefore no reads were trimmed The pairedend reads fromthe individual samples were aligned to the Sus scrofa genomeversion Sscrofa Pruitt using kallisto v0430Bray with default settings The normalized trimmedmean of Mvalues gene expression values were described usinga generalized linear model encompassing the eï¬ects of the MIAgroup MPA or CON levels sex Fe or Ma levels and MIAbysex interaction and analyzed using edgeR version in the R v environment Robinson Genessupported by transcripts per million TPM by each MIA“sex combination were analyzed to ensure adequate representationacross comparisonsFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaOrthogonal pairwise contrasts between MIA and sex groupswere evaluated in addition to testing for the eï¬ects of MIAbysex interaction and main eï¬ects of MIA and sex Thefour groups compared in the contrasts identified by treatmentfollowed by the sex levels are MPA_Fe MPA_Ma CON_Fe andCON_Ma The Pvalues were adjusted for multiple testing usingthe Benjamini“Hochberg false discovery rate FDR approachBenjamini and Hochberg categoriesamongtheMFand KEGG pathways The GeneFunctional Enrichment and NetworkInferenceapproaches were used to identifyTwo complementaryoverrepresented functionalgenesexhibiting diï¬erential expression across MIA and sex groupsCaetanoAnoll©s GonzalezPena et al2016ab Functional categories investigated included GeneBPs GO molecularOntology GO biological processesfunctionsSetEnrichment Analysisapproach implemented inthesoftware package GSEAP Subramanian was used to identify category overrepresentationwith gene over and underexpressed while considering allgenes analyzed The normalized enrichmentscore NESofin the Molecular Signature DatabaseMSigDB was calculated using the maximum deviation ofthe cumulative sum based on the signed and standardizedfold change The statistical significance ofthe enrichmentwas assessed using the FDRadjusted Pvalue computed from permutationscategoriesGSEAtheThe overrepresentation of functional categories was alsoevaluated among genes that exhibited a significant MIAbysex interaction or main eï¬ect using the Database forAnnotation Visualization and Integrated Discovery DAVID Huang The enrichment of Direct GOcategories in the DAVID database was assessed The Susscrofa genome was used as the background for enrichmenttesting and enrichmentis reported using the ExpressionAnalysis Systematic Explorer EASE score that was computedusing a onetailed jackknifed Fisher hypergeometric exacttest Functional categories were clustered based on geneannotation and the statisticalissummarized as the geometric mean of the log10 EASE scoresofthe categories Delfino Serao Delfino and RodriguezZas significance of clustersWeighted Gene Coexpression NetworkAnalysis and Gene Network VisualizationAn approach complementary to the identification of diï¬erentiallyexpressed genes was used to uncover coexpression networksusing Weighted Gene Coexpression Network AnalysisWGCNA version Langfelder and Horvath The input data were voomtransformed read count valuesgenerated using the limma package version Ritchie in R version Genes were filtered to removethose with low expression levels or no variation across samplesper developer recommendations The number of genes usedfor network analysis was genes Considering potentialfor interaction patterns a sexdependent softthresholdingpower was used to call for network topology analysis Thelowest power values that support a scalefree topology powerused were for the CON_MaMPA_Ma contrast and for the MPA_FeMPA_Ma contrast The Pearson correlationcoefficient ofthe normalized expression values was usedto identify modules of connected genes The minimummodule size was set to with the deepSplit set to and themergeCutHeight set to Module profiles were identifiedusing the correlation between the eigengene of each moduleand pig group Enrichment of functional categories among thegenes in each module profile was explored with DAVID using theSus scrofa genome as background and testing included an FDRmultiple test adjustmentFurther understanding of the impact of the MIAbysexinteraction was gained through the reconstruction of genenetworks using the BisoGenet package Martin inthe Cytoscape platform Shannon Information fromgene and protein interactions annotated in databases includingBIOGRID HPRD DIP BIND INTACT and MINT was usedto visualize relationships between genes Salwinski Alfarano Mishra Stark Kerrien Licata Networks highlightingdiï¬erences in gene levels associated with MIA within sex iethe contrasts MPA_MaCON_Ma and MPA_FeCON_Fe werecompared The network framework includes genes that exhibiteda significant MIAbysex interaction eï¬ect FDRadjusted P and are annotated to enriched functional categoriesThe framework genes were identified by full nodes with sizereflecting the diï¬erential expression level between the MPAand CON groups The network edges depict known molecularrelationships curated in the BisoGenet databases The frameworkgenes were connected through correlated genes listed in theBisoGenet database of molecular interactions that did not reachsignificant MIAbysex interaction eï¬ect The comparison ofthese networks oï¬ered insights into the simultaneous eï¬ect ofMIA across interacting genes and enabled the detection of sharedand distinct coregulation patterns between MPA and CONpigs across sexesRESULTSMaternal Immune Activation andSequencing MetricsThe diï¬erences between MPA and CON giltsin rectaltemperatures and daily diet consumption indicated the activationof the maternal immune system in response to PRRSV Thediï¬erence in body temperature between CON and MPA giltson GD was —¦C standard error —¦C P Thediï¬erence in feed refusal between CON and MPA gilts on GD was g standard error g P A significantincrease in rectal temperatures and decrease in feed intakeP was observed within h of inoculation and returnedto baseline levels within days for body temperature and within days for feed intake At days of age CON pigs were kgFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaheavier than MPA pigs standard error P whileno significant sex or interaction eï¬ects were detectedThe sequencing of the RNA samples produced billionsequenced reads and million pairedend reads per sampleThe number of reads was consistent across MIA and sexgroups coefficient of variation and the eï¬ects ofMIA sex and MIAbysex interaction were tested on genes that surpassed the minimum number of reads per MIA“sex combinationTranscriptome Changes Associated WithMaternal Immune Activation That AreSexDependentOverall genes exhibited a significant FDRadjusted P MIAbysex interaction eï¬ect and among these genes hada significant eï¬ect at FDRadjusted P The profile ofthese genes indicated that the eï¬ect of MIA diï¬ered betweenfemales and males Fortysix genes that presented a MIAbysex interaction eï¬ect are listed in Table together with theirexpression pattern and Pvalue The majority of the genes inTable including neurotensin NTS displayed a reversal inthe expression level between CON and MPA groups across sexesie opposite Log2[fold change] sign across sexes An extendedlist including genes that exhibited a MIAbysex interactioneï¬ect at FDRadjusted P is provided in SupplementaryFile S1 Table AAnother frequent pattern among the genes that displayed aMIAbysex interaction eï¬ect was characterized by a consistentexpression profile between CON and MPA across sexes albeitthe magnitude diï¬ered between sexes Table For exampleglycoprotein hormones alpha polypeptide CGA was overexpressed in CON relative to MPA but the diï¬erential washigher in males than in females Other genes presentingthis pattern included guanylatebinding protein GBP1transthyretin TTR aldehyde dehydrogenase family memberA2 ALDH1A2 hemoglobin subunit beta HBB and basichelixloophelix family member e22 BHLHE22GRPNotable is the significant MIAbysex interaction eï¬ecton genes associated with neuropeptides and hormones andgenes that participate in glutamatergic processes Genes underexpressed in MPA relative to CON males while presentingthe opposite pattern in females Table included NTS theneuropeptide gene proenkephalin PENK the neuropeptidegene gastrinreleasing peptidethe neuropeptiderelated gene vasoactive intestinal peptide receptor VIPR2corticotropin releasing hormone receptor CRHR2 neuronderived neurotrophic factor NDNF reelin RELN glutamatemetabotropic receptor GRM4 solute carrier family member SLC17A6 calcium voltagegated channel auxiliarysubunit alpha delta CACNA2D3 EFhand domain familymember D1 EFHD1 glutathione peroxidase GPX3parathyroid hormone receptor PTH1R thyroid hormoneresponsive THRSP and CGA The CGA gene codes for thealpha subunit protein of the hormones chorionic gonadotropinCG luteinizing hormone LH folliclestimulating hormoneFSH and thyroidstimulating hormone TSHFunctional and Network Analysis ofGenes That Exhibit SexDependentAssociations With Maternal ImmuneActivationThe genes expressing significant MIAbysex interaction eï¬ectswere analyzed for functional enrichment Table presentsthe clusters of most enriched and informative categoriesfrom the DAVID analysis and the complete list of categoriesis in Supplementary File S1 Table B The categories inTable encompass genes presenting the mostfrequentinteraction profile characterized by underexpression in CONfemales relative to males but overexpression in MPA femalesrelative to males These genes include KEGG Autoimmunethyroid diseaseCluster and BP brain developmentGO0007420 Cluster Enrichment results from GSEA complemented the findingsfrom DAVID Highly enriched informative categories amonggenes that have a MIAbysex interaction eï¬ect are presentedin Table and the extended list of categories is presented inSupplementary File S1 Table C The categories in Table in Table including ion homeostasissupport pathwaysTable and regulation of voltagegated calcium channelactivity processesenrichmentinteraction pathwayofthe neuroactiveand the hormoneactivity processesinclude genes such as CGA and VIPR2 that were identifiedin Table ligand receptorand neuropeptideTable Notablythethe diï¬erentialNetwork visualization furthered the understanding ofthe impact of MIA on the relationships among genes thatexhibited a significant MIAbysex interaction eï¬ect Thenetworks in Figures depictthe relationships betweengenes in the enriched neuroactive ligand receptor pathwaythat highlightexpression between CONand MPA in males and females ie CON_MaMPA_Maand CON_FeMPA_Ferespectively Red andrectangular nodesblueframework genes andthe known associations between genesedgesrepresentbased on curated databases of molecularinteractionsRed and blue nodes denote over or underexpressionof the gene in CON relative to MPA and the size is anthe diï¬erential expressioninverse logarithmic function ofPvalue Thediï¬erentialexpression pattern and connectivity among genes highlightsthe discrepancyelicited by MIAbetween the sexesin network modulescontrastsrepresentsimultaneousstudytheofTranscriptome Changes Associated WithMaternal Immune ActivationOverall genes exhibited diï¬erential FDRadjusted P expression between MPA and CON pigs irrespective of sexTable lists notable highly diï¬erentially expressed genesis in Supplementary File S1 Tableand the complete listD The majority of these genes were overexpressed in MPArelative to CON pigs Among the genes overexpressed in MPAcompared to CON pigs were islet amyloid polypeptide IAPPFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaTABLE Genes exhibiting significant FDRadjusted Pvalue maternal immune activationbysex interaction effectGene symbolPvalueaCON FeCON Ma MPA FeMPA MaCON FeMPA FeCON MaMPA MaCON FeMPA MaCON MaMPA FeRGS16CGAPOMCGPX3RELNVIPR2ANKRD34CGBP1GRM4CCDC136SLC17A6BTBD11TTRCACNA2D3CRHR2NDNFCXCL12USP43CCDC17KCNIP4CAMK2N2ALDH1A2GRPPENKSYT12PTH1RHBBESYT1EFHD1BHLHE22ZFP37SLC2A2THRSPNR4A3LOC396781C1QTNF1RAB27ANTSGVIN1SSTR1CCDC9BCCDC33CCDC162PPTHSYNPO2LCHGB5E115E115E115E115E115E115E115E115E1153E0911E0844E0850E0848E0728E0628E0662E0664E0671E0672E0696E0614E0515E0516E0529E0537E0567E0585E0596E0510E0412E0415E0431E0433E0444E0445E0456E0479E0479E0486E0488E0414E0314E0314E0315E0318E03ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’aLog2[fold change] between two maternal immune activationsex groups MPA PRRSVinduced maternal immune activation CON control Fe females Ma malesankyrin repeat domain ANKRD24interferoninducedtransmembrane protein IFITM1 and IFITM3 cathepsinC CTSC mitogenactivated protein kinase kinase MAP2K7heparan sulfateglucosamine 3sulfotransferase HS3ST5secreted phosphoprotein SPP1 immunoglobulin heavy chainIGHG and transforming acidic coiledcoilcontaining protein TACC1 Among the genes underexpressed in MPA relative toCON pigs are insulinlike growth factor IGF2 cellular retinoicacidbinding protein CRABP2 and aldehyde dehydrogenase family member A1 ALDH1A1Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaFunctional Analysis of Genes AssociatedWith Maternal Immune ActivationTable presents the top significant clusters of informativeenriched categoriesfrom the DAVID analysis of genesTABLE Most enriched DAVID clusters and supporting functional categoriesenrichment score ES among the genes presenting significant maternalimmune activationbysex interaction effectaCategory Category identifier and namePvaluebFDRPvalueCluster KEGGBPKEGGKEGGKEGGCluster BPBPBPBPBPCluster BPCluster BPBPES ssc05320Autoimmune thyroid diseaseGO000250ˆ¼Antigen processing andpresentation of peptide orpolysaccharide antigen via MHC class IIssc04514Celladhesion moleculesCAMsssc05323Rheumatoid arthritisssc05164Influenza AES GO0051050ˆ¼Positive regulation oftransportGO0051049ˆ¼Regulation of transportGO0050801ˆ¼Ion homeostasisGO0048878ˆ¼Chemical homeostasisGO0030001ˆ¼Metal ion transportES GO0048871ˆ¼Multicellular anismalhomeostasisES GO0061564ˆ¼Axon developmentGO0007420ˆ¼Brain development290E06190E03450E04340E01230E03320E02480E03200E02560E02170E01440E03350E01450E03130E02280E02450E02320E01360E01480E01570E01200E04370E01640E05130E03170E01310E01aBP biological process KEGG KEGG pathway bFalse discovery rate adjustedPvalueTABLE Enriched informative categories NES using GSEA among thegenes based on the overall maternal immune activationbysex interactionaCategory Category identifier and namebNES PvalueKEGGKEGGMFBPBPBPKEGGBPˆ’ 10E10ˆ’ 10E10ˆ’ 10E10ˆ’ 10E10ssc04080Neuroactive ligand receptorinteractionssc04912GnRH signaling pathwayGO0005179ˆ¼Hormone activityGO0006970ˆ¼Response to osmoticstressGO0019221ˆ¼Cytokine mediatedsignaling pathwayGO1901385ˆ¼Regulation of voltagegated calcium channel activityssc04020Calcium signaling pathway ˆ’ 12E01GO0085029ˆ¼Extracellular matrixˆ’ 18E01assemblyˆ’ 91E02ˆ’ 12E01cFDRPvalue83E0299E0222E0135E0154E0154E0154E0155E01aMF molecularfunction KEGG KEGG pathway BP biological processbNormalized enrichment score negative values indicate genes underexpressionin CON females relative to males but overexpression in MPA females relative tomales cFalse discovery rate adjusted Pvaluediï¬erentially expressed between MPA and CON groupsacross sexes the extended list of categories is presented inSupplementary File S1 Table E Some categories identifiedby the DAVID analysis are consistent with the categoriesdetected at more significant levels among the genes presentingan MIAbysex interaction eï¬ect Table and include theBP angiogenesisand KEGG autoimmunethyroid disease and Epstein“Barr virus infection pathwaysTable Also enriched Supplementary File S1 Table Ewere the BP homeostatic GO0042592 MF ion bindingGO0043167 and BP anatomical structure formation inmorphogenesis GO0048646GO0001525The GSEA enrichment results within the gene expressionpatterns of CON relative to MPA groups complemented thefindings from DAVID The most informative enriched categoriesare presented in Table and the extended list of categories ispresented in Supplementary File S1 Table F Enriched clustersof genes overexpressed in CON relative to MPA detected byGSEA were the BP enrichment of microtubule bundle formationGO0001578 and cilium morphogenesis GO0060271Transcriptome Differences BetweenSexes Independent of Maternal ImmuneActivationOverall genes were diï¬erentially expressed between malesand females FDRadjusted P These genes exhibiteda consistent diï¬erential expression between sexes irrespectiveof the MIA group The complete list of genes diï¬erentiallyexpressed between sexes at FDRadjusted P is available inSupplementary File S1 Table G and the majority were overexpressed in males relative to females Among the previousgenes excluding those that presented MIAbysex interactioneï¬ect a selection of informative genes is listed in Table Genesoverexpressed in males relative to females included eukaryotictranslation initiation factor 1A Ylinked EIF1AYleptinreceptor LEPR luteinizing hormone beta polypeptide LHBLIM homeobox LHX9 luteinizing hormone beta polypeptideLHB and immunoglobulin family member IGSF1Informative categories among the DAVID clusters ofenriched categoriesthe genes diï¬erentially expressedbetween sexes are listed in Table a complete list i

Thyroid_Cancer

"analyse the quality of information included in websites aimed at the public on COVID19Methods Yahoo Google and Bing search engines were browsed using selected keywords on COVID19first websites from each search engine for each keyword were evaluated Validated tools wereThe used to assess readability [Flesch Reading Ease Score FRES] usability and reliability LIDA tool andquality DISCERN instrument Nonparametric tests were used for statistical analysesResults Eightyfour eligible sites were analysed The median FRES score was range 00 Themedian LIDA usability and reliability scores were range 00 and 37range14 00 respectively Alow overall LIDA score was recorded for n of the websites The median DISCERN score 14 was found in websites The DISCERNwas range “ The DISCERN score of score was significantly associated with LIDA usability and reliability scores p and the FRES scorep Conclusion The majority of websites on COVID19 for the public had moderate to low scores with regardsto readability usability reliability and qualityPractice Implications Prompt strategies should be implemented to standardize online health informationon COVID19 during this pandemic to ensure the general public has access to good quality reliableinformationï¾ Elsevier BV All rights reserved Introductionfor The coronavirus COVID19 pandemic has become the greatestglobal health crisis of the st century [] During this pandemicthe demand information on COVID19 has skyrocketedInformation such as latest news updates on the pandemic itssymptoms prevention and mechanism of transmission are highlysought by the public [] On the other hand free access toinformation especially through social media which is accessed bythe majority [] has led to an increase in misinformation and panicassociated with COVID19 [] Although high quality healthinformation is known to be related to lower stress levels andbetter psychological health [] previous studies have shown thatonline information on many medical disorders to be of substandard quality []A previous study done on websites related to COVID19 hasreported substandard quality information that could potentiallymislead the public [] However this study has used a limitedsearch strategy and had not assessed some important areasincluding usability and reliability of the information Thereforethis topic remains a knowledge gap in COVID19 research []Therefore we conducted this study to analyse the current COVID websites targeting the general public in terms of qualityusability readability and reliability using a wide search strategyand validated instruments MethodsAbbreviations USA United States of America FRES Flesch reading ease scoreHONcode Health on the net Code of Conduct SPSS Statistical package for socialsciences NICE National Institute for Health and Care Excellence WHO WorldHealth anization Corresponding author at Department of Surgery Faculty of MedicineUniversity of Colombo PO Box Kynsey Road Colombo Western ProvinceSri LankaEmail addresses ravindrijayasinghegmailcom R Jayasingheranasigmcgmailcom S Ranasinghe umeshejayagmailcom U Jayarajahsanjeewasrgcmbaclk S SeneviratneYahoo Google and Bing were searched using the keywordsœsevere acute respiratory synœnovel coronavirusSARSCoV2 and œcoronavirus The searchdrome coronavirus2 œCOVID19was performed during the first week of May The details ofthe search strategy and the piloting process are provided in thesupplementary material File S1 []Two independent investigators with previous experience ofconducting similar studies assessed the selected websites []Prior to the assessment a pilot run was conducted to ensure101016jpec20200800107383991ï¾ Elsevier BV All rights reservedPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxxuniformity and accuracy The information on symptoms investigations public health measures and available treatmentmodalities were collected The accuracy of the content wasassessed using the national institute for health and care excellenceNICE guidelines on COVID19 [] A rating was given as all ornone based on congruence with the guidelinesValidated instruments were used to assess the quality ofwebsites Readability was assessed using the Flesch Reading EaseScore FRES [] The LIDA Instrument 2007Version12 wasused to analyse the content and the design of the websites usingthe usability and reliability domains [] The quality of thecontent was assessed using the DISCERN questionnaire which has questions in two separate groups [] The detailed assessmentcriteria and the scoring system is included in the supplementarymaterial File S1A website was classified as governmental if it was maintainedby the country™s public health authority If managed by privateinstitutions nongovernmental anizations or voluntary institutions independent from the government they were consideredas nongovernmental The online healthrelated websites arestandardized in terms of their credibility and reliability by onlinecertification sites We chose the Health on the Net code of conductHONcode which is the oldest and widely used out of the qualityevaluation tools available []Data analysis was performed using SPSS Version20 softwareand the associations were determined with non parametric testsA pvalue of was considered statistically significant ResultsOf the retrieved websites were excluded and thein the analysis Theremaining websites were characteristics of the websites are mentioned in Table included Half were governmental websites and only n were HON accredited websites The median FRES was range 00 10th12th grade readability level which is classified asfairly difficult to read Only three websites had a readabilityscore of above equivalent to 7th grade which is therecommended standardThe overall median LIDA score was range “ whilethe median LIDA usability and reliability scores were range 00 and range 00 respectively The median DISCERNscore was range “ which classifies websites as being ofœfair quality Excellent “ Good “ Fair “Poor “ Very poor “ However the top websitesTable A were of excellent qualityTable Website characteristicsWebsite Characteristics Frequency PercentageUsability Governmental websites Notforprofit and private websites HONcode accredited Readability score of above equivalent to 7th grade Readability Date of publication stated References mentioned Disclosure statement by authors Infographics Moderate Low Moderate 00 Low score Moderate 00 Low Used Textonly Reliability Table Correlation between DISCERN scores and other factorsDISCERN SCORELow Mean High 00 Mean Range 00 00 00 00 Range 00 00 00 00 N P valueP0001P0001P0001P P P LIDA Usability LIDA Reliability LIDA Overall FRES Score Government HON Certification No N No Yes Yes Significant correlations were observed between the DISCERNscore and the overall LIDA score as well as LIDA usability andreliability scores Table p HONcode certified websitessites obtained significantly higher DISCERN scores p Pertaining to the currency of information only publishers stated the date of the publication Most websites n did not declare the sources of evidence This was furtherestablished by the median reliability score of Nevertheless the authors have included a disclosure statement in mostn websitesœlowFigures A1 and A2 summarize the rating of websites onindividual criteria assessed by the DISCERN tool The specificinformation provided regarding COVID19 is shown in Fig More than half of the websites failed to discuss the treatmentoptions available n benefits or risks n and effects of no treatment n Furthermore potentialcomplications and prognosis were stated only in and websites respectively Discussion and conclusion DiscussionThis study has shown that still most of the websites on onlinehealth information on COVID19 are of suboptimal quality exceptfor a few credible sources of goodquality health informationNevertheless the websites ranked among the top according tothe DISCERN score Table A2 had high scores indicating thepotential for publishing credible highquality information onlinewhich would benefit the publicin turn causes panic which ranges Misinformation is a major concern during this pandemic aspeople fail to spend adequate time to critically analyse the onlineinformation This fromhoarding medical supplies to panic shopping and using drugswithout prescription with negative social and medical consequences [] Therefore measures implemented to ensure the qualityand accuracy of online information by the responsible authoritiesmay help negate these adverse consequencesinformation Stating the methods of content production with names of thecontributing authors may help increase the credibility of onlinehealth information while displaying the date of the publicationprovides an idea of the currency of the information Absence ofin over half of the websites was a majorsuch drawback especially for COVID19 where new information isgenerated almost daily Health authorities should therefore ensurethat the patient information websites provide the above information and certify websites based on such details so that the publiccan get information from trusted sources []Most users of the worldwide web only have an average level ofeducation and reading skills [] Guidance from the NationalPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxx Fig Website characteristics evaluated outside the DISCERN toolInstitute of Health NIH had shown that the readability should bebelow the level of seventh grade for the lay public to adequatelyunderstand the content [] However the median readabilitylevel was found to be equivalent to 10th12th grade readabilitySuch complexities with the readability of information mayincrease the risk of misunderstandings or misinterpretation Usingshort sentences in writing using the active voice using 12point orlarger font size using illustrations and nontextual media asappropriate and accompanying explanations with examples wouldbe helpful to overcome this problem []So far only a limited number of studies have been done to assessthe quality of health information websites related to COVID19 Thestudy by CuanBaltazar et al prior to February reported poorquality information with approximately of included websiteswith low DISCERN scores [] Our study done three months latershows similar results with only a minimal improvement in thequality of information Furthermore the Cuan Baltazar study hadseveral limitations which includes the limited search strategy andnoninclusion of key quality parameters including readabilityFurthermore out of the sites they had includedwere online news sites that are not considered as patientinformation websites In that study the HONcode seal waspresent only in n websites whereas in our study ofthe sites were HONcode certifiedThere were several limitations in this study Although mostpopular search engines were used in this study under defaultsettings they may produce variable results depending on manyfactors including geographical location and popularity of websitesat a given point of time The algorithms unique to those searchengines are subjected to constant change and therefore the exactresults of our study may not be reproducible However we believethe general patterns observed in our study are validproviding health information to the general public are to be ofsubstandard quality Practice implicationsTo improve the credibility of the content the websites shouldstate methods of content production and display the date of thepublication to give an idea about the currency of the informationTo improve the readability of the content the websites shouldincorporate more nontextual media write in short sentencesusing the activevoice and use larger font sizes The patientinformation websites should display scores of reliability qualityand readability as a guidance for its users Furthermore it is vitalfor medical regulatory authorities and the government to imposeregulations to ensure quality and to prevent the spread ofmisinformationAvailability of data and materialsOn reasonable request from the corresponding author the dataused in the above study can be made availableEthics approval and consent to participateUnnecessary in this type of studyInformed consent and patient detailsNot applicable in this type of studyFundingNone ConclusionCRediT authorship contribution statementThis study has shown the quality readability usability andreliability of the information on COVID19 on majority of websitesRavindri Jayasinghe Conceptualization Methodology Datacuration Writing original draft Visualization InvestigationPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxx“xxxValidation Formal analysis Resources Sonali RanasingheConceptualization Methodology Data curation Writing originaldraft Visualization Investigation Validation Formal analysisResources Umesh Jayarajah Conceptualization MethodologyData curation Writing original draft Visualization InvestigationValidation Formal analysis Resources Sanjeewa SeneviratneConceptualization Methodology Writing review editingSupervision Project administrationDeclaration of Competing InterestThe authors report no declarations of interestAcknowledgementsNone declaredAppendix A Supplementary dataSupplementary material related to this can be found in101016jversion at online the pec202008001References[] IJ Bes do Nascimento N Cacic HM Abdulazeem Novel Coronavirusinfection COVID19 in humans a scoping review and metaanalysis JClinical Med [] HT Le DN Nguyen AS Beydoun XTT Le TT Nguyen QT Pham NTK Ta QT Nguyen AN Nguyen MT Hoang Demand for health information onCOVID19 among Vietnamese Int J Environ Res Public Health [] C Wang R Pan X Wan Y Tan L Xu RS McIntyre FN Choo B Tran R Ho VK Sharma A longitudinal study on the mental health of general populationduring the COVID19 epidemic in China Brain Behav Immun [] CS Ho CY Chee RC Ho Mental health strategies to combat the psychologicalimpact of COVID19 beyond paranoia and panic Ann Acad Med Singapore “[] C Wang R Pan X Wan Y Tan L Xu CS Ho RC Ho Immediate psychologicalresponses and associated factors during the initial stage of the coronavirus disease COVID19 epidemic among the general population inChina Int J Environ Res Public Health [] RH Waidyasekera U Jayarajah DN Samarasekera Quality and scientificaccuracy of patientoriented information on the internet on minimallyinvasive surgery for colorectal cancer Health Policy Technol “[] R Jayasinghe S Ranasinghe U Jayarajah S Seneviratne Quality of patientoriented webbased information on oesophageal cancer J Cancer Educ In press[] JY CuanBaltazar MJ MuñozPerez C RobledoVega MF PÃrezZepeda ESotoVega Misinformation of COVID19 on the internet Infodemiology studyJMIR Public Health Surveill 2020e18444[] BX Tran GH Ha LH Nguyen GT Vu MT Hoang HT Le CA Latkin CS HoR Ho Studies of novel coronavirus disease COVID19 pandemic a globalanalysis of literature Int J Environ Res Public Health [] G Eysenbach C Köhler How do consumers search for and appraise healthinformation on the world wide web Qualitative study using focus groupsusability tests and indepth interviews Brit Med J “[] AS Prasanth U Jayarajah R Mohanappirian SA Seneviratne Assessment ofthe quality of patientoriented information over internet on testicular cancerBMC Cancer [] V Udayanga U Jayarajah SD Colonne SA Seneviratne Quality of thepatientoriented information on thyroid cancer in the internet Health PolicyTechnol [] National Institute for Health and Care Excellence Coronavirus COVID19 Accessed April wwwniceukcovid19[] Readable The Flesch Reading Ease and FleschKincaid Grade Level Accessed February readablecomblogthefleschreadingeaseandfleschkincaidgradelevel[] Minervation The Minervation Validation Instrument for Healthcare WebsitesLIDA Tool Accessed February httpwwwminervationcomwpcontentuploads201104MinervationLIDAinstrumentv12pdf[] Minervation Is the Lida Website Assessment Tool Valid Accessed February httpwwwminervationcomdoeslidawork[] Discern Online The DISCERN Instrument Accessed February httpwwwdiscernukdiscern_instrumentphp[] E Fahy R Hardikar A Fox S Mackay Quality of patient health information onthe Internet reviewing a complex and evolving landscape Australas Med J “[] J Kluger As Disinfectant Use Soars to Fight Coronavirus So Do AccidentalPoisonings Accessed April timecom5824316coronavirusdisinfectantpoisoning[] BX Tran AK Dang PK Thai HT Le XTT Le TTT Do TH Nguyen HQPham HT Phan GT Vu Coverage of health information by different sourcesin communities implication for COVID19 epidemic response Int J EnvironRes Public Health [] National Institutes of Health How to Write EasyToRead Health Materials Accessed April wwwscribdcomdocument261199628HowtoWriteEasyToReadHealthMaterialsMedlinePlus[] DM D™Alessandro P Kingsley J JohnsonWest The readability of pediatricpatient education materials on the world wide web Arch Pediatr AdolescMed “Please cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0c"

Thyroid_Cancer

"Long non‘coding RNA FOXD2‘AS1 regulates the tumorigenesis and progression of breast cancer via the S100 calcium binding protein A1Hippo signaling pathwayPEI HUANG1 and JINHUI XUE21Department of Pathology The First Affiliated Hospital of Zhengzhou University Zhengzhou Henan 2Department of Pathology Zhengzhou Central Hospital Affiliated to Zhengzhou University Zhengzhou Henan PR ChinaReceived November Accepted April 103892ijmm20204699Abstract Breast cancer is one of the most prevalent cancer types and is accompanied by a high incidence and mortality rate severely threatening women's health globally Long noncoding RNA forkhead box d2 adjacent apposite strand RNA lncRNA FOXD2‘AS1 has been identified to function as an oncogene in human cancers however it has rarely been investigated in breast cancer The aim of the present study was to investigate the role of FOXd2AS1 in breast cancer and to clarify the underlying mechanisms The expression of FOXD2‘AS1 in breast cancer cell lines was first quantified by reverse transcription‘quantitative PCR and the biological function of FOXD2‘AS1 was then determined Cellular proliferative ability was determined by Cell Counting kit‘ assay and wound healing and Transwell assays were conducted to assess the cell migratory and invasive ability corresponding protein expression levels were determined by western blot analysis In addition experimental animal models were established by the subcutaneous injection of MDA‘MB‘ cells into the right axillary lymph nodes of BALBc nude mice and the effects of FOXD2‘AS1 on tumor growth were observed The results indicated that FOXD2‘AS1 expression was upregulated in breast cancer cell lines and that FOXD2‘AS1 downregulation significantly inhibited the proliferation migration and invasiveness of MCF‘ and MDA‘MB‘ cells S100 calcium binding protein A1 S100A1 was also upregulated in breast cancer cell lines and was positively regulated by FOXD2‘AS1 Furthermore the inhibition of S100A1 and the overexpression of the serinethreonine‘protein kinase large tumor suppressor homolog LATS1 inhibited the FOXD2‘AS1‘induced Correspondence to Dr Pei Huang Department of Pathology The First Affiliated Hospital of Zhengzhou University East Jianshe Road Zhengzhou Henan PR ChinaE‘mail huangpei193163comKey words FOXD2‘AS1 S100 calcium binding protein A1 breast cancer large tumor suppressor homolog Hippocellular proliferation migration and invasiveness in breast cancer Experimental mouse models revealed that FOXD2‘AS1 downregulation significantly inhibited tumor growth and that the levels of phosphorylated p‘YAP and p‘LATS1 were upregulated by FOXD2‘AS1 knockdown indicating that the inhibition of FOXd2AS1 activated Hippoyesassociated protein signaling On the whole the findings of the present study suggest that the FOXD2‘AS1S100A1Hippo axis is involved in the tumorigenesis and progression of breast cancer In the future these may contribution to the identification of more effective breast cancer treatmentsIntroductionAs one of the most prevalent malignancies breast cancer is a primary cause of mortality among gynecological cancer cases and with increasing morbidity and mortality rates it poses a considerable threat to women's health worldwide In statistics from the American cancer Society estimated newly diagnosed cases and deaths from breast cancer in the United States The leading causes of the high death rate are distal metastasis and resistance to the existing treatments despite improvements in early diagnosis and systemic treatment the incidence of breast cancer and metastasisrelated mortality is steadily increasing Therefore there is an urgent need to elucidate the mechanisms responsible for the disordered cellular metastasis and to enhance our understanding of the tumorigenesis and development processes hence facilitating the identification of more efficient breast cancer treatmentsLong noncoding RNAs lncRNAs are a group of RNAs nucleotides in length which lack protein‘coding capacity Numerous studies have revealed that lncRNAs have versatile biological functions in pathological and physiological processes including tumorigenesis ‘ lncRNAs are considered to regulate the development of various types of cancer including breast cancer For instance LINC01089 is downregulated in breast cancer tissues and cell lines and LINC01089 overexpression increases tumor cell proliferation migration and invasiveness As an oncogene that regulates breast cancer cell proliferation and apoptosis hepatocellular 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERcarcinoma upregulated EZH2‘associated lncRNA is closely associated with the clinical progression of breast cancer These results indicate the indispensability of research on lncRNAs and breast cancerlncRNA forkhead box d2 adjacent apposite strand RNA FOXD2‘AS1 is a novel non‘coding RNA identified to be an oncogene in human cancers FOXD2‘AS1 has been shown to be upregulated in various types of cancer including glioma osteosarcoma and papillary thyroid cancer as well as breast cancer ‘ A previous study indicated that FOXD2‘AS1 participates in regulating the development of breast cancer via the miR‘‘5pPFN2 axis and that it may be a potential biomarker for the diagnosis and prognosis of breast cancer However to the best of our knowledge there are no additional data regarding the investigation of FOXd2AS1 in breast cancer and its effects and the underlying mechanisms on the regulation of breast cancer cell invasion and metastasis Thus the aim of the present study was to determine the role and potential mechanisms of action of FOXd2AS1 in breast cancer and to provide further support for its use in clinical diagnosis and treatmentMaterials and methodsDatasets The present study evaluated the expression level of FOXD2‘AS1 in breast cancer samples using The Cancer Genome Atlas TCGA dataset which was downloaded from the TCGA data portal tcga‘data‘nci‘nih‘govezxjtlueducn The TCGA data subset for breast cancer included normal samples and tumor samples The Mann‘Whitney test was used to determine statistically significant differences between normal and tumor samples P005 was considered to indicate a statistically significant differenceCell culture A human normal breast epithelial cell line MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ were purchased from the American Type Culture Collection ATCC The cells were incubated in RPMI‘ medium supplemented with fetal bovine serum FBS in a humidified incubator at ˚C CO2 Transfection To overexpress FOXd2AS1 an overexpression vector pcdNA FOXd2AS1 and its corresponding negative control vector pcDNA‘NC were synthesized by Shanghai GenePharma Co Ltd Short hairpin shRNAs targeting FOXD2‘AS1 nM shRNA‘FOXD2‘AS1‘ and shRNAFOXd2AS12 and a negative scramble control shRNA shRNA also purchased from Shanghai GenePharma Co Ltd were used to knock down FOXD2‘AS1 expression In addition pcdNALATS1 shRNAS100A11 and shRNAS100A12 were obtained from Shanghai GenePharma Co Ltd to overexpress LATS1 or to knock down S100A1 respectively The shRNA sequences were as follows shRNA‘FOXD2‘AS1‘ targets GGA CTC CAC TCT TCG CTT A shRNA‘FOXD2‘AS1‘ targets GCT TCC AGG TAT GTG GGA A shRNA‘S100A1‘ targets GAT CCG GAG ACC CTC ATC AAC GTG TTC TTC CTG TCA GAA ACA CGT TGA TGA GGG TCT CCT TTT TG shRNA‘S100A1‘ targets GAT CCG TGG ACT TCC AGG AGT ATG TGC TTC CTG TCA GAC ACA TAC TCC TGG AAG TCC ACT TTT TG Cells were transfected with pcDNA FOXD2‘AS1 nM pcdNALATS1 nM pcdNANc nM shRNAFOXd2AS11 ngµl shRNAFOXd2AS12 ngµl shRNAS100A11 ngµl shRNAS100A12 ngµl shRNA ngµl or co transfected with pcdNA FOXd2AS1 and pcdNALATS1 or cotransfected with pcDNA FOXD2‘AS1 and shRNA‘S100A1 using Lipofectamine® transfection reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's instructions Lipofectamine reagent was first mixed with vectors to form a reagent‘vector complex followed by incubation with cells at ˚C for h The transfection efficacy was assessed by reverse transcription‘quantitative PCR RT‘qPCR after h of transfectionRNA extraction and RT‘qPCR Total RNA was extracted from all cell lines using TRIzol® reagent Takara Bio Inc according to the manufacturer's instructions Total RNA was then reverse transcribed into cDNA using the PrimeScript„¢ RT Master kit and the mRNA expression levels were quantified using the SYBR Premix Ex Taq„¢ kit Both Takara Bio Inc with a Fast Real‘Time PCR System Applied Biosystems Thermo Fisher Scientific Inc The sequences of specific primers used for RT‘qPCR were as follows FOXDA‘AS1 forward '‘TGG ACC TAG CTG CAG CTC CA‘' and reverse '‘AGT TGA AGG TGC ACA CAC TG‘' S100A1 forward '‘GAG TAT GTG GTG CTT GTG GC‘' and reverse '‘CTT GGA CCG CTA CTC TTG CG‘' large tumor suppressor homolog LATS1 forward '‘ACC GCT TCA AAT GTG ACT GTG ATG CCA C CT‘' and reverse '‘CTT CCT TGG GCA AGC TTG GCT GAT CCT CT‘' and GAPDH forward '‘GCG AGA TCG CAC TCA TCA TCT ‘' and reverse '‘TCA GTG GTG GAC CTG ACC ‘' The data were displayed as ‘ΔΔCq values with GAPDH as the constitutive marker The PCR conditions were as follows ˚C for min cycles of ˚C for sec and ˚C for sec followed by ˚C for minCell Counting Kit‘ CCK‘ assay cell proliferation was determined using the Cell Counting Kit‘ assay CCK‘ Dojindo Molecular Technologies Inc Briefly cells were seeded into a ‘well plate and incubated for h at ˚C Following culture for the indicated periods of time and h µl of the CCK‘ reagent were added to each well and the plate was incubated at ˚C for a further h The optical density values at nm were then measured using a microplate spectrophotometer Thermo Fisher Scientific IncWestern blot analysis The total protein was extracted from the cells using RIPA lysis buffer Beyotime Institute of Biotechnology and quantified using a BCA protein assay kit Thermo Fisher Scientific Inc The same amount of each protein sample µg was subjected to SDS‘PAGE the proteins were then transferred onto PVDF membranes EMd Millipore and was blocked in nonfat milk for h at room temperature The membranes were then incubated with primary antibodies against cyclinE1 cat no ab33911 Abcam cyclin‘dependent kinase‘ CDK2 cat no ab32147 Abcam p21 cat no ab109520 Abcam matrix metalloproteinases MMP2 cat no ab92536 Abcam MMP9 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE was added to the lower 24well chamber After h cells on the upper surface were removed and cells attached to the lower surface were stained with crystal violet Beijing Solarbio Science Technology Co Ltd at room temperature for min The cells were viewed under a light microscope magnification x100 CKX41 Olympus Corporation and the invasive ability of the cells was determined using ImageJ software version by counting the number of cells attached to the lower surfaceCell cycle analysis The cell cycle distribution was determined by flow cytometry After being subjected to the indicated treatments the cells were collected and fixed in ethanol at ‘ËšC overnight The cells were then washed twice with PBS and incubated in the dark with RNase A and PI staining solution Roche Diagnostics at ˚C for min Finally the cell samples were analyzed using a FACSCalibur flow cytometer and CellQuest software version both from BD BiosciencesIn vivo experiments The present study was approved by the First Affiliated Hospital of Zhengzhou University and the animal experiments were performed according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals A total of 5weekold male BALBc nude mice were purchased from the Experimental Animal Center of Shanghai Institute for Biological Sciences and housed in a standard environment ˚C humidity h lightdark cycle with free access to food and water Each mouse was subcutaneously injected at the right axillary lymph nodes with 10x107 MdAMB468 cells which were stably transfected with either the shRNA negative control shRNA or shRNA‘FOXD2‘AS1 The weights and tumor volumes tumor volume x length x width2 of the mice were monitored every days until the mice were sacrificed At days after the injection all the mice were sacrificed by cervical dislocation that caused a sharp section of the spinal cord followed by an instantaneous cardiac arrest After the cessation of the heartbeat and respiratory arrest of the mice was confirmed the tumors were excised photographed and stored for the further investigationStatistical analysis data are presented as the means ± standard deviation SD from ‰¥ independent experiments and each experiment was conducted in triplicate The data were analyzed using SPSS statistical software version SPSS Inc and the differences among groups were analyzed using one‘way analysis of variance followed by Tukey's post hoc test P005 was considered to indicate a statistically significant differenceResultsFOXD2‘AS1 expression is upregulated in breast cancer cells The TCGA database cancergovtcga was used to identify the association between FOXD2‘AS1 and breast cancer by evaluating the expression profiles of FOXD2‘AS1 in breast cancer tissues and normal tissues The results of TCGA analysis revealed a significantly higher FOXd2AS1 expression in breast cancer tissues than in normal tissues Fig 1A Human Figure FOXD2‘AS1 is upregulated in breast cancer cells A The Cancer Genome Atlas TCGA database cancergovtcga was used to identify the association of FOXD2‘AS1 with breast cancer by collecting the profiles of FOXD2‘AS1 in breast cancer tissues and normal tissues P0001 vs normal samples B mRNA level of FOXD2‘AS1 in human normal breast epithelial cell line MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ was determined by RT‘qPCR P001 and P0001 vs MCF‘10A cells cat no ab38898 Abcam S100 calcium binding protein A1 S100A1 cat no Cell Signaling Technology Inc phosphorylated p‘yes‘associated protein YAP cat no Cell Signaling Technology Inc YAP cat no Cell Signaling Technology Inc serinethreonine‘protein kinase LATS1 cat no Cell Signaling Technology Inc p‘LATS1 cat no Cell Signaling Technology Inc mammalian STE20‘like protein kinase MST1 cat no Cell Signaling Technology Inc MST2 cat no Cell Signaling Technology Inc and GAPDH cat no ab8245 Abcam at ˚C overnight The membranes were washed with Tris‘buffered saline with Tween TBST and incubated with horseradish peroxidase HRP‘conjugated goat anti‘mouse IgG secondary antibody cat no sc‘ Santa Cruz Biotechnology Inc at room temperature for h The protein bands were visualized using an enhanced chemiluminescence kit Amersham Pharmacia Biotech and quantified using ImageJ software version National Institutes of HealthWound healing assay The cellular migration rate was determined using a wound healing assay The cells were seeded into a ‘well plate and cultured to confluence A wound was produced in each monolayer using a ‘µl pipette tip and the plate was washed times with PBS to remove detached cells The cells were then cultured in the fresh medium without FBS Following incubation for h the wound‘healing ability was assessed under a light microscope magnification x100 CKX41 Olympus Corporation and the widths of the wounds were measured at and hTranswell assay The cell invasive rate was determined with a Transwell assay cells 4x104well in serum‘free medium were placed in the upper chamber of each insert [which had been precoated with µl of Matrigel Bd biosciences at ˚C for h] and complete medium containing FBS 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 suppresses cell proliferation migration and invasion in breast cancer A and B Following transfection with shRNA‘FOXD2‘AS the mRNA level of FOXD2‘AS1 was measured by RT‘qPCR in MCF‘ and MDA‘MB‘ cells C and D CCK‘ assay was performed to determine cell proliferation following transfection E‘G Cell cycle distribution was determined and analyzed by FACS H and I The protein expression of cyclin E1 CDK2 and p21 was determined by western blot analysisnormal breast epithelial cell line MCF‘10A and human breast cancer cell lines McF7 MdAMB468 MdAMB453 and BT549 were also obtained to detect the mRNA levels of FOXD2‘AS1 The results revealed that FOXD2‘AS1 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure Continued Knockdown of FOXD2‘AS1 suppresses cell proliferation migration and invasion in breast cancer J Wound healing assay was performed to detect the migration of both MCF‘ and MDA‘MB‘ cells K and L Relative migration rate of MCF‘ and MDA‘MB‘ cells was quantified respectively M Transwell assay was performed to detect the invasion of both McF7 and MdAMB468 cells N and O Relative cell invasive rate of McF7 and MDA‘MB‘ cells was quantified respectively P MMP‘ and MMP‘ protein expression in MCF‘ transfected with shRNA‘FOXD2‘AS1 was detected and quantified Q MMP‘ and MMP‘ protein expression in MDA‘MB‘ transfected with shRNA‘FOXD2‘AS1 was detected and quantified P005 P001 and P0001 vs shRNA‘NC CDK2 cyclin‘dependent kinase‘ MMP matrix metalloproteinase expression was markedly upregulated in all breast cancer cells particularly in the MCF‘ ER‘positive breast cancer cell line and MdAMB468 cells triplenegative breast cancer cell line Fig 1B which were used for further experiments even 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 regulates the HippoYAP signaling pathway A The protein expression of HippoYAP signaling pathway‘related genes p‘YAP YAP p‘LATS1 LATS1 MST1 and MST2 was determined by western blot analysis in MCF‘ cells B The protein expression of HippoYAP signaling pathway‘related genes p‘YAP YAP p‘LATS1 LATS1 MST1 and MST2 was determined by western blot analysis in MDA‘MB‘ cells P005 P001 and P0001 vs shRNA‘NC LATS1 large tumor suppressor homolog MST mammalian STE20‘like protein kinase YAP yes‘associated protein though there may be some variability in the results between the cell lines These findings indicate that FOXD2‘AS1 is upregulated in breast cancerFOXD2‘AS1 knockdown suppresses breast cancer cell proliferation migration and invasiveness To further elucidate the role of FOXd2AS1 in breast cancer FOXd2AS1 was knocked down in both the McF7 and MdAMB468 cells Due to a higher transfection efficacy shRNA‘FOXD2‘AS1‘ referred to as shRNA‘FOXD2‘AS1 was subsequently used for breast cancer cell experimentation Fig 2A and B The results of CCK‘ assay indicated that FOXD2‘AS1 knockdown significantly inhibited the proliferative ability of the MCF‘ and MdAMB468 cells Fig 2c and d The cell cycle was then analyzed by flow cytometry which revealed that FOXd2AS1 knockdown increased the percentage of cells in the G1 phase whereas it decreased that in the S phase for both the MCF‘ and MDA‘MB‘ cells Fig 2E‘G Furthermore FOXd2AS1 knockdown decreased the protein expression levels of cyclin E1 and CDK2 and increased the expression of 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure S100A1 is upregulated in breast cancer cells A The mRNA level of S100A1 in MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ was determined by RT‘qPCR P005 P001 and P0001 vs MCF‘10A cells B and C In shRNA‘FOXD2‘AS1‘transfected McF7 and MdAMB468 cells the protein expression of Sl00A1 was detected by western blot analysis P005 and P0001 vs shRNA‘NC D and E Following transfection with pcDNA‘FOXD2‘AS1 the mRNA level of FOXD2‘AS1 in MCF‘ and MDA‘MB‘ cells was detected by RT‘qPCR P001 and P0001 vs pcDNA‘NC MCF‘ and MDA‘MB‘ cells were transfected with shRNA‘S100A1 and the protein expression and mRNA level of S100A1 in F and G MCF‘ and H and I MDA‘MB‘ cells were determined by western blot analysis and RT‘qPCR respectively P001 and P0001 vs shRNA‘NC S100A1 S100 calcium binding protein A1 p21 Fig 2H and I These findings indicate that FOXD2‘AS1 knockdown suppresses cellular proliferation by regulating the cell cycle specifically by preventing G1 to S phase progression Moreover FOXD2‘AS1 knockdown significantly decreased the migration rate Fig 2JL and the invasiveness Fig 2MO of the McF7 and MdAMB468 cells The 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure S100A1 mediates FOXD2‘AS1‘induced cell proliferation migration and invasion in breast cancer Following transfection with pcDNA‘FOXD2‘AS1 cells were transfected with shRNA‘S100A1 A and B CCK‘ assay was performed to determine the proliferation of the differently treated cells C Migratory ability of MCF‘ cells in the different treatment groups was determined by wound healing assay D Invasive ability of MCF‘ cells in the different treatment groups was determined by Transwell assay E Migratory ability of MDA‘MB‘ cells in the different treatment groups was determined by wound healing assay F Invasive ability of MDA‘MB‘ cells in the different treatment groups was determined by Transwell assay P0001 vs pcDNA‘NC P001 and P0001 vs FOXD2‘AS1 shRNA‘NC S100A1 S100 calcium binding protein A1 expression levels of MMP‘ and ‘ which are critical to the migration invasion and metastasis of breast cancer cells were both downregulated following FOXD2‘AS1 knockdown Fig 2P and Q indicating that FOXD2‘AS1 may enhance cellular migration and invasiveness by regulating MMP‘ and ‘FOXD2‘AS1 knockdown regulates the HippoYAP signaling pathway The HippoYAP signaling pathway is reportedly involved in the progression of breast cancer In the present study the levels of specific proteins involved in the YAPHippo signaling pathway were assessed in the MCF‘ and MdAMB468 cells following FOXd2AS1 knockdown Western blot analysis revealed that the levels of p‘YAP and p‘LATS1 were significantly upregulated while those of MST1 and were significantly downregulated by FOXD2‘AS1 knockdown Fig 3A and B Thus the results confirmed that FOXD2‘AS1 regulates the HippoYAP signaling pathway in breast cancer cellsS100A1 mediates FOXD2‘AS1‘induced breast cancer cell proliferation migration and invasiveness S100A1 is a 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure Overexpression of LATS1 inhibits FOXD2‘AS‘induced cell proliferation migration and invasion A and B mRNA level of LATS1 in MCF‘ and MDA‘MB‘ cells was detected by RT‘qPCR in LATS1‘overexpressing cells P0001 vs overexpression‘NC oe‘NC Following transfection with pcDNA‘FOXD2‘AS1 cells were transfected with pcDNA‘LATS1 to overexpress LATS1 C and D CCK‘ assay was performed to determine the proliferation of the differently treated cells E Migratory ability of MCF‘ cells in the different treatment groups was determined by wound healing assay F Invasive ability of MCF‘ cells in the different treatment groups was determined by Transwell assay G Migratory ability of MDA‘MB‘ cells in the different treatment groups was determined by wound healing assay H Invasive ability of MDA‘MB‘ cells in the different treatment groups was determined by Transwell assay P0001 vs pcDNA‘NC P0001 vs FOXD2‘AS1 shRNA‘NC LATS1 large tumor suppressor homolog calcium‘binding protein of the S100 protein family which is not only upregulated in but is also involved in the progression of ovarian cancer In the present study the expression of S100A1 was evaluated in the breast cancer cell lines indicating that S100A1 was significantly upregulated in breast cancer cells particularly in the MCF‘ and MDA‘MB‘ cells compared with the McF10A cells Fig 4A In McF7 and MdAMB468 cells transfected with shRNAFOXd2AS1 it was found that the protein expression level of S100A1 was downregulated Fig 4B and C To further investigate the role of S100A1 in FOXD2‘AS1‘mediated cellular proliferation migration and invasiveness breast cancer cells were transfected with an expression vector pcdNAFOXd2AS1 Fig 4d and E and shRNAS100A1 to inhibit S100A1 protein and mRNA expression Fig 4FI As shown in Fig 5A and B the overexpression of FOXd2AS1 significantly promoted the proliferation of McF7 and MdAMB468 cells which was subsequently reversed by the downregulation of S100A1 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 suppresses tumor progression of breast cancer in vivo A Each mouse was injected with 10x107 MdAMB468 cells which were stably transfected with either shRNA negative control or shRNA‘FOXD2‘AS1 subcutaneously at the right axillary lymph node Following sacrifice the tumors were excised and photographed B and C During the experiment the mouse weight and tumor volume tumor volume x length x width2 was monitored every days until the mice were sacrificed D Protein expression levels of p‘YAP YAP p‘LATS1 and LATS1 in the extracted tumors were determined by western blot analysis and were then quantified P0001 vs shRNA‘NC LATS1 large tumor suppressor homolog YAP yes‘associated protein Wound healing and Transwell assays demonstrated that FOXD2‘AS1 overexpression significantly increased MCF‘ cell migration and invasiveness respectively which were also reversed by the downregulation of S100A1 Fig 5C and D A similar result was observed in the MDA‘MB‘ cells Fig 5E and F These results suggest that S100A1 regulates the FOXd2AS1mediated proliferation migration and invasiveness of breast cancer cellsOverexpression of LATS1 inhibits FOXD2‘AS‘induced cell proliferation migration and invasiveness Since the HippoYAP signaling pathway is involved in the FOXd2AS1mediated characteristics of breast cancer cells HippoYAP signaling was further investigated for its regulatory role in breast cancer cell proliferation migration and invasiveness For this purpose LATS1 was overexpressed in the McF7 and MdAMB468 cells Fig 6A and B and the results of CCK‘ assay revealed that LATS1 overexpression significantly inhibited FOXD2‘AS1‘induced cellular proliferation Fig 6C and D Furthermore the results of wound healing and Transwell assays revealed that LATS1 overexpression significantly inhibited the FOXD2‘AS1‘induced migration and invasiveness of both the McF7 Fig 6E and F and MDA‘MB‘ cells Fig 6G and HFOXD2‘AS1 knockdown suppresses breast cancer tumor progression in vivo From the aforementioned results the role of FOXd2AS1 in both the McF7 and MdAMB468 cells was confirmed To explore the role of FOXD2‘AS1 in breast cancer in vivo mice were injected with MdAMB468 cells which were stably transfected with either an shRNA negative control or shRNA‘FOXD2‘AS1 Following sacrifice 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE the tumors were excised and photographed Fig 7A Tumors in the shRNA‘FOXD2‘AS1 group were the smallest in size directly reflecting the suppressive effect of FOXD2‘AS1 knockdown on tumor growth During the experiment body weights and tumor volumes were recorded every days Body weight increased at a slower rate in the shRNAFOXd2AS1 group and the tumor volumes of this group also increased at a slower rate than those in the other groups Fig 7B and C Additionally western blot analysis of the extracted tumor tissues indicated a significant increase in p‘YAP and p‘LATS1 expression in the shRNA‘FOXD2‘AS1 group Fig 7D which was consistent with the in vitro results These findings thus suggest that FOXD2‘AS1 knockdown suppresses the progression of breast cancer and regulates the HippoYAP signaling pathway in vivoDiscussionBreast cancer is one of the most common types of human tumors particularly among females The high rates of metastasis and recurrence typically result in the deterioration and death of patients with breast cancer Increasing evidence suggests that lncRNAs function as oncogenic or antitumor genes in various tumor types cells types and the microenvironment and that they may be used as effective and specific biomarkers for clinical diagnosis and prognosis Due to its oncogenic properties lncRNA FOXd2AS1 has been investigated in several malignant tumors In the present study FOXD2‘AS1 expression was found to be upregulated in breast cancer cell lines thus it was knocked down in MCF‘ and MdAMB468 cell to investigate its role in breast cancer FOXd2AS1 knockdown inhibited the proliferation migration and invasiveness of McF7 and MdAMB468 cells and inhibited tumor growth in vivo Notably S100A1 expression was also found to be upregulated in breast cancer cells and further investigation revealed that S100A1 was inhibited following FOXd2AS1 knockdown indicating that the expression of FOXd2AS1 and S100A1 was positively associated in breast cancer cells Subsequent experiments revealed that the overexpression of FOXD2‘AS1 significantly accelerated tumorigenesis by promoting cellular proliferation migration and invasiveness However the effects of FOXd2AS1 were reversed by the downregulation of S100A1 These results suggest that both FOXD2‘AS1 and S100A1 knockdown exert antitumor effects on the progression of breast cancer and that FOXD2‘AS1 may exert its oncogenic functions by regulating S100A1S100A1 is a calcium‘binding protein belonging to the S100 protein family which exhibit a range of biological properties surrounding cellular proliferation metastasis immune evasion and angiogenesis and are also involved in tumorigenesis For example S100A4 enhances p53dependent apoptosis and facilitates more aggressive tumor progression S100A6 has been reported to be upregulated in human osteosarcoma colorectal carcinoma and hepatocellular carcinoma which was mostly associated with its suppressive properties towards cancer cell migration and tumor metastasis Therefore S100 proteins play an important role in the development and progression of tumors highlighting the necessity to further understand their roles and potential underlying mechanisms In the present study S100A1 expression was found to be upregulated in breast cancer cell lines FOXd2AS1 overexpression was shown to accelerate breast cancer progression by promoting cellular proliferation migration and invasiveness and functional experiments demonstrated that the knockdown of S100A1 reversed the effects induced by FOXD2‘AS1 Furthermore S100A1 knockdown suppressed breast cancer progression by inhibiting the proliferation migration and invasiveness of McF7 and MDA‘MB‘ cells In agreement with these findings S100A1 has been reported to be overexpressed in ovarian cancer and to be associated with lymph mode metastasis the overexpression of S100A1 was shown to enhance cellular proliferation and migration whilst its inhibition exerted an opposite effect on ovarian cancer cells Moreover high tumor expression levels of S100A1 have been shown to be positively associated with decreased relapse‘free survival time in an endometrioid subtype of ovarian and endometrial cancers It was thus hypothesized that S100A1 functions as an important regulator in breast cancer and may therefore be a promising therapeutic target for this as well as other types of gynecological cancerThe Hippo pathway is an important signaling pathway that regulates cellular proliferation and apoptosis the activation of which is triggered by the phosphorylation of the large tumor suppressor kinases LATS1 and LATS2 The Hippo pathway is very complex as a number of kinases relay upstream signals to LATS to regulate this pathway The STE20 protein kinases MST12 as the core components of the Hippo pathway are considered responsible for the phosphorylation and activation of LATS12 YAP a downstream effector of the Hippo pathway is highly activated in various types of cancer and targeting YAP may effectively suppress tumorigenesis Both dysregulated Hippo signaling and aberrant YAP activation contribute to cancer progression In the present study FOXD2‘AS1 knockdown significantly increased the phosphorylation of YAP and LATS1 indicating that the Hippo signaling pathway was activated by FOXD2‘AS1 downregulation Notably it was found that MST12 expression levels were downregulated by

Thyroid_Cancer

Thyroid surgery in children in a single institution from Osama Ibrahim Almosallama Ali Aseerib Ahmed Alhumaida Ali S AlZahranic Saif Alsobhib Saud AlShanafeybFrom the aDepartment of Surgery College of Medicine Qassim University Buraidah Al Qassim Saudi Arabia bDepartment of Surgery King Faisal Specialist Hospital and Research Center Riyadh Saudi Arabia cDepartment of Medicine King Faisal Specialist Hospital and Research Center Riyadh Saudi Arabia Correspondence Dr Osama Ibrahim Almosallam Department of Surgery College of Medicine Qassim University PO Box Buraidah Al Qassim Saudi Arabia osama_iaahotmailcom ORCID orcid0000000290367564 Citation Almosallam OI Aseeri A Alhumaid A AlZahrani AS Alsobhi S AlShanafey S Thyroid surgery in children in a single institution from Ann Saudi Med Received January Accepted May Published August Copyright Copyright Annals of Saudi Medicine Saudi Arabia This is an access under the Creative Commons AttributionNonCommercialNoDerivatives International License CC BYNCND The details of which can be accessed at httpcreativecommons licensesbyncnd40Funding NoneBACKGROUND Data on thyroid surgery in children are scarceOBJECTIVE Analyze outcome data on thyroid surgery in a pediatric populationDESIGN Medical record reviewSETTING Tertiary health care institutionPATIENTS AND METHODS We collected demographic and clinical data on patients years or younger who had thyroid surgery in the period to Descriptive data are presentedMAIN OUTCOME MEASURES Indications for thyroidectomy thyroid pathology complications length of stay and radioactive iodine treatment and recurrencesSAMPLE SIZE RESULTS Of patients who underwent thyroidectomy procedures were females and the mean age at operation was years and were associated with multiple endocrine neoplasia type There was no history of radiation exposure Eightyone patients had fine needle aspiration FNA which correlated with the final histopathology in of cases Sixtysix patients had malignant cancer papillary of patients who had neck dissection had lymph node metastasis and had distant metastases to the lung Procedures included total thyroidectomy hemithyroidectomy completion and subtotal thyroidectomy Twentythree patients developed hypocalcemia permanent and had unilateral recurrent laryngeal nerve injury permanent Patients were followed up for a mean duration of months median months Of patients with thyroid cancer received radioactive iodine and had recurrence Malignancy is the commonest indication for thyroid surgery in children and FNA is highly diagnostic Hypocalcemia and recurrent laryngeal nerve injury are significant complications The recurrence rate in thyroid cancer is LIMITATIONS RetrospectiveCONFLICT OF INTEREST Noneoriginal ANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0cThyroid diseases requiring surgery are relatively uncommon in children compared to adults The prevalence of palpable thyroid nodules in children ranges from to Sporadic welldifferentiated thyroid cancer is the most common endocrine malignancy in children accounting for of pediatric cancers in the prepubertal age group and up to of cancers in adolescents aged “ year2 The most common indication for thyroid surgery in children varies among published studies but thyroidectomy for malignant conditions is rising38 Data in children throughout the world are relatively scarce The objective of this study was to analyze the clinical data and outcome of thyroid surgery in a large series of children treated at a single center at King Faisal Specialist Hospital and Research Center KFSHRC in RiyadhPATIENT AND METHODS With the approval of the Institutional Review Board IRB at KFSHRC the medical records of all patients years old and younger who underwent a thyroid surgery between and were retrospectively reviewed We elected to include patients up to the year to ensure a reasonable followup period Patients for the study were identified by a search of the operating room log for all procedures involving the thyroid gland for the specified age groupDemographic data clinical features and surgical outcomes were collected Specific data that were obtained included age at operation gender family history presenting symptoms history of radiation exposure presence of multiple endocrine neoplasia type MEN thyroid function test presence and size of thyroid nodules by ultrasound presence of lymph nodes metastasis or distant metastasis fine needle aspiration FNA cytology surgical procedure final histopathology and length of followup Outcomes analyzed were postoperative complications including transient or permanent hypocalcemia transient or permanent recurrent laryngeal nerve paralysis wound infection and hematoma length of stay and radioactive iodine treatment and recurrences Thyroid procedures in this series included hemithyroidectomy subtotal total and completion thyroidectomy Surgeries were performed by either an endocrine adult surgeon or a pediatric surgeon No intraoperative nerve monitoring was used Early in the series procedures were performed by adult endocrine surgeons but lately a combined approach was adopted where pediatric surgeons and adult endocrine surgeons collaborated in such cases proceduresthe normal range in our laboratory regardless of symptoms Transient hypocalcemia was identified if it lasted for less than months while permanent hypocalcemia was considered if the serum calcium level remained below normal range and the patient continued on calcium supplementation after months of the surgery All patients with a family history of MEN underwent genetic testing of the RET protooncogene to confirm the diagnosis All patients who underwent completion thyroidectomy had a preoperative and postoperative vocal cords assessment at the Otolaryngology clinic Descriptive data were generated and comparisons were conducted using the t test for continuous data and the chisquare or Fisher exact tests for proportionsRESULTSBetween and patients underwent surgical procedures patients underwent two procedures for thyroid disease at our institution Eighty patients were females The mean age at operation was years median years range years The most common indication for thyroidectomy was thyroid nodule which was present in of cases Table The mean SD size of thyroid nodules was mm There were cases associated with MEN syndromes The final pathology in two patients with MEN syndrome showed medullary thyroid cancer MTC while the remaining patients had prophylactic procedures before development of MTC None of the patients had a history of radiation exposure Eightyone patients FNA which correlated with the final histopathology in of cases There were three cases of toxic adenoma and one case of Graves™ disease which did not require FNA The remaining cases underwent FNA at another institution and FNA was not repeated at our institution or they came for completion thyroidectomy with documented pathology for malignancy after they had their first surgery in another hospitalThe most common diagnoses included papillary thyroid cancer and multinodular goiter or colloid Table Indications for thyroidectomy in patients IndicationNodulen MEN prophylaxisHyperthyroidismMultinodular goiterCompletion thyroidectomy Hypocalcemia was defined by calcium levels below Data are number original PEDIATRIC THYROID SURGERYANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0cnodule Table Surgical procedures included total thyroidectomy hemithyroidectomy completion thyroidectomy and subtotal thyroidectomy Neck dissection was performed in patients Operative complications were observed in patients The most common complication was hypocalcemia transient permanent and Table Thyroid pathology in the patientsPathologyn BenignNormal thyroid tissueColloid noduleCystAdenomaThyroiditisGraves™ diseaseThyroid cancerPapillaryFollicularMedullaryHurthleAnaplasticTotalData are number Table Benign and malignant lesions in patientsBenignn37Malignantn66 P value Age meanyearsGender malefemalePresence of noduleHypocalcemiaRecurrent laryngeal nerve palsyBleedinghematomaWound infectionTracheal injuryOverall complicationsMean length of stay daysMEN recurrent laryngeal nerve palsy transient permanent all were unilateral Table Of patients with malignant lesions had lymph node metastasis and patients had distant metastases to the lung None of the patients developed postoperative bleeding wound infection or tracheal injury Patients were followed up for a mean of months median range months radioactive iodine treatment was delivered to patients with malignant lesions patients had recurrences were local recurrences and were local and distant recurrences to the lung Three cases received radioactive iodine RAI before and after recurrence One case was low risk before recurrence so did not receive RAI until after recurrence One case had medullary thyroid cancer so did not receive RAI In the remaining five cases there was no clear data whether those patients received RAI before or only after a recurrence All local recurrences underwent resection except for one patient who was lost follow up There was no mortality in this study DISCUSSIONThe most common indication for thyroidectomy in this series was thyroid nodule which correlates with previously published reports in the pediatric population35 Children with thyroid nodules have an estimated fourfold higher risk of developing thyroid cancer compared to adults910 The high incidence of malignancy in this series suggests children with a thyroid nodule should be carefully evaluatedFNA is a valuablemethod for preoperative evaluation of thyroid nodules However there are limitations on the routine use of FNA in children including the need for sedation sampling errors and the limited availability of experienced cytopathologists11 Many previous studies reported high sensitivity and specificity of FNA in evaluating thyroid nodule in children1114 which correlate with our findingsOur data showed lymph node metastasis in of thyroid cancer cases which supports the notion that children with thyroid cancer frequently present with more extensive disease than adults Lymphnode involvement at diagnosis is seen in to of children compared with to of adults with differentiated thyroid cancer1523 Because our hospital is the largest referral center in Saudi Arabia especially for oncology cases this may explain the large number of lymph node and distant metastasis In this cohortThe most common complication reported after thyroidectomy in children is hypoparathyroidism with an incidence ranging between to which original PEDIATRIC THYROID SURGERYANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0ccorresponds with our results of which are reported as hypocalcemia in Table One study found that total thyroidectomy central and bilateral neck dissection Graves™ disease and malignancy were risk factors for hypocalcemia after thyroid surgery3 In this cohort postoperative hypocalcemia was noted more in malignant cases but it failed to reach statistical significance Moreover there was no significant difference between benign and malignant cases in terms of mean age gender distribution recurrent laryngeal nerve injury or overall complications a finding that was reported previously26 Multiple studies in recent years have found an inverse relationship between surgeon volume and complication rates2728 but similar data in the pediatric population is lacking One study found that highvolume endocrine surgeons have better outcomes and shorter lengths of stay and lower costs after thyroidectomy and parathyroidectomy in children compared to pediatric surgeons general surgeons or otolaryngologists29 Scheumann and colleagues also concluded that a collaborative approach between pediatric and endocrine surgeons would have better outcomes This has led other authors to suggest that a combined approach with endocrine and pediatric surgeons in addition to pediatric endocrinologists may optimize the care of children with surgical thyroid disease given the low number of pediatric patients4 Our data do not allow for comparisons of different approaches given the late adoption of the combined approach The recurrence rate for thyroid cancer in children after thyroidectomy has varied widely in reported studies ranging from to while it was in this cohort Only a few studies explored the predictors of recurrence Lymph node involvement multiple nodules male gender younger age histologic subtype and advanced tumor stage were risk factors associated with recurrence17233033 In this study of patients with malignant lesions received RAI Although there are conflicting data regarding the indications of postoperative RAI treatment in lowrisk patients the current recommendation is that lowrisk patients can be treated without RAI3436There are some limitations to this study The retrospective nature may affect the validity and quality of the data The small number of cases in some categories did not enable us to compare groups and explore predictors relative to these factors On the other hand this study adds to the scarce data on thyroid surgery in pediatric age group Malignancy is the commonest indication for thyroid surgery in children and FNA is highly diagnostic Hypocalcemia and recurrent laryngeal nerve injury are significant complications Cancerrelated death is extremely rare but recurrence is not uncommon and a significant number of patients with malignant cases received RAI treatmentoriginal PEDIATRIC THYROID SURGERYANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0cREFERENCES Trowbridge FL Matovinovic J McLaren GD Nichaman MZ Iodine and goiter in children Pediatrics Ries LAG Melbert D Krapcho M Stinchcomb DG Howlader N Horner MJ et al SEER Cancer Statistics Review “ Bethesda National Cancer Institute Based on November SEER data submission Chen Y[h] Masiakos PT Gaz RD Hodin RA Parangi S Randolph GW et al Pediatric thyroidectomy in a high volume thyroid surgery center Risk factors for postoperative hypocalcemia J Pediatr Surg Aug5081316 Wood JH Partrick DA Barham HP Bensard DD Travers HS Bruny JL et al Pediatric thyroidectomy a collaborative surgical approach J Pediatr Surg May4658238 Scholz S Smith JR Chaignaud B Shamberger RC Huang SA Thyroid surgery at Children™s Hospital Boston a 35year singleinstitution experience J Pediatr Surg Mar46343742 Josefson J Zimmerman D Thyroid nodules and cancers in children Pediatr Endocrinol Rev Sep611423 Hameed R Zacharin MR Changing face of paediatric and adolescent thyroid cancer J Paediatr Child Health LugoVicente H Ortiz VN Irizarry H Camps JI Pagán V Pediatric thyroid nodules management in the era of fine needle aspirationJ Pediatr Surg Mussa A De Andrea M Motta M Mormile A Palestini N Corrias A Predictors of Malignancy in Children with Thyroid Nodules J Pediatr Oct167488692 Amirazodi E Propst EJ Chung CT Parra DA Wasserman JD Pediatric thyroid FNA biopsy Outcomes and impact on management over years at a tertiary care center Cancer Cytopathol Partyka KL Huang EC2 Cramer HM Chen S Wu HH Histologic and clinical followup of thyroid fineneedle aspirates in pediatric patients Cancer Cytopathol Sinha CK Decoppi P Pierro A Brain C Hindmarsh P Butler G et al Thyroid Surgery in Children Clinical Outcomes Eur J Pediatr Surg Oct2554259 Kundel A Thompson GB Richards ML Qiu LX Cai Y Schwenk FW et al Pediatric Endocrine Surgery A 20Year Experience at the Mayo Clinic J Clin Endocrinol Metab February “ Jiang W Newbury RO Newfield RS Pediatric thyroid surgery and management of thyroid nodulesan institutional experience features and over a 10year period Int J Pediatr Endocrinol Burke JF Sippel RS Chen H Evolution of Pediatric Thyroid Surgery at a Tertiary Medical Center Surg Res “ AlQahtani KH Tunio MA Al Asiri M Aljohani NJ Bayoumi Y Riaz K et al Clinicopathological treatment outcomes of differentiated thyroid cancer in Saudi children and adults J Otolaryngol Head Neck Surg Nov Kluijfhout WP van Beek DJ Verrijn Stuart AA Lodewijk L Valk GD Van der Zee DC et al Postoperative Complications After Prophylactic Thyroidectomy for Very Young Patients With Multiple Endocrine Neoplasia Type Medicine Baltimore 20159429e1108 Raval MV Browne M Chin AC Zimmerman D Angelos P Reynolds M Total thyroidectomy for benign disease in the pediatric patient”feasible and safe J Pediatr Surg Stavrakis AI Ituarte PH Ko CY Yeh MW Surgeon volume as a predictor of outcomes in inpatient and outpatient endocrine surgery Surgery “ Sosa JA Bowman HM Tielsch JM Powe NR Gordon TA Udelsman R The importance of surgeon experience for clinical and economic outcomes from thyroidectomy Ann Surg “ Tuggle CT Roman SA Wang TS Boudourakis L Thomas D Udelsman R et al Pediatric endocrine surgery Who is operating on our children Surgery Dec144686977 Park S Jeong JS Ryu HR Lee C Park JH Kang S et al Differentiated Thyroid Carcinoma of Children and Adolescents27Year Experience in the Yonsei University Health System J Korean Med Sci Palmer BA Zarroug AE Poley RN Kollars JP Moir CR Papillary thyroid carcinoma in children risk factors and complications of disease recurrence J Pediatr Surg Wada N Sugino K Mimura T Nagahama M Kitagawa W Shibuya H et al Pediatric differentiated thyroid carcinoma in stage I risk factor analysis for disease free survival BMC Cancer D Danese Gardini A Farsetti A Sciacchitano S Andreoli M Pontecorvi A Thyroid carcinoma in children and adolescents Eur J Pediatr Astl J Chovanec M Lukes P Katra R Dvorakova M Vlcek P et al Thyroid carcinoma surgery in children and adolescents “ years experience surgery of pediatric thyroid lymph node metastases carcinoma Int J Pediatr Otorhinolaryngol Chaukar DA Rangarajan V Nair N Nadkarni MS Pai PS Dcruz AK et al Pediatric thyroid cancer J Surg Oncol Dzodic R Buta M Markovic I Gavrilo D Matovic M Milovanovic Z et al Surgical management of welldifferentiated thyroid carcinoma in children and adolescents years of experience of a single institution in Serbia Endocr J Scheumann GF Gimm O Wegener G Hundeshagen H Dralle H Prognostic significance and surgical management of locoregional in papillary thyroid cancer World J Surg Shi RL Qu N Yang SW Tumor size interpretation for predicting cervical lymph node metastasis using a differentiated thyroid cancer risk model Onco Targets Ther “ Zimmerman D Hay ID Gough IR Goellner JR Ryan JJ Grant CS et al Papillary thyroid carcinoma in children and adults longterm followup of patients conservatively treated at one institution during three decades Surgery Collini P Mattavelli F Pellegrinelli A Barisella M Ferrari A Massimino M Papillary carcinoma of the thyroid gland of childhood and adolescence Morphologic subtypes biologic behavior and prognosis a clinicopathologic study of sporadic cases treated at a single institution during a 30year period Am J Surg Pathol BorsonChazot Causeret S Lifante JC Augros M Berger N Peix JL Predictive factors for recurrence from a series of children and adolescents with differentiated thyroid cancer World J Surg Baumgarten HD Bauer AJ Isaza A MostoufiMoab S Kazahaya K Adzick NS Surgical management of pediatric thyroid disease Complication rates after thyroidectomy at the Children™s Hospital of Philadelphia highvolume Pediatric Thyroid Center Journal of pediatric surgery Oct Kurzawinski TR De Coppi P Thyroidectomy in Children InPediatric Surgery pp Springer Berlin Heidelberg Francis G Waguespack SG Bauer AJ Angelog P Benvenga S et al Management Guidelines for Children with Thyroid Nodules and Differentiated Thyroid Cancer The American Thyroid Association Guidelines Task Force on Pediatric Thyroid Cancer THYROID Volume Number original PEDIATRIC THYROID SURGERYANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0c'

Thyroid_Cancer

Segregation analysis of the BRCA2 c9227GT variant in multiple families suggests a pathogenic role in breast and ovarian cancer predispositionSimona Agata1 Silvia Tognazzo1 Elisa Alducci1 Laura Matricardi1 Lidia Moserle1 Daniela Barana2 Marco Montagna1Classification of variants in the BRCA1 and BRCA2 genes has a major impact on the clinical management of subjects at high risk for breast and ovarian cancer The identification of a pathogenic variant allows for early detectionprevention strategies in healthy carriers as well as targeted treatments in patients affected by BRCA associated tumors The BRCA2 c9227GT pGly3076Val variant recurs in families from Northeast Italy and is rarely reported in international databases This variant substitutes the evolutionary invariant glycine with a valine in the DNA binding domain of the BRCA2 protein thus suggesting a high probability of pathogenicity We analysed clinical and genealogic data of carriers from breastovarian cancer families in whom no other pathogenic variants were detected The variant was shown to cosegregate with breast and ovarian cancer in the most informative families Combined segregation data led to a likelihood ratio of of pathogenicity vs neutrality We conclude that c9227GT is a BRCA2 pathogenic variant that recurs in Northeast Italy It can now be safely used for the predictive testing of healthy family members to guide preventive surgery andor early tumor detection strategies as well as for PARP inhibitors treatments in patients with BRCA2associated tumorsNext to the hurdle of the bioinformatics processing of huge amount of sequencing data the clinical interpretation of sequence variants has become the most recent challenge of next generation sequencing NGS approaches Efforts are currently underway within international consortia such as the Evidencebased Network for the Interpretation of Germline Mutant Alleles ENIGMA to order and standardize a variety of methods that foster variants of uncertain significance VUS towards a benign or pathogenic classificationWhile pathogenic variants of the BRCA1 and BRCA2 genes account for about one fourth of all breast and ovarian cancer families1 VUS are the result of a smaller fraction of all tests “ and cannot be used for identification of predisposed family members as long as their clinical relevance is clearly defined In particular predictive testing within families is only recommended for variants with a probability of pathogenicity higher than ie class and according to a widely used 5tiered classification4 In the absence of a pathogenic variant healthy subjects of high risk families need to be managed according to the specific family history of the diseaseProbabilities of pathogenicity for variants occurring in the BRCA1 and BRCA2 genes were previously calculated based on variant location within splicing consensus sequences5 or crossspecies evolutionary conservation of each aminoacid positon6 These estimates were calibrated against large clinical data sets to generate a priori probabilities of pathogenicity reviewed in7 thus providing a hint for identification of those variants that might deserve further investigation1Immunology and Molecular Oncology Unit Veneto Institute of Oncology IOVIRCCS Padua Italy 2Oncology Unit Local Health and Social Care Unit ULSS8 Berica Montecchio Maggiore Italy email marcomontagnaiovvenetoitScientific RepoRtS 101038s41598020707290Vol0123456789wwwnaturecomscientificreports 0cFamily IDNumber of breast cancer cases age 3240b 4246b 50c 59c 4958b 4578b 43c 5570b Number of ovarian cancer cases ageNumber of breast cancer phenocopies ageOther tumors ageBOADICEANumber of other tested family members““ 57c 53c “““ 55c “ “ “ 5555b“ 55d“““ “““““Kidney “Central nervous system Kidney lung “““Ampulla of Vater ““Kidney prostate “Prostate thyroid Pancreas ““““““LRa“““““Table Characteristics of families carrying the BRCA2 c9227GT Numbers in bold refer to age at diagnosis in individuals specifically analysed for the c9227GT or obliged carries a LR likelihood ratio b Bilateral breast cancer c Subjects affected by breast and ovarian cancer d LCIS lobular carcinoma in a0situOn the other hand it has been suggested that additional proofs relying on œdirect evidences are necessary to reach a final posterior probability that fosters the variant from class including VUS4 to one of the extreme classes Using the multifactorial likelihood model several types of data sources can contribute to variant classification including family history of cancer cooccurrence in trans with known pathogenic variants breast cancer histopathological features and segregation9 Breast cancer histopathology provides little predictive power for BRCA2 variants as BRCA2associated and nonhereditary breast tumors display largely overlapping morphological and biochemical parameters10 Similarly cooccurrence with proven pathogenic variants is strongly predictive of neutrality Conversely in the absence of pathogenic variants it provides scant evidence for a classification towards pathogenicity Therefore at present the analysis of segregation of the variant with disease within families remains one of the most powerful and robust method to achieve a successful classification for class BRCA2 variants11Results and discussionDuring the molecular analysis of BRCA12 genes in more than breast andor ovarian cancer patients we identified families carrying the BRCA2 c9227GT variant All families were selected according to criteria approved by the Veneto Region and largely overlapping to those currently used in European countries see œMethods section for details Most of the families carrying the BRCA2 c9227GT variant showed typical BRCA2 tumor spectra with frequent bilateral breast tumors early age at first breast cancer diagnosis and presence of ovarian cancer in more than half of them Table a0Based on family histories of breast and ovarian cancer a high probability of occurrence of a BRCA1 or BRCA2 pathogenic variant was obtained in most of the families Table a0 In spite of these predictions neither clearly pathogenic variants nor other VUS were identified in addition to the BRCA2 c9227GT in any of these families Although screening of BRCA1 and BRCA2 genes was performed by different technical approaches over the time it always included the complete coding sequence as well as all exon“intron boundaries of both genes thus minimizing the possibility that pathogenic variants in BRCA1 or located in cis to the BRCA2 c9227GT variant might have been missed Since the analysis included only the BRCA1 and BRCA2 genes the presence of pathogenic variants in other highmoderate predisposition genes could not be excludedGlycine amino acid is an invariant position across twelve species from Pan troglodytes to Strongylocentrotus purpuratus see œMethods section for the complete list of species Comparison of the composition polarity and molecular volume of glycine vs valine highlights a moderate physicochemical difference corresponding to a Grantham distance12 of Using AlignGVGD1314 a widely used in silico prediction tool the combination of these features assigns this aminoacid substitution to category C65 which includes the most likely deleterious changes Glycine is located within the oligonucleotide binding3 motif OB3 of a larger domain specifically involved in ssDNA binding15 Altogether these data strongly favour a likely functional relevance of the pGly3076Val substitution According to previous estimates6 these observations provide a a priori probability of pathogenicityThe DNA binding motif is the most characterized functional domain of the BRCA2 protein and has been implicated in the homologous recombination activity necessary for the repair of DNA double strand breaks The relevance of the domain is emphasized by the high density of pathogenic missense variants mapping to this motif Accordingly using a homologydirected DNA break repair HDR functional assay Guidugli et a0al16 showed Scientific RepoRtS 101038s41598020707290Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Segregation of the BRCA2 c9227GT in family Carriers and non carriers are indicated by and ˆ’ signs respectively Tumor type is indicated below each symbol Numbers refer to current age and age at diagnosis for healthy and affected subjects respectively Proband is marked by the arrow LCIS lobular carcinoma in a0situthat of VUS located in this domain displayed an impaired ability to repair an ISce1induced DNA double strand break In particular when challenged by this method pGly3076Val showed an in a0vitro phenotype overlapping to those of pathogenic variants16 Using the classification guidelines from the American College of Medical Genetics ACMG17 data derived from wellestablished functional studies provide a strong evidence of pathogenicity PS3 category This feature combined with the absence of the c9227GT variant in control populations PM2 and with the in silico evidence of pathogenicity PP3 would move the variant to class likely pathogenic However it has to be noted that different functional assays though extremely powerful in some contexts can lead to inconsistencies depending on the specific experimental conditions Moreover they often lack proper validation in terms of sensitivity and specificity While efforts are currently in progress within the ENIGMA consortium to derive rules to include functional assays results into the multifactorial likelihood model18 at present further evidences are advisable to derive a final probability of pathogenicity to confidently support clinical management decisionsWe therefore evaluated segregation of c9227GT in a total of additional family members from of the families Likelihood scores were calculated by means of a cosegregation algorithm specifically designed for the evaluation of BRCA1 and BRCA2 class variants19 In addition to genotypes this method makes use of age of onset with penetrance used as a function of age of first and second breast cancer as well as ovarian cancer Based on the assumption of independence of all sources of evidence that are integrated into the multifactorial likelihood method œfamily history data were not used further in the analysis Cosegregation likelihood ratios are reported in Table a0 for families with at least family members genotyped Very similar results were obtained when the most informative families were evaluated by an alternative full likelihood Bayes factor algorithm11 data not shownA combined likelihood ratio of was obtained from the integration of all family scores generating a probability of pathogenicity higher than that definitely assigns this variant to class Segregation of c9227GT with disease was nearly complete with few exceptions In family the proband™s maternal cousin was negative for c9227GT and developed a lobular carcinoma in a0situ LCIS at age Fig a0 LCIS however has gradually moved from a rare form of breast cancer to a œmarker of increased cancer risk and it is commonly referred to as œlobular neoplasia As such it is not usually taken into consideration in computer modelling of mutation probability accordingly it was excluded from the calculation of the segregation likelihood ratio ie this subject was treated as a healthy one In contrast three phenocopies in family and were included in score calculations These three patients were third degree relatives of the closest carrier with “ healthy subjects interposed and at least two of them had a positive family history in the alternative parental branch Because of the genealogic distance from the proband likelihood ratios were only marginally lowered by these data and remained in favour of pathogenicity in each of these familiesConsidering all carrier family members mean age at first breast and ovarian cancer were and a0years respectively consistent with those reported for BRCA2 pathogenic variants20 Similarly the ratio of breast to ovarian cancer was in line with what expected for a pathogenic variant falling outside of the breast cancer cluster region BCCR and the ovarian cancer cluster region OCCR21Among the other tumors anecdotally reported as part of the BRCA2 spectrum an ampulla of Vater carcinoma occurred in a carrier from family Increased risk of gallbladder and bile duct tumors were initially observed among BRCA2 carriers22 Interestingly recent data apparently reinforce the association of the BRCA2 gene to this specific tumor type2324 Considering the rarity of the tumor it might represent a good predictor of pathogenicity for BRCA2 variants especially when associated with a family history of breast andor ovarian andor pancreatic cancer Renal cell cancers were observed in three subjects from independent families two of whom were obliged carriers of the BRCA2 variant Though a role for BRCA2 has been suggested in the kidney embryonic Scientific RepoRtS 101038s41598020707290Vol0123456789wwwnaturecomscientificreports 0cdevelopment of the zeppelin zebrafish mutant2526 renal cancer is only sporadically reported among BRCA2 carriers Therefore unless of a variantspecific effect the cases reported here remain a descriptive observationWith four entries in the ClinVar database27 during the last a0years record VCV0001262033 accessed on July the c9227GT variant has never been reported in international population databases such as œThe Genome Aggregation Database gnoma dbroad insti tute thus suggesting a geographically limited distribution with a higher prevalence in the Veneto Region of Italy This observation has important implications for sequence variants classification as the power of segregation analysis increases with the number of families studied While benign classification of commonly identified variants is more easily achieved by laboratories with a high throughput the large amount of tests increases the probability of identification of a cooccurrence with pathogenic variants local laboratories might retain a higher classification power for specific variants with a peculiar geographical distributionOf note the proband of family developed an invasive ductal breast cancer at age that progressed to a metastatic disease four years later She was therefore proposed a BRCA test in the context of a large clinical trial to apply for a PARPinhibitor treatment The BRCA test was centralized outside our Institute she turned out to be a carrier of the c9227GT œvariant of uncertain significance and she was therefore excluded from the trial This emphasizes the different consequences related to the inability to classify a VUS Indeed while in a family context this failure implies that all at risk subjects need to be œincluded into tailored surveillance strategies based on their family history this inability can represent an œexclusion criterion from specific treatments for the patient Importantly the list of BRCAassociated tumors that can benefit from PARP inhibitors treatment is rapidly growing and it now includes metastatic Her2negative breast cancer metastatic pancreatic cancer and metastatic castrationresistant prostate cancer in addition to high grade ovarian cancers28In conclusion our data demonstrate that the BRCA2 c9227GT variant cosegregates with disease in multiple families and shows a phenotypic expression falling within the classical BRCA2associated spectrum These findings combined with in silico predictions as well as functional impairment of the DNA double strand break repair provide definitive evidence for pathogenicity thus reliably moving the variant to class definitely pathogenic The BRCA2 c9227GT variant can therefore be safely used in families to identify predisposed family members and to guide riskreducing surgery as well as strict surveillance strategies Concurrently patients carrying the BRCA2 c9227GT variant can benefit from targeted treatments of PARPinhibitors sensitive tumorsMethodsSequence variants are described according to HGVS nomenclature guidelines varno menhgvs and the BRCA2 Refseq NM_0000593Families were identified during the molecular analysis of BRCA1 and BRCA2 genes offered to patients with personal andor family history of breast andor ovarian cancer according to selection criteria approved from the Veneto Region Briefly referral criteria included a a personal history of either of the following breast cancer before age bilateral breast cancer before age male breast cancer breast and ovarian cancer in the same patient triple negative breast cancer ie negative for estrogen receptor progesterone receptor and HER2 before age high grade ovarian cancer or b a family history including i two first degree relatives with bilateral breast cancer andor breast cancer before age or ii three first degree relatives affected by breast andor ovarian andor pancreatic cancerThe search for pathogenic variants was carried out on DNA extracted from peripheral blood Direct sequencing either Sanger sequencing or NGS Illumina MiSeq platform was used for the vast majority of the probands Major genomic rearrangements were analysed by multiplex ligationdependent probe amplification MLPA or NGSbased approaches Sophia DDM Sophia Genetics Only the specific variant under study was tested in the other family membersIn silico predictions were performed by means of the AlignGVGD program1314 freely available at agvgd hciutahedu Calculations were made using the largest number of alignments including the following species Homo sapiens Pan troglodytes Macaca mulatta Rattus norvegicus Canis familiaris Bos taurus Monodelphis domestica Gallus gallus Xenopus laevis Tetraodon nigroviridis Fugu rubripes and Strongylocentrotus purpuratusAll procedures were in accordance with the ethical standards of the Helsinki declaration and its later amendments Probands and family members who were tested for the BRCA2 c9227GT explicitly agreed to participate to the research project and signed an informed consent All experimental protocols were approved by the Ethics Committee of the Veneto Institute of Oncology IOVProbabilities to identify a pathogenic variant were computed using the breast and ovarian analysis of disease incidence and carrier estimation algorithm BOADICEA29Current age gender age of onset of the first and second breast cancer age of onset of ovarian cancer and genotype of members of families carrying the BRCA2 c9227GT variant were used to calculate likelihood ratios of the variant to be pathogenic vs neutral using an approach previously described for BRCA1 and BRCA2 variant cosegregation analysis19 Families with the highest pathogenicity likelihood were doublechecked using an alternative full likelihood Bayes factor approach available at analy zemyvar iantcoseg regat ionanaly sis11The overall likelihood was derived by the product of the likelihood ratios over the independent familiesReceived April Accepted July References Antoniou A C Easton D F Models of genetic susceptibility to breast cancer Oncogene “ Scientific RepoRtS 101038s41598020707290Vol1234567890wwwnaturecomscientificreports 0c Eccles D M et al BRCA1 and BRCA2 genetic testing”pitfalls and recommendations for managing variants of uncertain clinical Eggington J M et al A comprehensive laboratorybased program for classification of variants of uncertain significance in heredisignificance Ann Oncol “ tary cancer genes Clin Genet “ Plon S E et al Sequence variant classification and reporting recommendations for improving the interpretation of cancer susceptibility genetic test results Hum Mutat “ Vallée M P et al Adding in silico assessment of potential splice aberration to the integrated evaluation of BRCA gene unclassified variants Hum Mutat “ Tavtigian S V Byrnes G B Goldgar D E Thomas A Classification of rare missense substitutions using risk surfaces with genetic and molecularepidemiology applications Hum Mutat “ Lindor N M et al A review of a multifactorial probabilitybased model for classification of BRCA1 and BRCA2 variants of uncertain significance VUS Hum Mutat “ Goldgar D E et al Genetic evidence and integration of various data sources for classifying uncertain variants into a single model Goldgar D E et al Integrated evaluation of DNA sequence variants of unknown clinical significance Application to BRCA1 and Hum Mutat “ BRCA2 Am J Hum Genet “ Spurdle A B et al Refined histopathological predictors of BRCA1 and BRCA2 mutation status a largescale analysis of breast cancer characteristics from the BCAC CIMBA and ENIGMA consortia Breast Cancer Res Rañola J M O Liu Q Rosenthal E A Shirts B H A comparison of cosegregation analysis methods for the clinical setting Fam Cancer “ Grantham R Amino acid difference formula to help explain protein evolution Science “ Tavtigian S V et al Comprehensive statistical study of BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral J Med Genet “ Mathe E et al Computational approaches for predicting the biological effect of p53 missense mutations a comparison of three sequence analysis based methods Nucleic Acids Res “ Yang H et al BRCA2 function in DNA binding and recombination from a BRCA2DSS1ssDNA structure Science “ Guidugli L et al A classification model for BRCA2 DNA binding domain missense variants based on homologydirected repair activity Cancer Res “ Richards S et al Standards and guidelines for the interpretation of sequence variants a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology Genet Med “ Guidugli L et al Assessment of the clinical relevance of BRCA2 missense variants by functional and computational approaches Mohammadi L et al A simple method for cosegregation analysis to evaluate the pathogenicity of unclassified variants BRCA1 Am J Hum Genet “ and BRCA2 as an example BMC Cancer Kuchenbaecker K B et al Risks of breast ovarian and contralateral breast cancer for BRCA1 and BRCA2 mutation carriers JAMA JAMA Rebbeck T R et al Association of type and location of BRCA1 and BRCA2 mutations with risk of breast and ovarian cancer Breast Cancer Linkage Consortium Cancer risks in BRCA2 mutation carriers JNCI J Natl Cancer Inst “ Aburjania N Truskinovsky A M Overman M J Lou E Ampulla of Vater adenocarcinoma in a BRCA2 germline mutation carrier J Gastrointest Cancer “ Pinto P et al Analysis of founder mutations in rare tumors associated with hereditary breastovarian cancer reveals a novel association of BRCA2 mutations with ampulla of Vater carcinomas PLoS ONE e0161438 Drummond B E Wingert R A Scaling up to study brca2 the zeppelin zebrafish mutant reveals a role for brca2 in embryonic development of kidney mesoderm Cancer Cell Microenviron e1630 Kroeger P T et al The zebrafish kidney mutant zeppelin reveals that brca2fancd1 is essential for pronephros development Dev Landrum M J et al ClinVar improving access to variant interpretations and supporting evidence Nucleic Acids Res D1062“Biol “ D1067 Madariaga A Bowering V Ahrari S Oza A M Lheureux S Manage wisely poly ADPribose polymerase inhibitor PARPi treatment and adverse events Int J Gynecol Cancer “ Lee A J et al Boadicea breast cancer risk prediction model updates to cancer incidences tumour pathology and web interface Br J Cancer “ AcknowledgementsWe thank the probands and family members who contributed to the study We thank D Zullato for her expert technical assistance and Dr Maria Luisa Calabrò for critical revision of the manuscript The study was supported by × Istituto Oncologico Veneto research grantAuthor contributionsMM study design data analysis and writing of the manuscript SA BRCA12 screening LMo LMa genotyping of the c9227GT variant and data collection EA ST DB oncogenetic counselling and patients recruitment all authors reviewed and approved the manuscriptCompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to MMReprints and permissions information is available at wwwnaturecomreprintsPublisher™s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsScientific RepoRtS 101038s41598020707290Vol0123456789wwwnaturecomscientificreports 0c Access This article is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate if changes were made The images or other third party material in this article are included in the article™s Creative Commons license unless indicated otherwise in a credit line to the material If material is not included in the article™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this license visit httpcreat iveco mmons licen sesby40 The Authors Scientific RepoRtS 101038s41598020707290Vol1234567890wwwnaturecomscientificreports 0c'

Thyroid_Cancer

"researchWhat are the implications of using individual and combined sources of routinely collected data to identify and characterise incident site specific cancers a concordance and validation study using linked English electronic health records dataHelen Strongman Rachael Williams2 Krishnan Bhaskaran1To cite Strongman a0H Williams a0R Bhaskaran a0K What are the implications of using individual and combined sources of routinely collected data to identify and characterise incident site specific cancers a concordance and validation study using linked English electronic health records data BMJ 202010e037719 101136bmj 2020037719 –º Prepublication history and additional material for this paper are available online To view these files please visit the journal online http dx bmj Received February Revised April Accepted July Authors or their employers Re use permitted under CC BY Published by BMJFor numbered affiliations see end of Correspondence toDr Helen Strongman helen strongman lshtm ac ukObjectives To describe the benefits and limitations of using individual and combinations of linked English electronic health data to identify incident cancersDesign and setting Our descriptive study uses linked English Clinical Practice Research Datalink primary care cancer registration hospitalisation and death registration dataParticipants and measures We implemented case definitions to identify first site specific cancers at the most common sites based on the first ever cancer diagnosis recorded in each individual or commonly used combination of data sources between and We calculated positive predictive values and sensitivities of each definition compared with a gold standard algorithm that used information from all linked data sets to identify first cancers We described completeness of grade and stage information in the cancer registration data setResults gold standard cancers were identified Positive predictive values of all case definitions were ‰¥ and ‰¥ for the four most common cancers breast lung colorectal and prostate Sensitivity for case definitions that used cancer registration alone or in combination was ‰¥ for the four most common cancers and ‰¥ across all cancer sites except bladder cancer using cancer registration alone For case definitions using linked primary care hospitalisation and death registration data sensitivity was ‰¥ for the four most common cancers and ‰¥ for all cancer sites except kidney oral cavity and ovarian cancer When primary care or hospitalisation data were used alone sensitivities were generally lower and diagnosis dates were delayed Completeness of staging data in cancer registration data was high from minimum in and in for the four most common cancersConclusions Ascertainment of incident cancers was good when using cancer registration data alone or in combination with other data sets and for the majority Strengths and limitations of this study –º This is the first study to present comprehensive information on the implications of using different individual and combinations of linked electronic health data sources in England to identify cases of the most common incident cancers –º Using a gold standard algorithm that combined all available data from multiple sources as a comparator we were able to estimate both positive predictive values and sensitivity values for a range of pragmatic case definitions –º We described similarities and differences in values between age groups sexes and calendar years the impact of choice of sources on diagnosis dates and mortality rates and completeness of stage and grade in cancer registration data –º A key limitation was that our gold standard algorithm is not validated and may be affected by differences in clinical diagnosis and coding of invasive cancers between data sourcesof cancers when using a combination of primary care hospitalisation and death registration dataINTRODUCTIONThe Clinical Practice Research Datalink CPRD provides de identified primary care data linked to additional secondary health data sources under a well governed framework1 Use of linked data helps researchers to answer more epidemiological questions and increase study quality through improved exposure outcome and covariate classification2 In the field of cancer epidemiology CPRD primary care data linked to Hospital Episode Statistics Admitted Patient Care Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access data HES APC Office of National Statistics ONS mortality and National Cancer Registration and Analysis Service NCRAS cancer registration data are used to analyse factors contributing to the risk of cancer and the consequences of cancer and its treatment Use of linked data reduces the sample to the common source population and data coverage period for each included data set and has cost and logistical implications which are greatest for NCRAS data Research teams therefore commonly choose not to use all available linked data3 Cancer epidemiology studies can also be conducted using NCRAS and HES APC data provided by National Health Service NHS Digital and Public Health England PHE without linkage to CPRD primary care data4 This provides national coverage at the expense of the detailed health data that are available in primary care recordsValidation studies assessing concordance between CPRD GOLD HES APC and NCRAS data have estimated high positive predictive values PPVs for CPRD GOLD data and varying proportions of registered cancers that are not captured in CPRD GOLD and HES APC5“ The most up to date analysis by Arhi et al included the five most common cancers and all papers focussed on concordance between CPRD GOLD only and NCRAS existing evidence therefore does not provide a complete assessment of the benefits and limitations of using different combinations of data sources within the context of practical study designs National data are available describing completeness of data fields within the cancer registry data in each collection year9 and over time for all cancers combined4 missingness for individual years has been associated with age comorbidities and Clinical Commissioning Groups10 We aim to describe and compare the benefits and limitations of using different combinations of linked CPRD primary care data HES APC ONS mortality and NCRAS cancer registration data for conducting cancer epidemiology studies Our analyses focus on incident cancer ascertainment as it is a common and important outcome in cancer epidemiology and it is more difficult to distinguish between secondary recurrent and primary cancers at a second site in these data sets We have compared definitions of the most common cancers based on the first ever cancer recorded in individual or combinations of data sets with a gold standard definition comparing information from all four data sets We also describe the availability of stage grade and treatment variables over time in the cancer registration data for the CPRD linked cohort This reflects real life study design and will help researchers to decide which combination of data sources to use for future studiesMETHODSStudy design and settingWe completed a concordance study using linked2 English CPRD GOLD HES APC ONS mortality and NCRAS data CPRD GOLD data were extracted from the January monthly release and the 13th update to CPRD™s linked data The study period was from January to December with December matching the end of the NCRAS coverage periodThe CPRD GOLD database includes de identified records from participating general practices in the UK who use Vision software1 General practice staff can record cancer diagnoses using Read codes or in free text comments boxes though the latter are not collected by CPRD Diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care CPRD GOLD data are linked to HES APC ONS mortality and NCRAS through a trusted third party for English practices that have agreed to participate in the linkage programme2 HES APC data are collected by NHS Digital to co ordinate clinical care in England and calculate hospital payments12 Admissions for and related to cancer diagnoses are recorded using International Classification of Diseases version ICD10 codes National cancer registration data are collected by NCRAS which is part of PHE in accordance with the Cancer Outcomes and Services Data set13 which has been the national standard for reporting of cancer in England since January Data include ICD10 codes to identify the cancer site and more detailed information such as stage and grade ONS mortality data includes dates and causes of deaths registered in England recorded using ICD10 codesParticipants exposures and outcomesOur underlying study population included male and female patients registered in CPRD GOLD practices who were eligible for linkage to HES APC NCRAS and ONS mortality data and had at least days of follow up between January and December Start of follow up was defined as the latest of the current registration date within the practice and the CPRD estimated start of continuous data collection for the practice up to standard date End of follow up was determined as the date the patient left the practice ONS mortality date of death or practice last collection dateIdentification and classification of cancer codesWe used code lists to classify cancer records in each of CPRD GOLD HES APC and ONS mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https data Incompletely specified cancer codes could be mapped to cancer site eg ICD10 code C689 œMalignant neoplasms of urinary an unspecified was considered consistent with both bladder and kidney cancer For NCRAS we accessed coded records for the most common cancers We included cancers recorded in the clinical or referral file for CPRD GOLD cancers recorded in any diagnosis field for HES APC and the underlying or Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c accessFigure Gold standard algorithm to identify incident site specific cancers using all data sources HES Hospital Episode Statistics NCRAS National Cancer Registration and Analysis Service ONS Office of National Statisticsmost immediate cancer cause of death in ONS mortality dataCancer case definitions based on individual sources and combinations of sourcesWe developed alternative cancer case definitions mirroring those commonly used in epidemiology studies based on identifying the first malignant cancer excluding administrative codes and benign tumours recorded in various combinations of data sources NCRAS alone NCRAS and HES APC all sources CPRD GOLD HES APC and ONS mortality CPRD GOLD alone HES APC alone Multiple malignant cancers recorded on the index date in CPRD GOLD or HES APC were reclassified as multiple site cancer and were not considered as individual site cancer records for positive predictive value and sensitivity calculations multiple codes recorded in different sources on the same date were reclassified as the site identified in the NCRAS data if available and as multiple site cancer if not For each case definition we only examined the first malignant cancer per individual where this occurred within the study period and at least year after the start of follow upGold standard cancer case definitionWe developed a gold standard algorithm that classifies incident records of the most common cancers by comparing the first malignant cancer identified in each individual source figure Cancers recorded in NCRAS alone with no contradictions ie records for first cancers at different sites were considered true cases whereas cancers recorded in HES APC alone or GOLD alone required internal confirmation within that source in the form of another code for cancer consistent with the same site or with site unspecified within months and no contradictory codes eg for cancers at other sites in this period Where cancer records were present in data source we considered a site specific cancer to be a true case a if it was recorded as the first cancer in NCRAS and the total number of data sources with records for cancer at that site was equal to or greater than the number of data sources with contradictory records ie records for first cancers at different sites or b where the cancer was not present in NCRAS if there were more data sources in total with records for cancer at that site than data sources with contradictory recordsWe used NCRAS data to identify stage grade and treatment where available in the cancer registry only cohort Binary surgery chemotherapy and radiotherapy variables were derived using individual records of treatment from the first year after diagnosisStatistical analysisFor each cancer site and each individual or combined data source we combined our applied study definitions Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access with our gold standard definition to classify each applied study definition as a true positive false positive or false negative recordWe used these categories to calculate sensitivity and positive predictive value overall and stratified by age categories to and calendar year and sex We calculated differences in diagnosis dates for true positives by subtracting the gold standard index date from the index date for each source and combination of sourcesWe used Kaplan Meier methods to describe mortality over time for cancers identified using each definition The ONS mortality death date was used for these analysesWe used the NCRAS only definition to calculate proportions of patients with complete stage and grade and recorded cancer treatment modalities over timePatient public involvementPatients and the public were not involved in conceiving designing or conducting this study and will not be consulted regarding the dissemination of study resultsRESULTSOf research quality patients in the CPRD GOLD January build were eligible for linkage to HES ONS mortality and NCRAS data in set were excluded due to unknown sex Of the remainder and had at least year of follow up between January and December and were included in the study population Using the gold standard algorithm incident cases of cancer were identified The number of patients identified with each cancer is presented in online supplementary appendix table Half n82 of these patients were male aged to aged to and aged or olderFigure presents PPVs for each case definition comparing the first recorded cancer in each combination of data sources with the gold standard algorithm When using NCRAS data alone to of cancers were confirmed by the algorithm for out of cancer sites the NCRAS only case definition gave the highest PPV Case definitions using data sources not including NCRAS generally had lower PPVs ranging from to for individual cancer sites For the four most common cancers breast lung colorectal and prostate PPVs were at least for all case definitions Minimal differences in PPVs were observed between age groups years and sexes online supplementary appendix figures “Figure presents sensitivity values for each case definition Sensitivity was generally higher for the case definitions that included NCRAS data ranging from to for individual cancer sites except bladder cancer identified using NCRAS data alone and ‰¥ for the four most common cancers breast lung colorectal and prostate Sensitivity was also generally high for definitions using a combination of CPRD GOLD HES APC and ONS mortality data ranging from to ‰¥ for the four most common cancers Sensitivity was lower for case definitions that used CPRD GOLD alone range to for individual cancer sites or HES APC alone range to Sensitivity values for CPRD GOLD alone and HES APC alone increased slightly in younger patients and more recent years no differences were observed between men and women online supplementary appendix figures “ Post hoc analysis suggested that the low sensitivity of CPRD GOLD only definitions for kidney cancer sensitivity n false negatives was driven by missing n1136 or incompletely specified urinary an cancer codes n1108 in CPRD GOLD rather than contradictory information about the first cancer record n625 These incompletely specified codes are less likely to be used for bladder cancers n85 than kidney cancers n1108 Bladder cancers that were not recorded in NCRAS data n3445 were commonly recorded in both HES APC and CPRD GOLD n2228 or in HES APC only with a subsequent unspecified or bladder cancer record in HES APC within months n995 Table describes the number of days median IQR and 5th95th percentile lag between the date of incident cancers from the gold standard definition and the date of cancer arising from each case definition ie the first record within the specific combinations of data sources used Case definitions using NCRAS alone and combinations of ‰¥ data sources captured cancers close to the gold standard date median lag ‰¤ days for all cancer sites whereas median lags were generally longer for the case definitions using CPRD GOLD alone and HES APC aloneFigure describes mortality over time following incident cancer diagnoses ascertained from each case definition Minimal differences in mortality were observed between cancers identified from different case definitions Where variability was observed cancers identified using CPRD GOLD only had the lowest mortality rates eg kidney cancer and cancers identified using HES APC only or NCRAS only had higher mortality rates eg prostate cancer and bladder cancer respectivelyFigure describes completeness of grade and stage for cancers identified using NCRAS only Recording of grade was highly variable between cancers with gradual increases in completeness over time Completeness of staging information was low in earlier calendar years but improved substantially from around especially for the four most common cancers minimum in and in Post hoc logistic regression models adjusted for year and cancer site indicated that completeness of stage and grade were associated with each other and these variables were least complete in patients aged stage data was more complete for higher grade tumours whereas grade data was more complete for lower stage tumours online supplementary appendix figure Online supplementary appendix figure describes recording of treatment modalities identified using NCRAS Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c accessFigure Positive Predictive Value of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm Percentage of incident cancers defined using the first ever record in each combination of sources confirmed by a gold standard algorithm that considers confirmatory and contradictory data from each source Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites CNS central nervous system NHL Non Hodgkin's lymphoma CPRD Clinical Practice Research Datalink HES APC Hospital Episode Statistics Admitted Patient Care data NCRAS National Cancer Registration and Analysis Service ONS Office of National StatisticsStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access Figure Sensitivity of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm Percentage of incident cancers identified using the main gold standard algorithm that considers confirmatory and contradictory data from each source that are identified using the first ever record in each combination of sources Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites CNS central nervous system NHL Non Hodgkin's lymphoma CPRD Clinical Practice Research Datalink HES APC Hospital Episode Statistics Admitted Patient Care data NCRAS National Cancer Registration and Analysis Service ONSOffice of National StatisticsStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0celitnecrep ht“htRQI inadeMelitnecrepRQI inadeM ht“htelitnecrep ht“ht inadeMRQIelitnecrep ht“htCPASEH DLOGDRPC ytil atromSNOdna CPASEH DLOGDRPC CPASEHdnaSARCN secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emTi l ebaT access recnaC lanoitaN SARCN atad eraC tneitaPdetti mdA scitsitatS edospEi latipsoH CPASEH knil ataDhcraeseR ecitcarP lacniilC DRPC metsys suovren lartnec SNCl tluafed ybnoitinfieddradnats dog eht sa emas eht si etad ssongad sa nwohs tonnoitinfied secruos ll iiA setis recnac nommoc tsom ruoFDCiI nosrev sesaesDi fonoitacfissaCliscitsitatS lanoitaN rof ecfifO SNOi atadnoitartsgerrecnac ecvreS ssyanAdna liinoitartsgeRi lanoitanretnI eht imorf sedoc gndnopserroc ot gndrocca deredro era setis recnaCi snoitinfiedde ilii ppa dna etad ssongaddradnats dog nam neewteb syadil fo rebmuN ot ot ““ ot ot ot Cl amoeym epitluMl ot Ci ameakueL““““““““““““ ot ot ot ot ot ot ot ot ot ot ot ot “ot “““““““ ot ot ot ot ot ot ot ““““““““““““““““““““ ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ““““““““““““““ˆ’““““““ ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ““““““““““““““““““ inadeMRQI ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep ht“ht““““““““““““““SARCN inadeMRQI ot ot ot ot ot ot ot ot ot ot ot ot ot ot “ ot ot ot ot “““““C ytivac larOC laegahposeOC hcamotS CC latcerooClrecnaC amonaeml tnangilaMC saercnaPC gnuLC suretUC etatsorPC seiravOC yendKiC tsaerBCi xvreCCC SNCnarBiC reddaBlC amohpmyl s'inkgdo HnoNC idoryhTC revLiStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access Figure Mortality following first ever record of cancer in each combination of sources Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites CNS central nervous system CPRD Clinical Practice Research Datalink HES APC Hospital Episode Statistics Admitted Patient Care data NCRAS National Cancer Registration and Analysis Service NHL Non Hodgkin's lymphoma ONS Office of National StatisticsStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c accessFigure Completeness of grade and stage for cancers identified using NCRAS data only Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites Grading information is not applicable to brainCNS sarcoma or haematological cancers and not required by in the national data standard COSD for prostate cancer Core staging is not applicable to haematological and gynaecological cancers Other types of staging are recommended by COSD CNS central nervous system COSD Cancer Outcomes and Services Data set NCRAS National Cancer Registration and Analysis Service NHL Non Hodgkin's lymphomaStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access only Missing records may indicate that the patient did not receive that treatment modality or that the treatment modality was not recordedDISCUSSIONStatement of principal findingsWe investigated the use of different sources of electronic health record data to identify incident cancers For all case definitions using individual or combined data sources a minimum of of incident site specific cancers were confirmed using the gold standard algorithm this rose to of the four most common cancers Use of cancer registration data alone or in any combination of data sources captured at least of site specific cancers identified by the gold standard algorithm excepting bladder cancer and of cases for the four most common cancers Combining CPRD GOLD HES APC and ONS mortality data captured at least of site specific cancers excepting kidney oral cavity and ovarian cancers and captured of cases for the four most common cancers Sensitivity was much more variable when using primary care or hospital data alone and dropped to when identifying bladder cancers using cancer registration data alone Use of primary care or hospital data alone resulted in a small lag in identifying cancers of interest compared with the gold standard dates but other case definitions captured cancers close to the gold standard date Finally while we observed minimal changes in PPVs and sensitivities between and completeness of NCRAS cancer registration stage and grade data increased markedly from onwards for specific cancer types demonstrating that initiatives to improve data can have a profound impact on the quality of the data4 Completeness of cancer treatment recording was difficult to assess due to the absence of a missing categoryStrengths and weaknesses of the studyThe main strength of this study is that we have developed a gold standard algorithm using the entirety of the evidence available from CPRD to demonstrate the impact of choice of data sets in identifying incident cancers for real life studies We have also assessed the value of using NCRAS cancer registration data to measure stage grade and cancer treatment modalitiesA limitation of the study is that our gold standard algorithm is not validated We feel that we were justified in pre weighting NCRAS data as more reliable that other data sources as NCRAS is a highly validated data set that matches merges and quality checks data from multiple sources4 We did not consider NCRAS to be the outright gold standard as it is plausible that NCRAS does not identify all tumours diagnosed and treated in primary and secondary care For most cancer sites our gold standard algorithm identified a small proportion of cancers that are recorded in HES APC CPRD GOLD or ONS mortality data but not in NCRAS These tumours may have been diagnosed and coded as invasive in primary or secondary care but not by NCRAS been incorrectly coded in HES APC CPRD GOLD or ONS mortality data not have been notified to NCRAS eg tumours treated in private hospitals or be the result of linkage errors between the data sets The proportion of cancers identified in HES APC but not in NCRAS is particularly high for bladder cancer This is likely to be the result of difficulties inconsistencies and changes in the pathological definition and coding of cancers over time in NCRAS which are greatest for bladder cancer4 This explanation is supported by the higher mortality rates that we observed in bladder cancer cases identified in NCRAS compared with other data sources To identify incident cancers we required months of research quality follow up in CPRD GOLD prior to inclusion in the study Previous research has demonstrated that historic data is generally incorporated within the patient record with this time frame15 The identification of first ever cancers will also have been affected by different lengths of follow up data available in linked data sources as NCRAS data collection started in HES APC in and ONS mortality data in and by the inclusion of all diagnostic codes in HES APC assuming that the first ever primary or secondary record identified incident cancer Reassuringly PPVs for liver and brain cancer were high for all individual and combinations of data sets suggesting that these were not unduly misclassified as primary incident cancers despite being common sites for metastases Requiring internal confirmation within months for cancers recorded in CPRD GOLD alone in our GOLD standard definition is more likely to discount cancers with poorer prognoses and those recorded in the last months of follow up Our data cut only included NCRAS data for the top cancers earlier cancers at other sites will have been missed in this studyIt is also important to note that as the gold standard algorithm uses data recorded after the first record of the cancer site in any source index date it cannot be used to identify outcomes in applied studies and follow up of cohort studies with cancer as an exposure would need to start at least months after diagnosis our first ever cancer record in any source definition would be more appropriate for most studiesStrengths and weaknesses in relation to other studies discussing important differences in resultsThe most up to date study describing concordance between linked CPRD GOLD HES APC and NCRAS data sets demonstrated that to of the five most common cancers recorded in CPRD GOLD are not confirmed in either HES APC or cancer registration data and to of registered cancers are not recorded in CPRD GOLD8 For cancers recorded in both sources the diagnosis date was a median of to days later in CPRD GOLD than in the cancer registration data Using CPRD GOLD alone to identify these Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0ccancers marginally over represented younger healthier patients and identified to fewer deaths in the first years after diagnosis Use of HES APC only identified a higher proportion of patients with the correct diagnosis date than CPRD GOLD but over represented older patients and those diagnosed through the emergency route The majority of registered cancers were picked up using both CPRD GOLD and HES APC ranging from for lung cancer to for breast cancer Previous research demonstrated similar results with substantial differences between cancer types5 Additionally a study using data from to found that using HES data in addition to NCRAS data identified an additional and of surgically treated colorectal lung and breast cancer cases respectively16Our study is consistent with these results and provides more complete and practical evidence of the strengths and limitations of using individual and combinations of linked data sets to identify and characterise the most common incident cancersWe have also demonstrated the added value of using cancer registration data to measure stage and grade of incident cancers from about onwards Levels of data completeness of staging information in the CPRD extract in were similar to those reported by the United Kingdom and Ireland Association of Cancer Registries UKAICR9Meaning of the study possible explanations and implications for clinicians and policymakersUse of NCRAS cancer registration data maximised the proportion of cases confirmed as true positive based on all available linked information and captured the highest proportion of true positive cases highly complete staging and grading information is available from this source from approximately Case definitions based on a combination of CPRD GOLD HES APC and ONS mortality data also had acceptable validity for the majority of cancer sites including the four most common cancersThese findings

Thyroid_Cancer

"fundamental influences of sex and gender as modifiers of the major causes of death and morbidity We articulate how the genetic epigenetic and hormonal influences of biological sex influence physiology and disease and how the social constructs of gender affect the behaviour of the community clinicians and patients in the healthcare system and interact with pathobiology We aim to guide clinicians and researchers to consider sex and gender in their approach to diagnosis prevention and treatment of diseases as a necessary and fundamental step towards precision medicine which will benefit men™s and women™s healthIntroductionWhat clinicians know about the diagnosis treatment and prevention of disease originates from studies mostly done on male cells male mice and men1 Historically for multiple reasons including the purported safety of women and their offspring women of childbearing age were excluded from clinical trials As a result medical research and care have been centred on male physiology The assumption was that male and female cells and animals were biologically identical and evidencebased medicine was defined by clinical trials done predominantly in men1 In the US National Institutes of Health NIH mandated the inclusion of women in NIHfunded clinical trials but many investigators did not follow this mandate and many of those who did include women did not analyse the results by sex23 minimising the effectiveness of this policy Preclinical research and drug development studies have also predominantly used male animal models and cells4“ It is not surprising that a US Government Accountability Office report found that eight of the ten prescription drugs withdrawn from the market between and œposed greater health risks for women than for men Most funding agencies from Europe and North America have implemented policies to support and mandate researchers to consider sex and gender at all levels of medical research8 Still the field of sexbased biology and medicine is often viewed as a specialised area of interest rather than a central consideration in medical research Essential for the success of clinical care and translational science is awareness by clinicians and researchers that the diseases they are treating and studying are characterised by differences between women and men in epidemiology pathophysiology clinical manifestations psychological effects disease progression and response to treatmentThis Review explores the role of sex biological constructs and gender social constructs as modifiers of the most common causes of death and morbidity and articulates the genetic biological and environmental determinants that underlie these differences We aim to guide clinicians and researchers to better understand and harness the importance of sex and gender as genetic wwwthelancetcom Vol August biological and environmental modifiers of chronic disease Ultimately it is a necessary and fundamental step towards precision medicine that will benefit women and menSex as a genetic modifier of biology and diseaseSex differences in disease prevalence manifestation and response to treatment are rooted in the genetic differences between men and women Genetic sex differences start at conception when the ovum fuses with a sperm cell carrying an X or a Y chromosome resulting in an embryo carrying either XX or XY chromosomes This fundamental difference in chromosome complement eg genes outside the testisdetermining SRY gene generates ubiquitous sex differences in the molecular makeup of all male and female cells9 First the Y chromosome carries genes that exhibit subtle functional differences from their Xlinked homologues eg ZFY vs ZFX and UTY vs UTX and also carries genes with no homologue at all eg SRY In addition in men the X chromosome carries only maternal imprints ”ie epigenetic modifications made by the parent in generating the sex cells”which alter the expression of genes in the offspring As women have X chromosomes from both parents they carry maternal and paternal imprints which target a different set of genes Random inactivation of one of the X chromosomes in female cells which prevents sex differences in X chromosome gene dosage causes another degree of sex difference in gene expression As some of these Xlinked genes escape inactivation in women those genes are often expressed at higher levels in women than in men9 Sexspecific gene expression due to genomic Search strategy and selection criteriaWe searched PubMed for papers published in English between Jan and June using œsex or œgender and the name of the disease of interest as search terms Although we tried to cite seminal studies when necessary because of space limitation representative reviews were often selectedLancet “Diabetes Discovery SexBased Medicine Laboratory Section of Endocrinology John W Deming Department of Medicine Tulane University School of Medicine and Southeast Louisiana Veterans Health Care System Medical Center New Orleans LA USA Prof F MauvaisJarvis MD Barbra Streisand Women™s Heart Center CedarsSinai Smidt Heart Institute Los Angeles CA USA Prof N Bairey Merz MD National Heart Lung Institute Imperial College London London UK Prof P J Barnes MD Department of Pharmacology and Department of Neurology College of Medicine Center for Innovation in Brain Science University of Arizona Tucson AZ USA Prof R D Brinton PhD Department of Medical Epidemiology and Biostatistics and Center for Gender Medicine Karolinska Institutet Stockholm Sweden Prof JJ Carrero PhD Channing Division of Network Medicine and the Division of Pulmonary and Critical Care Medicine Department of Medicine Brigham and Women™s Hospital Harvard Medical School Boston MA USA D L DeMeo MD Neuroscience Institute and Department of Biology Geia State University Atlanta GA USA Prof G J DeVries PhD Department of Psychiatry University of Colorado School of Medicine Anschutz Medical Campus Aurora CO USA Prof C N Epperson MD Division of Oncology Department of Medicine Washington University School of Medicine St Louis MO USA Prof R Govindan MD W Harry Feinstone Department of Molecular Microbiology and Immunology The Johns Hopkins Bloomberg School of Public Health Baltimore MD USA Prof S L Klein PhD Department of Biomedical Metabolic and Neural Sciences University of Modena andReview 0cReggio Emilia Azienda OspedalieroUniversitaria di Modena Ospedale Civile di Baggiovara Modena Italy Prof A Lonardo MD Department of Psychiatry Department of Psychology and Department of Obstetrics Gynecology University of Illinois at Chicago Chicago IL USA Prof P M Maki PhD Department of Neurology McGovern Medical School University of Texas Health Science Center Houston TX USA Prof L D McCullough MD Berlin Institute of Gender Medicine Charit”Universittsmedizin Berlin Berlin Germany Prof V RegitzZagrosek MD Department of Cardiology University Hospital Z¼rich University of Z¼rich Switzerland Prof V RegitzZagrosek Center for Women™s Health Research Divisions of General Internal Medicine and Cardiology University of Colorado School of Medicine Aurora CO USAimprinting extends to autosomes as well and these imprinted genes exhibit sexspecific and tissuespecific expression in humans10 Thus fundamental sex differences deriving directly from genetic heterogeneity between the X and Y chromosome complements and parentoforigin inher itance exist at the molecular level in all human cells These sex differences persist throughout life and are independent of sex hormones figure Arguably the greatest source of differences between men and women comes from the Y chromosomal SRY gene which directs the development of a testis in men The ensuing developmental surge of testicular testosterone permanently masculinises the reproductive tract and the anisation of brain circuits affecting male behaviour at puberty1112 In humans the first surge occurs at the end of the first trimester of pregnancy Because it alters cellular gene expression and tissue structure in multiple ans of men via epigenetic mechanisms this testosterone surge is also paramount in programming sex differences in physiology and susceptibility to diseases that will manifest in adulthood After this initial testicular testosterone surge gonadal hormone concentrations remain low until puberty which triggers lasting sex differences in circulating oestrogens and testosterone concentrations After puberty cells with androgen or AFemale sexBRandom X chromosomeinactivation and escapeXXXXXXXXXXXXXXXXXXXY chromosome complementMale sexSRYCTesticular testosterone surgeFetal testisTestosteroneOHOGenetic diï¬erences of male and female cellsEpigenetic programming of male cellsFigure Genetic causes of sex differencesA Genetic sex differences start with cells carrying either XX or XY chromosome complement eg genes outside the testisdetermining SRY gene which generates ubiquitous sex differences in the molecular makeup of all male and female cells B Random inactivation of one X chromosome in female cells causes another level of sex differences in gene expression Some Xlinked genes escape inactivation in female individuals and have a higher expression in female than male individuals C The Y chromosomal SRY gene directs the development of a testis in male individuals which produces a surge of testicular testosterone at the end of pregnancy The testosterone surge programmes cellular gene expression and tissue structure in multiple ans of male individuals via epigenetic remodelling The combination of these genetic and developmental events programmes sex differences in physiology and susceptibility to diseases that will manifest in adulthoodoestrogen receptors will be affected differ ently in men and women The combination of all genetic and hormonal causes of sex differences aforementioned culminates in two different biological systems in men and women that translate into differences in disease predisposition manifestation and response to treatment Therefore sex is an important modifier of physiology and disease via genetic epigenetic and hormonal regulations figure Gender as a determinant of patients™ and doctors™ behaviour and as a modifier of health disease and medicineGender according to the Global Health definition refers to the socially constructed norms that impose and determine roles relationships and positional power for all people across their lifetime13 Gender interacts with sex the biological and physical characteristics that define women men and those with intersex identities13 Gender is not a binary term It includes the understanding that in many people traits of masculinity or femininity coexist and are expressed to different degrees Gender attributes are fluid more than two thirds of women and men report genderrelated characteristics traditionally attributed to the opposite sex1415 In transgender people gender identity differs with the sex they were assigned at birth So far transgender people have generally been underrepresented in clinical studies to date although this underrepresentation is changing Gender is an equally important variable as biological sex in human health and influences the behaviour of communities clinicians and patients1415 Gender roles represent the behavioural norms applied to men and women in society which influence individuals™ everyday actions expectations and experiences including diet perceived stress smoking and physical activity and affect health and disease susceptibility Gender identity describes the fluidity of how a person perceives oneself as a woman or a man which affects feelings and behaviours Gender relations refer to how we interact with or are treated by people on the basis of our ascribed gender Institutionalised gender reflects the distribution of power between men and women in the political educational and social institutions in society and shapes social norms that define perpetuate and often justify different expectations and opportunities for women and men1617 As such the distribution of genderrelated charac teristics within populations of men and women can influence health differently than biological sex Together these gender constructs determine access to health care helpseeking behaviours and individual use of the healthcare system Being perceived as a man or a woman triggers different responses from clinicians who might diagnose and suggest interventions differently according to gender As such gender largely determines the use of preventive measures and referral for or acceptance of invasive therapeutic strategies Genderrelated behaviours contribute to risk exposure and preventive behaviour in several wwwthelancetcom Vol August Review 0cdiseases This postulation is well exemplified in the cardiovascular field in which women often underestimate their risk compared with men and seek consultation later than men in the clinic for treatment of myocardial infarction1819 In the GENESISPRAXY prospective study mortality year after an acute coronary event was more strongly associated with gender than with biological sex1415 Similarly control of cardio vascular risk factors hypertension diabetes depres sive symptoms was better predicted by gender than by biological sex1415 Therefore including a gender dimension in clinical studies and practice will contribute to the understanding of different clinical manifestations and outcomes of diseases in women and men1617 Although beyond the scope of this Review it is also important to consider that regarding health and disease gender intersect with race or ethnicity and age20“ Sex and gender are fundamentally and frequently reciprocally interrelated in biology and disease24 Sex influences behaviours eg towards more aggressive or caring phenotypes On the other hand genderrelated behaviours eg smoking lifestyle perceived stress and pain and nutritional habits might produce epigenetic modifications that modulate gene expression and biological phenotypes Figure summarises how sex and gender are interrelated in biology and diseaseSex and gender differences in major chronic diseasesHaving established the importance of sex and gender in disease we will summarise their influences on the most common causes of death and debilitating diseases in the USA as an example figure Note that these sex and gender disparities are relevant to other highincome countries as well as lowincome and middleincome countries where the burden of these diseases becomes increasingly like those in highincome countries26 In most diseases efforts to separate the effects of sex and gender are still incomplete so that we just refer to the differences among women and men Because current knowledge about pathophysiology diagnosis and treatment of disease is primarily based on men as representative of the human species this Review focuses on how women differ from men We discuss some key aspects regarding the dimensions of men in a dedicated sectionHeart diseaseEpidemiology pathogenesis manifestations and diagnosisHeart disease is the leading cause of death in the USA In heart disease accounted for · of all deaths for men and for · of all deaths for women figure Ischaemic heart disease and heart failure are major contributors to heart disease mortality and have important sex and gender differences For example heart failure disproportionately contributes to coronary heart disease mortality in women27 potentially due to undiagnosed ischaemic heart disease in women The strength of the association with cardiovascular risk factors differ by sex Biological sexSex chromosomesEpigeneticeï¬ectsSex hormonesOHOHOHOBehaviour of patients and doctorsSocietyGender constructsLifestyleNutritional habitsExercisePerceived stressSmokingDiseasePathophysiologyManifestationResponse to treatmentDisease perceptionHelpseeking behaviourUse of health careDecision makingTherapeutic responseSex and gender diï¬erences in health disease and medicineFigure Interrelation between sex and gender in health diseases and medicineBiological sex causes sex differences through genetic and hormonal influences in disease pathophysiology clinical manifestations and response to treatment Sex also influences behaviours towards more aggressive or caring phenotypes On the other hand genderrelated behaviours eg smoking lifestyle perceived stress and nutritional habits produce epigenetic modifications that modulate the expression of biological sex Gender constructs determine patients™ perception of disease helpseeking behaviour and individual use of health care Gender constructs also influence decision making and trigger different therapeutic responses from providers biased by genderSystolic blood pressure and hypertension smoking and diabetes are associated with higher hazard ratios for myocardial infarction in women than in men28Ischaemic heart disease is the most recognised example for integrating the concept of gender and sex which shape divergent or distinct disease outcomes Compared with men women suffering from ischaemic heart disease are older this difference is historically believed to be due to the protection of endogenous oestrogens29 although contemporary study refutes this simplistic explanation30 and associations cannot be inferred to be causation Still women suffering from ischaemic heart disease are underdiagnosed3132 and less likely to have a prehospital diagnosis of myocardial infarction33“ The reasons for this disparity reflect the intersection between sex and gender First biological sex differences exist in the pathogenesis of ischaemic heart disease Whereas men are more likely to be affected by obstructive coronary artery disease of large vessels than women coronary microvascular dysfunction36 leading to chronic myocardial ischaemia without obstructive coronary artery disease has a higher prevalence in women than men37 A metaanalysis reported that following acute myocardial infarction both sexes most often presented with chest pain but compared with men women were more likely to present with pain between the shoulder blades nausea or vomiting and shortness of breathProf J G Regensteiner PhD Department of Medicine Department of Paediatrics and Department of Neuroscience Washington University School of Medicine St Louis MO USA Prof J B Rubin MD Center for the Study of Sex Differences in Health Aging and Disease Geetown University Washington DC USA Prof K Sandberg PhD Division of Gastroenterology Duke University Medical Center Durham NC USA A Suzuki MD and Durham VA Medical Center Durham NC USA A SuzukiCorrespondence to Prof Franck MauvaisJarvis Diabetes Discovery SexBased Medicine Laboratory Section of Endocrinology John W Deming Department of Medicine Tulane University School of Medicine New Orleans LA USA fmauvaistulaneeduwwwthelancetcom Vol August Review 0cMale individualsOther·Heart disease·protection might disappear after menopause45 By contrast testosterone induces adverse cardiac remod elling in the male heart44Chronic liver disease ·Influenza and pneumonia ·Suicide ·Alzheimer™s disease ·Type diabetes ·Stroke ·CPD ·Injuries ·Female individualsOther·Septicaemia ·Chronic kidney disease ·Influenza and pneumonia ·Type diabetes ·Injuries ·Alzheimer™s disease ·Stroke ·CPD ·Cancer·Heart disease·Cancer·Figure Percent distribution of the ten leading causes of death by sex USA Adapted from Heron25 CPDchronic pulmonary diseaseSecond a gender bias appears to be responsible for the absence of recognition of ischaemic heart disease presentation in women38 Men and women with ischaemic heart disease who score high on feminine roles and personality traits on questionnaires designed to ascertain aspects of gender are at an increased risk of recurrent ischaemic heart disease independent of female sex39Heart failure affects of adults aged years and older and more women than men in absolute numbers4041 Heart failure occurs at an older age and with less ischaemic causes in women than in men However hypertension and diabetes predispose older women to heart failure to a greater extent than men Heart failure with preserved ejection fraction a form of heart failure with normal systolic function is twice as prevalent in women as in men By contrast heart failure with reduced ejection fraction affects more men than women Women who have heart failure with preserved ejection fraction have smaller and stiffer hearts than men Inflammation and the resulting fibrosis play a sexspecific role in the pathogenesis of heart failure with preserved ejection fraction Under stress premenopausal women™s hearts develop less inflammation resulting in less fibrosis than men™s hearts4243 This difference is partially driven by sex hormones as oestrogens produce antiinflammatory actions on endothelial and immune cells and promote cardioprotective effects in premenopausal women44 This Response to treatmentCompared with men women suffering from ischaemic heart disease are less likely to receive evidencebased treatment3132 and when suffering from acute myocardial infarction they are less likely to receive reperfusion33“ An STelevation myocardial infarction registry revealed that compared with men women exhibit delayed reperfusion leading to higher mortality46 Women suffering from acute myocardial infarction treated by male emergency physicians have a higher mortality rate than those treated by female physicians38 Additionally male physicians are more effective at treating female patients with acute myocardial infarction when they work with female colleagues and when they have experience in treating female patients38 This treatment disparity between women and men can be corrected by improving emergency recognition of STelevation myocardial infarction in women and acceleration of percutaneous coronary intervention which equilibrates gender mortality47Guidelines for the treatment of heart failure are similar for women and men24 However evidence suggests that optimal survival in women occurs with lower doses of β blockers angiotensin receptor blockers and angiotensin converting enzyme inhibitors than in men48 Finally fewer women undergo heart transplantation than men although women are more frequently donors suggesting a referral bias could exist41CancersEpidemiology pathogenesis manifestations and diagnosisCancers are the second leading cause of death dominated by lung cancer accounting for · of deaths in men and · of deaths in women figure More men develop cancer than women49 With few exceptions eg meningioma thyroid cancer lung cancer in nonsmokers nonreproductive cancers exhibit a male predominance though for some cancers oropharynx larynx oesophagus and bladder the male versus female incidence ratios can be higher than A male predominance in cancers that affect both sexes is evident around the world in all races and at all ages50 Survival is also shorter for men than women across multiple cancer types The higher cancer risk in men is partially explained by gender constructs like dietary habits or risk behaviours such as smoking and alcohol consump tion51 It is unlikely to be the only cause After appropriate adjustment for these risk factors adult men still have a higher cancer risk than women52 Moreover a similar male bias in incidence and survival is seen in paediatric cancers before puberty and the adoption of highrisk cancerpromoting behaviours eg smoking53The universal male predominance in cancer incidence and differential outcomes argues for a fundamental role wwwthelancetcom Vol August Review 0cin inutero tumour suppressors of sex in addition to gender in cancer biology Sexspecific biology includes genetic differences XX vs XY chromosomes the incomplete Xinactivation in female individuals which results in biallelic expression of Xencoded female cells54 Y chromosomeencoded oncogenes such as the RNAbinding motif on Y chromosome in male cells55 and the chromatin remodelling effects of testicular testosterone in male cells56 These mechanisms have an influence on several of the hallmarks of cancer57 including metabolism growth regulation58 angiogenesis and immunity which all contribute to cancer predisposition59 A crucial example is the male predisposition to glioblastoma which is the most common form of brain cancer In glioblastoma there is a cellintrinsic predisposition of male astrocytes a subtype of glial cell to malignant transformation60 After puberty sex hormones produce additional epigenetic and acute effects on cells that further influence sex disparities in cancer For example the increased frequency and aggressive phenotype of hepatocellular carcinoma in male individuals has been linked to the stimulatory effects of androgens in male individuals and the protective effects of oestrogens in female individuals61 Importantly the biology of cancer is not the same across histological and genetic diagnostic groups or even within single histol ogical subtypes Thus the interaction between sex gender and cancer mechanisms cannot be expected to be constant Take colon cancer the second leading cause of cancerrelated death for example Although women have a lower overall incidence of colon cancer than men they have a higher incidence of rightsided colon cancers which have the worst outcomes62 Tumours from women with rightsided colon cancers exhibit a distinct molecular signature of energy metabolism compared with those of women with leftsided colon cancers63 This molecular signature is not observed when comparing tumours from men with rightsided colon cancers to men with leftsided colon cancers Thus overall the male predisposition to cancer is probably the consequence of genetic program ming of male cells and the effect of sex hormones after puberty interacting with genderspecific behaviours to establish cancer risksResponse to treatmentIn the future cancer prevention and treatment will be improved by sexspecific and genderspecific approaches For example immune checkpoint inhibitors can improve survival for men with advanced melanomas and nonsmallcell lung cancers more than for women64 The molecular subtyping of glioblastomas based on sexspecific transcriptomes has the potential to enhance chemotherapy in a sexspecific manner65 In colon cancer sex differences in xenobiotic metabolism regulatory networks might underlie greater treatment response in women than men and require modification of approaches for men with colon cancer66 Other elements of cancer metabolism are also ripe for novel sexspecific targeting in treatment Cellular nutrient partitioning is sexually dimorphic so approaches such as ketogenic diets or glutaminase inhibition might be associated with substantial sexspecific responses67 Furthermore data from models of development ageing and cancer all indicate that molecular pathways such as the enzyme phosphatidylinositol 3kinase and tumour suppressors such as p53 and retinoblastoma protein are sexually dimorphic and require sexspecific targeting545968Chronic pulmonary diseaseEpidemiology pathogenesis manifestations and diagnosisChronic pulmonary disease is the third leading cause of death for women · of deaths and the fourth for men · figure It is mostly accounted for by chronic obstructive pulmonary disease and to a lesser extent by asthma Chronic obstructive pulmonary disease is characterised by irreversible airflow limitation and is associated with previous exposure to smoking or air pollutants Women are overrepresented among individuals with chronic obstructive pulmonary disease especially among those with earlyonset disease or those who have never smoked69 Women are also twice as likely to have chronic obstructive pulmonary disease with chronic bronchitis and women with severe chronic obstructive pulmonary disease have as much emphysema as men countering the misconception of emphysema as a male form of chronic obstructive pulmonary disease70 The female lung is more susceptible to chronic obstructive pulmonary disease than the male lung and women develop symptoms of the disease at a younger age with less tobacco exposure than men71 The Genetic Epidemiology of Chronic Obstructive Pulmonary Disease COPDGene study72 suggests that earlyonset chronic obstructive pulmonary disease might originate in utero in susceptible women from alterations in lung development potentiated by maternal asthma and smoking genetic factors or hormonal influences Future studies should focus on the contribution of maternally inherited factors such as mitochondrial and X chromosome genes to understand disease pathogenesis It is important to consider gender constructs as well Smoking advertising campaigns targeting women rose in the 1960s and the resulting higher smoking rates influenced women™s risk for developing chronic obstructive pulmonary disease73 From a disease severity vantage point chronic obstructive pulmonary disease exacerbation rates are also higher in women than men especially at a younger age74 Additionally despite the burden of symptomatology and increased rates of hospitalisations and deaths women with chronic obstructive pulmonary disease are often misdiagnosed and disproportionally suffer from comorbid conditions including anxiety and depression Therefore physicians should consider chronic obstructive pulmonary disease in the differential diagnosis of women with pulmonary symptoms regardless of tobacco or pollutant exposure historieswwwthelancetcom Vol August Review 0cAsthma characterised by variable airflow obstruction and chronic airway inflammation also affects men and women differently Asthma is more prevalent in prepubertal boys than girls Regarding asthma both male biological sex lung development and atopy and male gender constructs related to outdoor play and indoor pet exposure are factors contributing to the devel opment of asthma and sex versus gender contributions could be difficult to separate75 F

Thyroid_Cancer

Lenvatinib inhibits tyrosine kinases including vascular endothelial growth factor VEGF receptor fibroblast growth factor receptor platelet derived growth factor receptor alpha RET proto oncogene and KIT proto oncogene receptor tyrosine kinase We assessed the efficacy and safety of lenvatinib in patients with metastatic colorectal cancer after failure of standard chemotherapiesPatients and methods This was an label single centre single arm phase study Eligible patients had unresectable metastatic colorectal adenocarcinoma refractory or intolerant to fluoropyrimidine irinotecan oxaliplatin trifluridinetipiracil anti VEGF therapy and anti epidermal growth factor receptor therapy for tumours with wild type RAS Patients were treated with oral lenvatinib at mg one time a day in day cycles until disease progression or unacceptable toxicity The primary endpoint was centrally assessed disease control rate Secondary endpoints included safety response rate progression free survival and overall survival The planned sample size was patients to expect a disease control rate of with a threshold disease control rate of one sided alpha of and power of Results Between October and January patients were enrolled and had received or ‰¥ lines of prior chemotherapy for metastatic disease respectively The median number of lenvatinib cycles was range “ The centrally assessed disease control rate was CI to one sided p00001 patients had a partial response and had a stable disease Median progression free survival was months CI to Median overall survival was months CI to The most common grade ‰¥ adverse events were hypertension thrombocyt ia increased alanine aminotransferase and anorexia eachConclusions Lenvatinib showed promising clinical activity and was tolerated in patients with metastatic colorectal cancer after failure of standard chemotherapiesTrial registration number UMIN CTR UMIN000023446 and JAMCCT CTR JMA IIA00261InTRoduCTIonThe combination of cytotoxic chemotherapy with a molecular targeted agent has significantly Key questionsWhat is already known about this subject –º No studies have previously reported the efficacy and safety of lenvatinib monotherapy in patients with metastatic colorectal cancer refractory to standard chemotherapiesWhat does this study add –º Lenvatinib showed promising antitumour activity with acceptable toxicity for heavily pretreated patients with metastatic colorectal cancer refractory to standard chemotherapies –º No unexpected safety signals were observed and toxicities were manageable with dose modification interruptions and supportive medicationsHow might this impact on clinical practice –º Further prospective randomised studies are warranted to evaluate the efficacy of lenvatinib in patients with metastatic colorectal cancer refractory to standard chemotherapiesimproved the survival of patients with unresectable metastatic colorectal cancer1“ From results of recent clinical trials trifluridinetipiracil and regorafenib are recognised as new treatment options for patients with metastatic colorectal cancer refractory or intolerant to standard therapies6 Nevertheless the prognosis of patients which are refractory or intolerant to standard chemotherapies is poor and there are still an unmet medical needs for these patients especially for those who are in a good performance status and eligible for further therapiesLenvatinib is an oral multitargeted tyrosine kinase inhibitor of the vascular endothelial growth factor receptor VEGFR “ fibroblast growth factor receptors “ platelet derived growth factor receptor alpha RET and KIT8 Preclinical studies have shown that Iwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776 0c accesslenvatinib not only interferes the interaction between cancer cells and endothelial cells but also inhibits tumour growth10 Several phase trials of patients with solid tumours in the USA11 Europe12 and Japan13 showed that the optimum dosage of lenvatinib was mg one time a day in a day cycleA total of patients were enrolled in four phase studies of lenvatinib monotherapy of whom had colorectal cancer Disease control rate DCR was achieved in out of patients including one with a partial response which continued for weeks mg two times a day for weeks of a week cycle Grade palmar plantar erythrodysesthesia was reportedly much lower in of patients treated with lenvatinib for thyroid cancer in a Japanese population of the SELECT trial than that of reported in a Japanese population of CORRECT trial using regorafenib for metastatic colorectal cancer15 These results suggested that lenvatinib may have a potential for improving the outcomes of patients with unresectable metastatic colorectal cancer who have already received conventional chemotherapy with a fluoropyrimidine irinotecan and oxaliplatinWe conducted a single centre phase study to evaluate efficacy and safety in patients with metastatic colorectal cancer failing to standard therapiesPaTIenTs and meTHodsstudy design and patientsThis study was a single arm phase study conducted at National Cancer Center Hospital Tokyo Japan The inclusion criteria were histological diagnosis of colorectal adenocarcinoma excluding carcinoma of the appendix and the anal canal unresectable metastatic disease an Eastern Cooperative Oncology Group performance status of or an age of “ years no previous treatment with regorafenib or lenvatinib sufficient oral intake adequate an and bone marrow function at least one measurable lesion in accordance with the Response Evaluation Criteria in Solid Tumors RECIST version refractory or intolerant to fluoropyrimidine irinotecan oxaliplatin therapy and antiepidermal growth factor receptor therapy for tumours with wild type RAS and no systemic therapy for at least weeks weeks if any investigational drug had been administered before study enrolment The exclusion criteria were provided in the online supplementary materialtrifluridinetipiracil anti VEGF All patients provided written informed consentProceduresPatients received lenvatinib at mg one time a day in day cycles orally until disease progression or unacceptable toxicity The dose was reduced to mg mg mg mg and mg if a patient had an intolerable grade or grade adverse event Treatment was discontinued if a dose interruption was required for more than consecutive daysTumour response was assessed by the independent radiological review committee based on the CT or MRI performed at baseline every weeks for weeks and every weeks thereafter until confirmed objective disease progression Safety assessments including laboratory tests were done at screening days and of cycle and days and of the subsequent cycles Urinalysis thyroid function prothrombin time international normalized ratio PT INR and tumour markers both carcinoembryonic antigen and carbohydrate antigen “ were measured at screening and on day of each treatment cycle Adverse events were recorded from the first day of the protocol treatment to days after the last dose of study medication and graded using the National Cancer Institute Common Terminology Criteria for Adverse Events version Blood sampling for biomarker analyses was done at baseline on days and and at the end of treatment Plasma levels of angiopoietin2 were measured by the Human Angiopoietin2 Quantikine ELISA Kit RD Systems Minneapolis USAoutcomesThe primary endpoint was centrally assessed DCR which was defined as the proportion of patients with a complete response partial response or stable disease persisting for more than weeks from the initiation of study treatment according to RECIST version A complete response and partial response were needed to be confirmedThe secondary endpoints were the objective response rate ORR proportion of patients who had a complete response or partial response progression free survival PFS time from the enrolment until investigator assessed disease progression or death overall survival OS time from the enrolment until death due to any cause and adverse events The incidence of adverse events was calculated based on the information of the worst grade of each adverse event experienced in each patient Relative dose intensity which is unprespecified outcome was calculated as the proportion of the actual cumulative dose divided by planned cumulative dose mg times treatment daysstatistical analysisFor this single arm study the required sample size of patients provided power to reject the null hypothesis of DCR ‰¤ with expectation that of patients would have a disease control one sided α of Considering the possibility of a few ineligible patients we planned to recruit patientsThe final analysis was planned approximately months after enrolment of the last patient We included all eligible patients in the efficacy analysis and all patients receiving a least one dose of lenvatinib in the safety analyses For the primary analysis binomial test was performed and the centrally assessed DCR was estimated with CI using the Clopper and Pearson method which corresponds to one sided α of We also estimated the investigator assessed DCR a supplementary analysis of the primary Iwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776 0cTable Baseline patient characteristicsCharacteristicsTable ContinuedOverall N CharacteristicsOverall N access Median range   Continued Intolerant Wild type Mutant RAS mutational status BRAF mutational status Wild type Mutant UnknownMSI status MSS Unkown There is an overlapping This number includes patients with the RAS wild type and patient with mutant RASECOG Eastern Cooperative Oncology Group EGFR epidermal growth factor receptor MSI Microsatellite instability MSS Microsatellite stableendpoint and ORR with CIs using the same method We estimated the median time and month and year probability of OS and PFS with the Kaplan Meier method The CIs for the median time were calculated using Brookmeyer and Crowley method The CIs of month and year survival probabilities were calculated based on the Greenwood™s formula HRs and CIs were estimated by Cox regression We did subgroup analyses divided by prespecified baseline patient and disease characteristic variables including RAS status for DCR PFS and OS We also did a prespecified exploratory analysis of potential predictive biomarkers in blood samples We did all analyses with SAS V94ResulTsPatient characteristicsBetween October and January patients with unresectable metastatic colorectal cancer were enrolled All patients were eligible and received the study medication Table summarises the baseline characteristics of all enrolled patients The median number of previous lines of palliative chemotherapy was range “ and patients had received or ‰¥ prior lines of chemotherapy for metastatic disease respectively The data cut off date was January with median follow up of months IQR “efficacyThe centrally assessed DCR was CI to one sided p00001 two patients had a partial response and had a stable disease including unconfirmed PR table figure A total of patients had a reduction in target lesion size from baseline figure Time on treatment for all patients is ‰¥ ‰¥ Male Female months ‰¥ months Right sided colon Left sided colorectum Lung Liver Lymph node PeritoneumAge years Sex ECOG performance status Primary site Number of metastatic site Metastatic an Time from start of first line chemotherapy Number of previous palliative chemotherapy Previous chemotherapy and reason for discontinuation Fluoropyrimidine Refractory IntolerantOxaliplatin Irinotecan TAS102 trifluridinetipiracil Angiogenesis inhibitor Anti EGFR inhibitor Refractory Intolerant Refractory Intolerant Refractory Refractory Intolerant Refractory IntolerantIwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776 0c accessTable Best response to treatmentComplete responsePartial responseStable diseaseProgressive diseaseNot evaluableDisease control rate CIResponse rate CICentral assessmentn30 to to Investigator assessmentn30 to to shown in online supplementary figures and Events for PFS were recorded in all patients and median PFS was months CI to figure All deaths were recorded median OS was months CI to with a month and year OS of CI to and CI to figure safetyPatients received the study treatment for four cycles at median range “ The median relative dose intensity was IQR “ Dose interruptions and reductions were required in and patients respectively The major treatment related adverse events ‰¥ for dose reduction were proteinuria patients palmar plantar erythrodysesthesia patients diarrhoea patients hypertension patients fatigue patients and thrombocyt ia patients The reasons for treatment discontinuation of all patients were disease progression in patients and adverse events in patients gastrointestinal perforation and grade proteinuria in of each After treatment with lenvatinib patients received a subsequent treatment online supplementary table Most patients only had mild grades “ adverse events table The most common grade ‰¥ adverse events were hypertension patients thrombocyt ia patients increased alanine aminotransferase and anorexia patients each No clear relationship was found between the incidence of lenvatinib associated adverse event of any grade and baseline body surface area online supplementary table Serious adverse events occurred in four patients including Figure Waterfall plot analysis of maximum percentage change from baseline in measurable target lesions Response Evaluation Criteria in Solid Tumors version central reviewIwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776 0c accessFigure Kaplan Meier curves of A progression free survival PFS by investigator assessment and B overall survival OS in all patients n30five treatment associated events anorexia in two and gastrointestinal perforation central venous catheter related bloodstream infection caused by Staphylococcus aureus and nausea in each one in each of four patients all patients recovered from these adverse eventssubgroup analysisIn patients with wild type RAS the median PFS was months CI to and that was months CI to in patients with mutant RAS online supplementary figure In patients with wild type RAS the median OS was months CI CI to and months CI to in patients with mutant RAS online supplementary figure Plasma angiopoietin2 levels were decreased by lenvatinib treatment in almost all patients and increased at the Iwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776time of treatment discontinuation online supplementary table With a first quartile cut off point17 the eight patients with a first quartile or lower level of angiopoietin2 had a median OS of months CI to months compared with months CI to in the patients with higher than a first quartile level of angiopoietin2 HR CI to online supplementary figure Patients with a first quartile or less level of angiopoietin2 had a median PFS of months CI to compared with months CI to in the patients with more than a first quartile level of angiopoietin2 HR CI to online supplementary figure 0c accessTable Treatment related adverse events occurring in ‰¥ patients N30Any gradeGrade ‰¥Treatment related adverse eventHypertensionProteinuriaThrombocyt iaFatigueHypothyroidismWeight lossHoarsenessPalmar plantar erythrodysesthesia syndromeAnorexiaDiarrhoeaMucositis oralSerum AST increasedSerum creatinine increasedAST Aspartate transaminase dIsCussIonPatients with metastatic colorectal cancer with disease progression after three or more lines of therapy have limited treatment options In this label single arm phase study of patients with previously treated metastatic colorectal cancer lenvatinib demonstrated manageable toxic effects and promising antitumour activity A total of out of patients had disease control including with partial responses Moreover patients experienced reduction in measurable tumour size The overall toxicity profiles were similar to that reported for lenvatinib across a spectrum of advanced malignant neoplasmsTwo recent international phase studies reported that regorafenib or trifluridinetipiracil provided significant improvements in DCR PFS and OS compared with placebo in patients with metastatic colorectal cancer after failure of standard chemotherapies DCR median PFS months median OS months in the CORRECT study and DCR median PFS months median OS months in the RECOURSE study6 Interestingly the present single arm phase study of lenvatinib revealed favourable DCR and median PFS values in patients with metastatic colorectal cancer compared with those in the regorafenib or trifluridinetipiracil study Moreover about half of the patients received post study treatment which led to a favourable OSThe lenvatinib safety profile in this study was similar to the published safety profiles of lenvatinib for thyroid cancer and hepatocellular carcinoma in the Japanese population18 Moreover we found no unexpected or off target safety signals The most common adverse events were hypertension proteinuria thrombocyt ia and fatigue while the most case of grade or hypertension and proteinuria required treatment interruption and dose reduction While the target population for thyroid cancer or hepatocellular carcinoma that showed efficacy for lenvatinib was first line setting20 this study targeted patients receiving salvage line therapy Most patients with metastatic colorectal cancer in the salvage line setting had grade or proteinuria and hypertension at baseline because of the long term prior treatment with anti VEGFVEGFR treatment whereas the occurrence of grade hypertension was significantly higher compared with that of regorafenib in a similar study population in the CORRECT CONCUR and CONSIGN trials7 It was manageable by dose reduction or interruption but it may be necessary to consider the starting dose in the future Although palmar plantar erythrodysesthesia is a not life threatening toxicity these adverse events have a significant impact on treatment schedules and quality of life in treated patients Grade ‰¥ palmar plantar erythrodysesthesia has been observed in and of patients treated with lenvatinib in this study and the SELECT Japanese population15 respectively while in patients treated with regorafenib in the CORRECT Japanese population16 To date the clear mechanism of palmar plantar erythrodysesthesia by VEGF receptor tyrosine kinase inhibitors is not known but it has been reproduced that palmar plantar erythrodysesthesia by lenvatinib is well tolerated Overall it is suggested that lenvatinib might be a favourable treatment option in terms of toxicitiesSeveral preclinical studies demonstrated that VEGF targeted treatment affects immune suppression by promoting the expansion of suppressive immune cell populations such as regulatory T cells and myeloid derived suppressor cells24 Several clinical studies suggested that modulation of VEGF mediated immune suppression via angiogenesis inhibition could potentially augment the immunotherapeutic activity of anti programmed cell death PD1 antibody26 Regorafenib and nivolumab showed antitumour activity in patients with metastatic colorectal cancer including those with microsatellite stable tumours in a phase study28Angiopoietin2 a relatively novel regulator of angiogenesis that acts through the TEK tyrosine kinase endothelial Tie2 receptor has been identified as a potential prognostic biomarker for some types of cancer Although the baseline Ang2 level was a predictive biomarker in patients with thyroid cancer in the SELECT trial17 it did not become a reliable biomarker of lenvatinib response in this study Prior treatment with anti VEGFVEGFR antibodies probably had an effect on baseline angiopoietin2 levels because the study population was refractory to standard treatment in this study The decrease in angiopoietin2 levels was observed after treatment therefore it may be an indicator of treatment responseThe limitations of our study include its small size which could limit the interpretation of the subgroup analyses Iwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776 0cand the absence of a comparison group However the level of clinical benefit in the form of confirmed responses observed in this study was remarkable in the historical context of other clinical trials done in heavily pretreated patients with metastatic colorectal cancer Moreover most of the patients in our study had left sided tumours which were known to have a better prognosis compared with right sided tumoursIn conclusion lenvatinib provided promising activity with prolonged survival relative to the anticipated median PFS in heavily pretreated patients with metastatic colorectal cancer The safety profile of lenvatinib was similar to that in other tumour types with no new safety signals recorded Based on these findings further investigation of lenvatinib with anti PD1 antibody or other novel combinations with the potential to build on the benefit of lenvatinib is currently taking place NCT03797326 and NCT04008797acknowledgements The authors thank the patients and their families the members of the Clinical Research Support Office for their support with data collection and running the study and NAI incorporated for editing a draft of this manuscriptContributors All authors conceived and designed the study and drafted and revised the manuscript for publication SI NO HS YH AT KK TH NB and YY collected data AK GO MK and KN analysed the data and managed data and study progress All authors interpreted the data and approved the final version of the manuscriptFunding The study was supported by the Project Promoting Clinical Trials for Development of New Drugs and Medical Devices Japan Medical Association from the Japan Agency for Medical Research and Development Grant Number JP18lk0201037 and by Eisai CoCompeting interests SI has received research grants from Eisai and Merck Biopharma TH has received research grants from Eisai and honoraria from Merck Serono YY has received honoraria from EisaiPatient consent for publication Not requiredethics approval The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines The study protocol was approved by the National Cancer Center Institutional Review Board T4329Provenance and peer review Not commissioned externally peer revieweddata availability statement Data are available upon reasonable request Proposals should be directed to siwasa ncc go jp The data will be available for achieving aims in the approved proposal access This is an access article distributed in accordance with the Creative Commons Attribution Non Commercial CC BY NC license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited any changes made are indicated and the use is non commercial See a0http creativecommons licenses by nc oRCId idsSatoru a0Iwasa http orcid Yasuhide a0Yamada http orcid RefeRences Saltz LB Clarke S Díaz Rubio E et a0al Bevacizumab in combination with oxaliplatin based chemotherapy as first line therapy in metastatic colorectal cancer a randomized phase III study J Clin Oncol “ Tabernero J Yoshino T Cohn AL et a0al Ramucirumab versus placebo in combination with second line FOLFIRI in patients with metastatic colorectal carcinoma that progressed during or after first line therapy with bevacizumab oxaliplatin and a fluoropyrimidine raise a randomised double blind multicentre phase study Lancet Oncol “ access Van Cutsem E Tabernero J Lakomy R et a0al Addition of aflibercept to fluorouracil leucovorin and irinotecan improves survival in a phase III randomized trial in patients with metastatic colorectal cancer previously treated with an oxaliplatin based regimen J Clin Oncol “ Yamazaki K Nagase M Tamagawa H et a0al Randomized phase III study of bevacizumab plus FOLFIRI and bevacizumab plus mFOLFOX6 as first line treatment for patients with metastatic colorectal cancer WJOG4407G Ann Oncol “ Price TJ Peeters M Kim TW et a0al Panitumumab versus cetuximab in patients with chemotherapy refractory wild type KRAS exon metastatic colorectal cancer ASPECCT a randomised multicentre label non inferiority phase study Lancet Oncol “ Mayer RJ Van Cutsem E Falcone A et a0al Randomized trial of TAS102 for refractory metastatic colorectal cancer N Engl J Med “ Grothey A Van Cutsem E Sobrero A et a0al Regorafenib monotherapy for previously treated metastatic colorectal cancer correct an international multicentre randomised placebo controlled phase trial Lancet “ Matsui J Yamamoto Y Funahashi Y et a0al E7080 a novel inhibitor that targets multiple kinases has potent antitumor activities against stem cell factor producing human small cell lung cancer H146 based on angiogenesis inhibition Int J Cancer “ Matsui J Funahashi Y Uenaka T et a0al Multi Kinase inhibitor E7080 suppresses lymph node and lung metastases of human mammary breast tumor MDA MB231 via inhibition of vascular endothelial growth factor receptor VEGF R and VEGF R3 kinase Clin Cancer Res “ Wiegering A Korb D Thalheimer A et a0al E7080 lenvatinib a multi targeted tyrosine kinase inhibitor demonstrates antitumor activities against colorectal cancer xenografts Neoplasia “ Hong DS Kurzrock R Wheler JJ et a0al Phase I dose escalation study of the multikinase inhibitor lenvatinib in patients with advanced solid tumors and in an expanded cohort of patients with melanoma Clin Cancer Res “ Boss DS Glen H Beijnen JH et a0al A phase I study of E7080 a multitargeted tyrosine kinase inhibitor in patients with advanced solid tumours Br J Cancer “ Yamada K Yamamoto N Yamada Y et a0al Phase I dose escalation study and biomarker analysis of E7080 in patients with advanced solid tumors Clin Cancer Res “ Nakamichi S Nokihara H Yamamoto N et a0al A phase study of lenvatinib multiple receptor tyrosine kinase inhibitor in Japanese patients with advanced solid tumors Cancer Chemother Pharmacol “ Kiyota N Schlumberger M Muro K et a0al Subgroup analysis of Japanese patients in a phase study of lenvatinib in radioiodine refractory differentiated thyroid cancer Cancer Sci “ Yoshino T Komatsu Y Yamada Y et a0al Randomized phase III trial of regorafenib in metastatic colorectal cancer analysis of the correct Japanese and non Japanese subpopulations Invest New Drugs “ Tahara M Schlumberger M Elisei R et a0al Exploratory analysis of biomarkers associated with clinical outcomes from the study of lenvatinib in differentiated cancer of the thyroid Eur J Cancer “ Takahashi S Kiyota N Yamazaki T et a0al A Phase II study of the safety and efficacy of a0lenvatinib in patients with advanced thyroid a0cancer Future Oncol “ Yamashita T Kudo M Ikeda K et a0al REFLECT a phase trial comparing efficacy and safety of lenvatinib to sorafenib for the treatment of unresectable hepatocellular carcinoma an analysis of Japanese subset J Gastroenterol “ Kudo M Finn RS Qin S et a0al Lenvatinib versus sorafenib in first line treatment of patients with unresectable hepatocellular carcinoma a randomised phase non inferiority trial Lancet “ Schlumberger M Tahara M Wirth LJ et a0al Lenvatinib versus placebo in radioiodine refractory thyroid cancer N Engl J Med “ Li J Qin S Xu R et a0al Regorafenib plus best supportive care versus placebo plus best supportive care in Asian patients with previously treated metastatic colorectal cancer concur a randomised double blind placebo controlled phase trial Lancet Oncol “ Van Cutsem E Martinelli E Cascinu S et a0al Regorafenib for patients with metastatic colorectal cancer who progressed after standard therapy results of the large single arm label phase IIIB CONSIGN study Oncologist “Iwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776 0c access Ott PA Hodi FS Buchbinder EI Inhibition of immune checkpoints and vascular endothelial growth factor as combination therapy for metastatic melanoma an overview of rationale preclinical evidence and initial clinical data Front Oncol Kato Y Tabata K Kimura T et a0al Lenvatinib plus anti PD1 antibody combination treatment activates CD8 T cells through reduction of tumor associated macrophage and activation of the interferon pathway PLoS One 201914e0212513 Taylor MH Lee C H Makker V et a0al Phase IbII trial of lenvatinib plus pembrolizumab in patients with advanced renal cell carcinoma endometrial cancer and other selected advanced solid tumors J Clin Oncol “ Makker V Rasco D Vogelzang NJ et a0al Lenvatinib plus pembrolizumab in patients with advanced endometrial cancer an interim analysis of a multicentre label single arm phase trial Lancet Oncol “ Fukuoka S Hara H Takahashi N et a0al Regorafenib plus nivolumab in patients with advanced gastric GC or colorectal cancer CRC an label dose finding and dose expansion phase 1B trial REGONIVO EPOC1603 JCO Iwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776 0c'

Thyroid_Cancer

Deoxyshikonin Inhibits Viability andGlycolysis by Suppressing theAktmTOR Pathway in Acute MyeloidLeukemia CellsHuijuan Wu   Hongmian Zhao   and Li Chen Telemedicine and Connected Health Center Huaihe Hospital of Henan University Kaifeng China Department ofHematology Huaihe Hospital of Henan University Kaifeng ChinaDeoxyshikonin was reported to exhibit an antitumor effect in colorectal cancer Howeverno studies are available to illustrate the effect of deoxyshikonin on acute myeloid leukemiaAML The effects of deoxyshikonin on viability apoptosis caspase37 activity andcytochrome Cyt C expression were evaluated by Cell Counting Kit8 assay flowcytometry analysis caspase37 activity assay and western blot analysis respectivelyGlucose consumption and lactate production were measured to determine the glycolysislevel The expression of pyruvate kinase M2 PKM2 was detected by quantitativerealtime polymerase chain reaction and western blot analysis The results showed thatdeoxyshikonin inhibited cell viability and increased the apoptotic rate the caspase37activity and the Cyt C protein level in AML cells in a dosedependent manner Additionallydeoxyshikonin concentrationdependently decreased glucose consumptionlactateproduction and PKM2 expression in AML cells Deoxyshikonin inactivated the proteinkinase B Aktmammalian target of the rapamycin mTOR pathway The activation ofthe AktmTOR pathway reversed the effects of deoxyshikonin on viability apoptosisand glycolysis in AML cells In deoxyshikonin dampened the viability and theglycolysis of AML cells by suppressing PKM2 via inactivation of the AktmTOR signalingEdited byCyrus KhandanpourUniversity Hospital M¼nster GermanyReviewed byPeng YangShanxi University ChinaHaili WuShanxi University ChinaHongyu ZhouKunming Medical University ChinaCorrespondenceHuijuan Wuhjwu297163com These authors share first authorshipKeywords deoxyshikonin glycolysis PKM2 the AktmTOR signaling acute myeloid leukemiaSpecialty sectionThis was submitted toHematologic Malignanciesa section of the journalFrontiers in OncologyReceived March Accepted June Published August CitationWu H Zhao H and Chen L Deoxyshikonin Inhibits Viability andGlycolysis by Suppressing theAktmTOR Pathway in Acute MyeloidLeukemia CellsFront Oncol 103389fonc202001253INTRODUCTIONAcute myeloid leukemia AML the most prevalent form of acute leukemia in adults is anaggressive malignancy derived from hemopoietic progenitor cells and with poor survival rate andfrequent relapse posing a threat to the health and life of aï¬ected patients AML is characterizedby rapid growth impaired apoptosis and abnormal clonal accumulation of hematopoietic stemcells in the bone marrow due to various genetic and epigenetic changes eventually leading to bonemarrow failure There were an estimated new cases diagnosed with AML and mortalities due to AML according to the statistics in Presently the efficacy of the standardchemotherapy for AML patients remains suboptimal owing to drug resistance and high clinicalrelapse rate In this regard searching for novel antileukemia drugs is urgently required toeï¬ectively improve the outcome of patients with AMLShikonin 58dihydroxy2[1S1hydroxy4methylpent3en1yl]naphthalene14 dionea naturally occurring naphthoquinone extracted from the oriental traditional medical herbFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML Cellsand suppressed glycolysis in AML cells Mechanistically the antiAML eï¬ect of deoxyshikonin was mediated via repressing PKM2by inactivation of the AktmTOR pathwayMATERIALS AND METHODSCell Cultivation and TreatmentAML cell lines including THP1 and HL60 were purchasedfrom American Tissue Culture Collection ATCC ManassasVA USA and routinely cultured in Roswell Park MemorialInstitute1640 medium HyClone South Logan UT USAconjugated with heatinactivated fetal bovine serum ExCellBio Shanghai China and penicillinstreptomycin SigmaAldrich St Louis MO USA The cells were fostered at —¦Cin a drippy environment flushed with air plus CO2Deoxyshikonin purity Tauto Biotech Shanghai Chinawas dissolved in dimethyl sulfoxide SigmaAldrich St LouisMO USA as a reserve solution and diluted into diï¬erentconcentrations and µgml The THP1and HL60 cells were exposed to diverse doses of deoxyshikoninfor h The Aktoverexpressing plasmid pcDNAAkt andthe empty vectorpcDNA control were obtained fromRibobio Guangzhou China Transfection was performed usingLipofectamine reagent Invitrogen Carlsbad CA USACell Counting Kit8 AssayCell Counting Kit8 CCK8 Beyotime Shanghai China wastaken to evaluate the viability of AML cells THP1 and HL60cells were seeded into 96well plates at — cellswell anddealt with various concentrations of deoxyshikonin and µgml for h or incubated with µgmldeoxyshikonin in the presence or the absence of µM 740YP Tocris Bioscience Shanghai China an activator of theAktmTOR signaling pathway After treatment for h µl ofCCK8 solution was added to each well followed by incubationfor another h The optical density of each well was recorded at awavelength of nm using a microplate reader Thermo FisherScientific Waltham MA USAFlow Cytometry Analysis for ApoptosisAfter the treatments as aforementioned annexin V FITCand propidium iodide kitKeyGen Nanjing China wasimplemented to analyze the apoptosis of THP1 and HL60 cellsCell apoptotic rate was detected by means of a flow cytometerFACScan BD Biosciences San Diego CA USAtheastreatmentsCaspase37 Activity AssayFollowingaforementioned ApoONEHomogeneous CaspaseGlo Assay kit Promega MadisonWI USA was adopted to measure the caspase37 activityof THP1 and HL60 cells referring to the manufacturer™sdescription Finally M2000 Infinite Pro instrument TecanTrading AG Maennedorf Switzerland was used to determinethe luminescenceFIGURE Chemical structure of deoxyshikoninLithospermum erythrorhizon Sieb et Zucc has been extensivelyused for the treatment of many diseases including burnssore throats HIV1 infection and macular eruption Currently clinical and pharmacological propertiesstudieshave demonstrated that shikonin and its derivatives exhibitvarious biological activities such as immune regulation andantithrombotic anti‚ammatory antioxidative and antiglycolytic activities An increasing number of researchesreveal that shikonin derivatives have garnered much researchinterest due to its limited toxicity and stronger antitumoractivities in miscellaneous cancers Interestingly a previousinvestigation proved that deoxyshikonin its chemical structureis shown in Figure a derivative of shikonin exhibited an antitumor eï¬ect in colorectal cancer However no studies areavailable to illustrate the eï¬ect of deoxyshikonin on AMLAerobic glycolysis also known as the Warburg eï¬ect is wellrecognized as a metabolic pathway in the rapidly proliferatingcancer cells for the regeneration of energy and the biosynthesisof macromolecules even in the presence of sufficient oxygen Aerobic glycolysis has been extensively accepted as an importantcharacteristic of tumor cells including AML eventually resultingin increased glucose consumption and lactate production Pyruvate kinase PK is a ratelimiting glycolytic enzymePyruvate kinase M1 PKM1 is expressed in normal diï¬erentiatedtissues whereas pyruvate kinase M2 PKM2 is expressedin cancer cellsleading to increased glycolysis As weall know the protein kinase B Aktmammalian target ofrapamycin mTOR pathway widely existing in cells is one ofthe most important survival signaling pathways that participatein the regulation of diverse physiological processes such as cellgrowth apoptosis and metabolism It has been reportedthat the AktmTOR pathway is a positive regulator of PKM2expression Previous studies suggested that shikonininhibited glycolysis by suppression of PKM2 expression Therefore we hypothesized that deoxyshikonin inhibitedviability and glycolysis suppressing pyruvate kinase M2 via theAktmTOR pathway in acute myeloid leukemia cellsIn the present study we assessed the eï¬ects and the underlyingmechanisms of deoxyshikonin on viability apoptosis glycolysisand PKM2 expression in AML cells These results revealed thatdeoxyshikonin treatment inhibited viability induced apoptosisFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsDetermination of Glucose Consumptionand Lactate ProductionAfter the treatments as aforementioned the culture medium ofTHP1 and HL60 cells was collected for the measurement ofglucose and lactate levels using a glucose uptake colorimetricassay kit SigmaAldrich and a lactic acid assay kit JianchengBioengineering Institute Nanjing China respectivelyPKM2 Activity AssayPKM2 activity was detected by the lactate dehydrogenasecoupled assay as described earlier Five microliters of wholecelllysate was utilized in the assay The absorbance at awavelength of nm was measured using a microplate readerThermo Fisher Scientificicecold radioimmunoprecipitation assayWestern Blot AnalysisThe treated THP1 and HL60 cells were collected and rinsedwith phosphatebuï¬ered salinefollowed by the additionoflysis buï¬erBeyotime containing mM phenylmethylsulfonyl fluorideSigmaAldrich A total of µg of protein samples wasfractionated on sodium dodecyl sulfatepolyacrylamide gelelectrophoresis prior to electrotransfer onto polyvinylidenedifluoride membranes Millipore Bedford MA USA Afterblocking with defatted milkTrisbased saline with Tween atroom temperature for h the membranes were immunoblottedovernight at —¦C with corresponding primary antibodiesagainst cytochrome C Cyt C Cell Signaling TechnologyInc Danvers MA USA phosphorylated Akt pAkt Ser473Abcam Cambridge MA USA AktAbcam glycogensynthase kinase3β GSK3β Abcam phosphorylated GSK3βpGSK3β Ser9 Abcam mTOR Abcam phosphorylatedmTOR pmTOR Ser2448 Abcam p70 ribosomal S6 kinasep70S6K Cell Signaling TechnologyInc phosphorylatedp70S6K pp70S6K Thr389 Cell Signaling Technology Inceukaryotic translation initiation factor 4Ebinding protein 4EBP1 Cell Signaling Technology Inc phosphorylated4EBP1 p4EBP1 Thr70 Cell Signaling TechnologyIncPKM2 Abcam and βactin Abcam and then incubated withhorseradish peroxidaseconjugated secondary antibodies CellSignaling Technology Inc for h at room temperature Lastlyan enhanced chemiluminescence kit Amersham PharmaciaPiscataway NJ USA was implemented to examine the antigen“antibody complexes βActin was used as a loading control Theprotein bands were visualized by VersaDoc imaging systemBioRad Hercules CA USAQuantitative RealTime PCRRNAiso Plus TaKaRa Dalian China was used to extracttotal RNA from treated THP1 and HL60 cells and theconcentration of extracted RNA was measured using a NanoDrop spectrophotometer Thermo Fisher Scientific Reversetranscription was carried out using the PrimeScript RT ReagentKit Takara Dalian China SYBR Green Taq Mix TaKaRawas then implemented to detect PKM2 mRNA expressionon the StepOnePlus qPCR system Thermo Fisher Scientificwith βactin as an endogenous control The thermocyclingconditions were displayed as follows —¦C for s followedby cycles of —¦C for s —¦C for s and —¦C for sThe primers were as follows PKM2 ²GCTG CCAT CTACCACT TGC3² forward and ²CCAG ACTT GGTG AGGACGAT T3² reverse GAPDH ²ATGT CGTG GAGT CTACTGGC3² forward and ²TGAC CTTG CCCA CAGC CTTG² reverse The ˆ’\x01\x01Ct method was taken to quantify theexpression level of PKM2 mRNAStatisticsAll data are shown as mean ± standard deviation of threeindependent experiments Statistical assays were determinedusing SPSS software IBM Corp Armonk NY USA withStudent™s ttest or oneway ANOVA followed by Dunnett™s test P were regarded as statistically significantRESULTSDeoxyshikonin Inhibited the Viability ofAML CellsTo clarify the antitumor activity of deoxyshikonin in AMLcells CCK8 was taken to evaluate cell viability after THP and HL60 cells were exposed to a series of deoxyshikoninconcentrations and µgml for h Asshown in Figures 2AB cell viability was significantly declinedin a concentrationdependent manner in THP1 and HL60 cellsin response to deoxyshikoninDeoxyshikonin Enhanced the Apoptosis ofAML CellsThe eï¬ect of deoxyshikonin on the apoptotic consequences ofAML cells was investigated by annexin VFITC apoptosis assayAs a result deoxyshikonin treatment led to a concentrationdependent increase of apoptotic rate in THP1 Figure 3Aand HL60 cells Figure 3B In line with the results of theflow cytometry analysis an elevation of caspase37 activityin deoxyshikonintreated THP1 Figure 3C and HL60 cellsFigure 3D was observed The mitochondrial protein Cyt Cis known as the initiating factor of mitochondrial apoptosispathway The expression of apoptotic marker Cyt C in THP1and HL60 cells was further detected by western blot analysisThe results demonstrated that deoxyshikonin concentrationdependently increased Cyt C protein level in THP1 Figure 3Eand HL60 cells Figure 3F relative to the control group Theseresults suggested that deoxyshikonin facilitated the apoptosis ofAML cellsDeoxyshikonin Suppressed Glycolysis inAML CellsTo further characterize the eï¬ect of deoxyshikonin on glycolysisin AML cells we measured glucose consumption and lactateproduction in AML cells These results demonstrated thatdeoxyshikonin exposure decreased glucoseconsumptionFigures 4AB and lactate production Figures 4CD in THP1Frontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin dosedependently inhibited the viability of acute myeloid leukemia cells THP1 A and HL60 B cells were administrated with variousdoses of deoxyshikonin and µgml for h and cell viability was then assessed by CCK8 assay P compared to the control groupand HL60 cells in a dosedependent manner We concluded thatdeoxyshikonin suppressed glycolysis in AML cellsenzymeratelimitingan importantDeoxyshikonin Decreased the ExpressionLevel of PKM2 in AML CellsA previous study reported that shikonin suppressed tumoraerobic glycolysis through suppressing the activity of PKM2in regulatingcellular glycolysis Accordingly we supposed whetherdeoxyshikonin had an inhibitory eï¬ect on PKM2 expression Asexpected the quantitative realtime polymerase chain reactionqRTPCR results showed that the mRNA levels of PKM2were suppressed following the addition of deoxyshikonin in adosedependent manner in THP1 Figure 5A and HL60 cellsFigure 5B The western blot results showed that deoxyshikonintreatment inhibited the protein levels of PKM2 in a dosedependent manner in THP1 Figure 5C and HL60 cellsFigure 5D We also found that deoxyshikonin suppressedPKM2 activity in THP1 Figure 5E and HL60 cells Figure 5FThese results suggested that deoxyshikonin decreased theexpression level of PKM2 in AML cellsDeoxyshikonin Inactivated the AktmTORPathway in AML CellsThe aberrant AktmTOR signaling has been demonstrated tobe associated with the tumorigenesis of miscellaneous cancersincluding AML We further determined the ‚uence ofdeoxyshikonin on the AktmTOR pathway in AML cells Asdemonstrated by western blot analysis deoxyshikonin treatmentrestricted the phosphorylation of Akt GSK3β mTOR p70S6Kand 4EBP1 in a concentrationdependent manner but causedno noticeable change on the total protein levels of Akt GSK3β mTOR p70S6K and 4EBP1 in THP1 and HL60 cellsFigures 6A“C indicating that deoxyshikonin inactivated theAktmTOR pathway in AML cellsActivation of the AktmTOR PathwayReversed the Effects of Deoxyshikonin onViability and Apoptosis of AML CellsTo figure out whether the AktmTOR pathway was involved inmediating the antitumor eï¬ects of deoxyshikonin on AML cellsTHP1 and HL60 cells were treated with µgml deoxyshikoninin the presence or the absence of 740YP for h The 740YP treatment alone resulted in a notable enhancement of pAkt and pmTOR expressions but produced little alternationon Akt and mTOR protein levels in THP1 Figure 7A andHL60 cells Figure 7B suggesting the activation of AktmTORsignaling by 740YP The CCK8 assay presented thatthedeoxyshikonin treatmentinduced viability reduction in THP Figure 7C and HL60 cells Figure 7D was eï¬ectivelyameliorated following the addition of 740YP The increaseof apoptotic rate in deoxyshikonintreated THP1 Figure 7Eand HL60 cells Figure 7F was significantly abolished aftercotreatment with deoxyshikonin and 740YP Moreover caspase activity was enhanced in THP1 Figure 7G and HL60 cellsFigure 7H in response to deoxyshikonin which was attenuatedfollowing the addition of 740YP Furthermore the protein levelof Cyt C in the deoxyshikonin 740YP cotreatment groupin THP1 Figure 7I and HL60 cells Figure 7J was reducedwhen compared with that of the deoxyshikonin treatment groupCollectively these results suggested that the activation of theAktmTOR pathway reversed the eï¬ects of deoxyshikonin on theviability and the apoptosis of AML cellsActivation of the AktmTOR PathwayReversed the Effects of Deoxyshikonin onGlycolysis and PKM2 Expression in AMLCellsThe 740YP treatment overturned the reduction of glucoseconsumption Figure 8A and lactate production Figure 8Bmediated by deoxyshikonin in THP1 and HL60 cells ThedeoxyshikoninsuppressedPKM2 mRNA expression in THP1 and HL60 cells whichsignificantlytreatmentaloneFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin concentrationdependently promoted the apoptosis of acute myeloid leukemia cells THP1 and HL60 cells were treated with increasingdoses of deoxyshikonin and µgml followed by detection of the apoptotic rate caspase37 activity and Cyt C protein level by annexin VFITC apoptosisassay AB caspase37 activity assay CD and western blot analysis EF respectively P compared to the control groupFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin concentrationdependently suppressed glycolysis in acute myeloid leukemia cells After THP1 and HL60 cells were administrated withincreasing doses of deoxyshikonin and µgml for h glucose consumption AB and lactate production CD were then measured P compared to control the groupwas restored by the combined treatment of deoxyshikoninand 740YP Figure 8C Moreover 740YP resisted thedeoxyshikonininduced decrease of PKM2 protein levelFigures 8DE and PKM2 activity Figure 8F in THP1 andHL60 cells These results suggested that the activation of theAktmTOR pathway reversed the eï¬ects of deoxyshikonin onglycolysis and PKM2 expression in AML cells To confirmthe abovementioned results THP1 and HL60 cells weretransfected with Aktoverexpressing plasmid pcDNAAkt toactivate the AktmTOR pathway As shown in Figures 9ABthe ratios of pAktAkt and pmTORmTOR were increased h after transfection in THP1 and HL60 cells The CCK8assay showed that deoxyshikonin treatmentcaused viabilityreduction in THP1 and HL60 cells was attenuated aftertransfection with pcDNAAkt Figure 9C The increase ofapoptotic rate in deoxyshikonintreated THP1 and HL60 cellswas significantly abolished after transfection with pcDNAAktimpairedthe deoxyshikonin treatmentcaused decrease of glucoseconsumption Figure 9E and lactate production Figure 9Fin THP1 and HL60 cells The deoxyshikonin treatmentinhibited the expression of PKM2 mRNA Figure 9G andprotein Figures 9HI and the activity of PKM2 Figure 9Jwhereas these eï¬ects were attenuated after transfection withpcDNAAkt These results confirmed that the activation ofFigure 9D Transfection with pcDNAAktthe AktmTOR pathway reversed the eï¬ects of deoxyshikoninon viability apoptosis glycolysis and PKM2 expression inAML cellsDISCUSSIONIn spite of significant improvements in therapeutic interventionsof AML the prognosis of patients suï¬ering from AML remainsunfavorable and the 5year survival rate of AML patients islower than accompanied by a high mortality rate Forthe high mortality there is a great need to identify eï¬ectivealternative therapeutic agents specifically targeting AML It iscommonly reckoned that natural products have the potentialto induce apoptosis in cancer cells including AML and maytherefore be essential sources for anticancer drugs becauseof their extensive biological activities and limited side eï¬ects Shikonin and its derivatives the predominant typeof naphthoquinone derivatives extracted from the root ofLithospermum erythrorhizon Sieb et Zucc have been welldocumented to possess a wide range of pharmacologicalactivities including antitumor activity by suppression of cellproliferation For instance shikonin potently depressed theviability and the metastasis of triplenegative breast cancercells by reversing the epithelialtomesenchymal transition viaFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin decreased the expression level and the activity of PKM2 in acute myeloid leukemia cells THP1 and HL60 cells were exposed toincreasing doses of deoxyshikonin and µgml for h A“D The mRNA and protein levels of PKM2 were determined by qRTPCR and western blotrespectively EF PKM2 activity was detected by the lactate dehydrogenasecoupled assay P compared to the control groupFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Deoxyshikonin inactivated the AktmTOR pathway in AML cells A“C THP1 and HL60 cells were treated with different doses of deoxyshikonin and µgml for h and the protein levels of pAkt Akt pGSK3β GSK3β pmTOR mTOR pp70S6K p70S6K p4EBP1 and 4EBP1 were measured bywestern blot analysis P compared to the control groupglycogen synthase kinase 3βregulated repression of βcateninsignaling Additionally βdimethylacrylshikonin suppressedcell viability and induced mitochondriadependent apoptosisin human lung adenocarcinoma cells via the activation of thep38 signaling pathway Acetylshikonin another shikoninderivative significantly inhibited the anchorageindependentgrowth of pancreatic cancer cells by suppressing the nuclearfactorkappa B signaling pathway More importantly itwas previously demonstrated that deoxyshikonin exhibited antiproliferative and proapoptotic activities in colorectal cancercells through the phosphoinositide 3kinase PI3KAktmTORpathway Nevertheless whether deoxyshikonin showed anantitumor activity in AML remained far from being addressedTo our knowledgetime to demonstratethat deoxyshikonin dampened the viability of AML cellsin a dosedependent manner Meanwhile we found thatexposure to deoxyshikonin led to a concentrationdependentthis is the firstincrease of the apoptotic rate caspase37 activity and CytC protein levelin AML cells The increase of caspase activity is an important indicator of apoptosis These resultssuggested that deoxyshikonin exerted an antitumor activityin AML cells The eï¬ects of deoxyshikonin on the viabilityand the apoptosis of normal bone marrow stromal HS5cells were also evaluated in this study The results showedthat deoxyshikonin at µgml did not aï¬ect HS5 cellviability and apoptosis Supplementary Figure suggestingthat deoxyshikonin was selectively toxic to cancer cells but notto normal cellsIt has been proven that disruption of aerobic glycolysisrestricts cancer carcinogenesis suggesting that elevated aerobicglycolysis facilitates tumor development and oncogenesis Our study provided evidence that deoxyshikonin inhibitedglycolysis in AML cells as demonstrated by decreased glucoseconsumption and lactate production Moreover we found thatFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Activation of the AktmTOR pathway reversed the effects of deoxyshikonin on the viability and the apoptosis of acute myeloid leukemia cells AB Theprotein levels of pAkt Akt pmTOR and mTOR in the THP1 and HL60 cells treated with 740YP for h were detected by western blot analysis THP1 and HL60cells were treated with µgml deoxyshikonin in the presence or the absence of µM 740YP for h followed by assessment of cell viability CD apoptosisEF caspase37 activity GH and Cyt C protein level IJ by CCK8 assay flow cytometry analysis caspase37 activity assay and western blot analysisrespectively P compared to the control group P compared to the deoxyshikonin treatment groupdeoxyshikonin inhibited the expression of PKM2 in AML cellsPKM2 a critical ratelimiting enzyme of aerobic glycolysis isproposed to play a crucial role in glycolysis process and cancerprogression The robust expression of PKM2 has beenobserved in various human cancers which facilitates cancercell proliferation and growth These findings togetherrevealed that deoxyshikonin inhibited glycolysis in AML cells bysuppressing PKM2It has been demonstrated that the AktmTOR signalingnetwork is constitutively activated and associated with thedevelopment of several types of cancers including AML Overactivation of the AktmTOR signaling is involvedin the elevated aerobic glycolysis of cancer cellstherebycontributing to cancer cell survival and growth Thusthe AktmTOR pathway may be regarded as a promisingtherapeutic target for cancer treatment To elucidate themolecular mechanism underlying the antitumor eï¬ects ofdeoxyshikonin we detected the ‚uence of deoxyshikoninon the AktmTOR signaling in AML cells It was shownthat deoxyshikonin impeded the activation of the AktmTORsignaling in AML cells In the restoration assay activation ofthe AktmTOR signaling by 740YP or pcDNAAkt plasmidabolished the antitumor eï¬ect of deoxyshikonin in AML cellsTaken togetherthese results suggested that deoxyshikoninFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Activation of the AktmTOR pathway reversed the effects of deoxyshikonin on glycolysis and PKM2 expression in acute myeloid leukemia cells THP1and HL60 cells were exposed to µgml deoxyshikonin or together with µM 740YP for h AB Glucose consumption and lactate production in thesupernatants of treated THP1 and HL60 cells were measured C The mRNA level of PKM2 in treated THP1 and HL60 cells was estimated by quantitative realtimepolymerase chain reaction DE The protein level of PKM2 in the treated THP1 and HL60 cells was estimated by western blot F PKM2 activity was detected bythe lactate dehydrogenasecoupled assay P compared to the control group P compared to the deoxyshikonin treatment groupFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML CellsFIGURE Transfection with pcDNAAkt reversed the effects of deoxyshikonin on viability apoptosis glycolysis and PKM2 expression in acute myeloid leukemiacells AB The Aktoverexpressing plasmid pcDNAAkt and empty vector pcDNA control were transfected into THP1 and HL60 cells After h the protein levelsof pAkt Akt pmTOR and mTOR were detected by western blot analysis Transfected THP1 and HL60 cells were exposed to µgml deoxyshikonin for hfollowed by assessment of cell viability C apoptosis D glucose consumption E and lactate production F as well as PKM2 mRNA G protein HI and activityJ P compared to the pcDNA group P compared to the pcDNA deoxyshikonin groupFrontiers in Oncology wwwfrontiersinAugust Volume 0cWu et alDeoxyshikonin Inhibits AML Cellsdampened the viability and the glycolysis of AML cells bysuppressing PKM2 via inactivation of the AktmTOR signalingThe main defect ofthis study is that experiments wereonly performed on two AML cell lines THP1 and HL60and no primary AML cells were tested The heterogeneityof AML is therefore not taken into account Future studiesshould explore the role of deoxyshikonin using primaryAML cellsCONCLUSIONTo sum up our study provided the first evidence thatdeoxyshikonin exerted antitumor and antiglycolytic activitiesin AML cells by suppressing PKM2 via inactivation of theAktmTOR signaling Our study provided novel insights intothe antitumor and antiglycolytic activities of deoxyshikonin inAML Deoxyshikonin may be a promising anticancer candidateagent in AML cellsREFERENCES Kavanagh S Murphy T Law A Yehudai D Ho JM Chan S et al Emergingtherapies for acute myeloid leukemia translating biology into the clinic JCIInsight 101172jciinsight95679 Papaemmanuil E Gerstung M Bullinger L Gaidzik VI Paschka PRoberts ND et al Genomic classification and prognosis in acute myeloidleukemia N EnglJ Med “ 101056NEJMoa1 Siegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin “ 103322caac21442 Krug U Berdel WE Gale RP Haferlach C Schnittger S MullerTidow C etal Increasing intensity of therapies assigned at diagnosis does not improvesurvival of adults with acute myeloid leukemia Leukemia “ 101038leu201625 Gao H Liu L Qu ZY Wei FX Wang SQ Chen G et al Antiadenovirus activities ofshikonin a component of Chinese herbalmedicine in vitro Biol Pharm Bull “ 101248bpb Li W Zhang C Ren A Li T Jin R Li G et al Shikonin suppressesskin carcinogenesis via inhibiting cell proliferation PLoS ONE 10e0126459 101371journalpone0126459 Andujar I Rios JL Giner RM Recio MC Pharmacological properties ofshikonin a review of literature since Planta Med “ 101055s00331350934 Li W Liu J Zhao Y PKM2 inhibitor shikonin suppresses TPAinducedmitochondrial malfunction and proliferation of skin epidermal JB6 cells MolCarcinog “ 101002mc21988 Boulos JC Rahama M Hegazy MF Eï¬erth T Shikonin derivatives “for cancer prevention and therapy Cancer Lett 101016jcanlet201904033 Zhu Y Zhong Y Long X Zhu Z Zhou Y Ye H et al Deoxyshikoninisolated from Arnebia euchroma inhibits colorectal cancer by downregulating the PI3KAktmTOR pathway PharmBiol “ Liberti MV Locasale JW The Warburg eï¬ect how does it benefit cancer cellsDATA AVAILABILITY STATEMENTAll datasets generated for this study are included in theSupplementary MaterialAUTHOR CONTRIBUTIONSHW conducted the experiments and participated in theconception and the design of the study HZ conducted theexperiments and performed the analysis LC contributed toanalyzing the data and drafting the manuscript All authorscontributed to the and approved the submitted versionSUPPLEMENTARY MATERIALThe Supplementary Materialonline202001253fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncat Xu D Liang J Lin J Yu C PKM2 a potential regulator of rheumatoid arthritisvia glycolytic and nonglycolytic pathways Front Immunol 103389fimmu201902919 Gong T Cui L Wang H Wang H Han N Knockdown of KLF5 suppresseshypoxiainduced resistance to cisplatin in NSCLC cells by regulating HIF1αdependent glycolysis through inactivation of the PI3KAktmTOR pathway JTransl Med 101186s1296701815432 Nemazanyy I Espeillac C Pende M Panasyuk G Role of PI3K mTOR andAkt2 signalling in hepatic tumorigenesis via the control of PKM2 expressionBiochem Soc Trans “ 101042BST20130034 Zheng YL Li L Jia YX Zhang BZ Li JC Zhu YH et al LINC01554mediated glucose metabolism reprogramming suppresses tumorigenicityin hepatocellular carcinoma via downregulating PKM2 expression andinhibiting AktmTOR signaling pathway Theranostics “ 107150thno28992 Zhao X Zhu Y Hu J Jiang L Li L Jia S et al Shikonin inhibits tumor growthin mice by suppressing pyruvate kinase M2mediated aerobic glycolysis SciRep 101038s4159801831615y Tang J Ren YG Zhao J Long F Chen J Jiang Z Shikonin enhancessensitization of gefitinib against wildtype EGFR nonsmall cell lung cancervia inhibition PKM2stat3cyclinD1 signal pathway Life Sci “ 101016jlfs201805012 Popescu NC Cheng SY Chromosomal localization of the gene for a humancytosolic thyroid hormone binding protein homologous to the subunitof pyruvate kinase subtype M2 Somat Cell Mol Genet “ 101007BF01233100 Martelli AMF McCubreyEvangelisti C ChiariniJA Thephosphatidylinositol 3kinaseAktmTOR signaling network as a therapeutictarget in acute myelogenous leukemia patients Oncotarget “ 1018632oncotarget114 Yang D Zhang X Zhang X Xu Y The progress and current status ofimmunotherapy in acute myeloid leukemia Ann Hematol “ 101007s002770173148x Wang X Feng Y Chinese medicines induce cell death the molecular andcellular mechanisms for cancer therapy Biomed Res Int Trends Biochem Sci “ 101016jtibs201512001 Wang ZY Chen Z Acute promyelocytic leukemia from highly fatal to highly Hanahan D Weinberg RA Hallmarks of cancer the next generation Cellcurable Blood “ 101182blood200707102798 “ 101016jcell201102013 Herst PM Howma

Thyroid_Cancer

obesity and ethnicity alter gene expression in skinJeanne M Walker18 Sandra Garcet28 Jose O Aleman34 Christopher E Mason5 David Danko5 Simone Zuffa6 Jonathan R Swann67 James Krueger2 Jan L Breslow3 peter R Holt3Obesity is accompanied by dysfunction of many ans but effects on the skin have received little attention We studied differences in epithelial thickness by histology and gene expression by Affymetrix gene arrays and PCR in the skin of obese BMI “ and normal weight BMI “ postmenopausal women paired by age and ethnicity Epidermal thickness did not differ with obesity but the expression of genes encoding proteins associated with skin blood supply and wound healing were altered In the obese many gene expression pathways were broadly downregulated and subdermal fat showed pronounced inflammation There were no changes in skin microbiota or metabolites African American subjects differed from European Americans with a trend to increased epidermal thickening In obese African Americans compared to obese European Americans we observed altered gene expression that may explain known differences in water content and stress response African Americans showed markedly lower expression of the gene encoding the cystic fibrosis transmembrane regulator characteristic of the disease cystic fibrosis The results from this preliminary study may explain the functional changes found in the skin of obese subjects and African AmericansObesity defined as a body mass index BMI greater than a0kgm21 has become a major epidemic in industrial and emerging countries The prevalence of obesity has doubled since the 1980s and it is now estimated that million adults worldwide are obese2 Obesity affects many ans of the body and it is this an dysfunction that leads to excess mortality and morbidity3 Much attention has focused on the consequences of obesity in the heart liver and pancreas and other ans in which increased inflammation and oncogenesis become apparent4 Less attention has been paid to the effects of obesity on the skinObesity increases psoriasis5 which can be ameliorated with weight loss and cutaneous infections6 Since diabetes is common in obesity disorders such as fibroepithelial polyps and acanthosis nigricans also occur in the skin of obese subjects78 Moreover physiologic changes found in obese skin include increased transepidermal water loss with lower capacitance dry rough textured skin with pronounced erythema and reduced microvascular reactivity Altered collagen formation and increased delayedtype hypersensitivity have also been reported9Adipocyte depots that exist adjacent to the epidermis have distinct morphology and physiologic characteristics and are termed dermal or subdermal adipose tissue In addition to the principal role for dermal adipocytes in lipid storage and thermal insulation10 they also promote skin immunity11 wound healing and hair follicle cycling12 Obesity is accompanied by inflammatory immune changes in subcutaneous and visceral adipose tissues13 but the role of inflammatory changes within the adipose layer of the skin has received little attention Furthermore obesity is associated with increased circulating leptin levels which appear to independently affect dermal cell proliferation and hair growth14 In addition the microanisms that live on the skin surface also affect skin immunity11 so that it is important to analyse the skin microbiome comparing obese and normal individuals1The Rockefeller University Hospital New York NY USA 2Laboratory of Investigational Dermatology The Rockefeller University New York NY USA 3Laboratory of Biochemical Genetics and Metabolism The Rockefeller University New York NY USA 4Laboratory of Translational Obesity Research New York University Langone Health New York NY USA 5Weill Cornell Medical College New York NY USA 6Department of Metabolism Digestion and Reproduction Imperial College London London UK 7School of Human Development and Health Faculty of Medicine University of Southampton Southampton UK 8These authors contributed equally Jeanne M Walker and Sandra Garcet email walkerjrockefelleredu holtprockefellereduScientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cIn view of the profound clinical and physiologic changes described in the skin in obesity it would not be surprising also to find biologically important molecular changes The present study was designed to compare gene expression in skin of healthy obese and nonobese subjects and to evaluate the potential importance of parallel changes in the microbiome and metabolites found on the adjacent skin surface and in the adipose tissue immediately below the skinMaterials and methodsSubjects Participants were recruited from the surrounding community through advertisements and from the Rockefeller University subject repository Eligible were healthy obese BMI “ a0kgm2 and nonobese BMI “ a0kgm2 postmenopausal women between the ages of and a0years The two groups were matched by age ± a0years and defined by selfreported ethnicity and by skin colour Exclusions were unstable weight change within the past three months HIV infection weight loss surgery inflammatory bowel disease history of malignancy other than nonmelanoma skin cancer in the previous a0years generalizable or systemic skin diseases history of a bleeding disorder current anticoagulant therapy or regular NSAID use current weight control medication or hypoglycaemic therapy individuals taking oestrogenprogesterone hormones and current tobacco smokers Also excluded were candidates with fasting blood glucose a0mgdl liver function tests ALT AST alkaline phosphatase greater than times the upper limit of normal ULN abnormal thyroid function test or serum creatinine — ULNFourteen obese subjects were screened two refused skin biopsies one was withdrawn due to an intercurrent inflammatory illness and one was not postmenopausal by our criteria Ten obese subjects met our inclusion criteria and underwent skin swab collections and punch biopsy Twenty nonobese subjects were screened Two subjects refused to undergo punch biopsy one was withdrawn due to an intercurrent illness two with a BMI outside the required range one withdrew consent one was excluded with a history of keloid formation one with a low platelet count one with uncontrolled hypertension One nonobese subject who underwent skin punch biopsy was not included in the analysis because we were unable to find an age and ethnicitymatched obese subject These obese and agematched ethnicitymatched nonobese postmenopausal women completed all aspects of the study Fig a0 Six participants were European American and four were African Americans in each group Postmenopausal women were chosen to exclude effects of the menstrual cycle upon study end points and to exclude gender effectsBased on preliminary data from a previous study comparing skin from seven obese and six nonobese postmenopausal women there was a variation of in a set of RTPCR genes unpublished data Assuming the same variation and proportion of differentially expressed genes to be we calculated that a sample size of n subjects per group matched by age and ethnicity would provide power at a falsediscovery rate to detect the expected number of differentially expressed genes based on a threshold of twofold changesDesign and setting This was an label comparison of a group of postmenopausal obese women and postmenopausal nonobese women who were agematched ± a0years and racematched Screening comprised a complete history and physical examination and fasting blood testing for complete blood count sedimentation rate comprehensive chemistry panel lipid panel thyroid function tests hepatitis C antibody uric acid and haemoglobin A1C Observing Good Clinical Practice guidelines all participants read and signed an informed consent document approved by the Institutional Review Board and the Advisory Committee for Clinical and Translational Science at The Rockefeller University Protocol JWA0921Procedure methods Anthropometric measurements Body weight was measured daily to the nearest a0kg using a ScaleTronix scale Welch Allyn Skaneateles Falls NY with precision of ± a0kg Subjects were weighed in a hospital gown after an overnight fast and postvoiding Height was measured to the nearest a0cm at baseline with a Seca216 stadiometer Hamburg Germany in a0cm increments Body mass index BMI was calculated as kgm2 using the NIH Standard Metric BMI calculatorBlood collection and analysis Fasting blood samples were analysed in the Clinical Pathology Laboratory of the Memorial SloanKettering Cancer Center for complete blood count electrolytes glucose creatinine blood urea nitrogen liver function Creactive protein sedimentation rate and uric acid Research serum samples were drawn pre and post intervention aliquoted and stored at ˆ’ a0°C for subsequent analysisSkin swabbing Subjects were permitted to shower but did not wash the planned biopsy area over the midlower abdomen with soap for a0days before the biopsyFor microbiome analysis two areas of skin approximately — a0cm were swabbed using the eSwab collection and preservation system for aerobic anaerobic and fastidious bacteria Copan Diagnostics Marietta CA Swabs were labelled sealed separately in the provided tubes and immediately stored at ˆ’ a0°C For metabolome analysis two different areas of skin approximately — a0cm were swabbed using salinemoistened sterile cottontipped applicators The tips were cut sealed in separate sterile collection tubes and immediately stored at ˆ’ a0°CSkin microbiome The DNA extraction protocol was adapted from the Maxwell RSC Buccal Swab DNA kit Catalogue number AS1640 Promega Corporation Madison WI Briefly a0μl of lysis buffer and a0μl of Proteinase K was mixed and added to each swab tube Swab tubes were then incubated for a0min at C using a Thermo Fisher water bath removed from the tubes and fluid was transferred to well of the Maxwell RSC Cartridge The swab head was centrifuged using a ClickFit Microtube Cat V4741 and extracted fluid was added to the corresponding well of Maxwell Cartridge and eluted in a0μl of provided elution bufferScientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Consort flow chart of eligible subjectsExtracted DNA was taken forward to the Nextera Flex protocol by Illumina Briefly a0μl of extracted DNA was taken into library prep protocol and run with cycles of PCR Libraries were cleaned up with a left sided size selection using a bead ratio of 08x The right sided size selection was omitted Libraries were then quantified using a Thermo Fisher Qubit Fluorometer and an Advanced Analytical Fragment Analyzer Libraries were sequenced on an Illumina HiSeqPE — at the Weill Cornell Epigenomics CoreAll bioinformatic analysis was performed on Weill Cornell Medicine™s Athena compute cluster a highperformance grid compute system Secondary analysis was performed on a Linux and MacOS systems Unless otherwise noted programs were run with default settingsRaw sequence data were processed with AdapterRemoval v217 to remove low quality reads and reads with ambiguous bases15 Subsequently reads were aligned to the human genome hg38 including alternate contigs using Bowtie2 v230 fast preset16 Read pairs where one or both ends mapped to the human genome were separated from read pairs where neither mate mapped Read pairs where only one mate mapped were discarded Hereafter we refer to the read sets as human reads and nonhuman readsTaxonomic profiles were generated by processing nonhuman reads with KrakenUniq v032 with a database based on all draft and reference genomes in RefSeq Microbial bacteria fungi virus and archaea ca March KrakenUniq identifies kmers that are unique to taxa in a database Reads are broken into kmers and searched against this database Finally the taxonomic makeup of each sample was given by taking the proportion reads which were assigned to each clade KrakenUniq counts the number of unique marker kmers assigned to each taxon and we filtered taxa with fewer than unique markers17We performed differential abundance testing over microbial species using the ALDEx2 R package ALDEx2 performs variance stabilization read counts using a centred log ratio transformation that models samples as Scientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cDirichletMultinomial distributions over taxa then compares taxonomic abundances across groups18 Comparison of abundances across groups was done with a Wilcoxon rank sum test and Benjamini Hochberg Correction for multiple hypothesis testingDimensionality reduction of taxonomic profiles was performed with Principal Coordinates Analysis PCA based on a matrix of JensenShannon Divergences JSD between samples Analysis of intersample beta diversity was performed using the same matrix of JSD Intrasample alpha diversity was measured by finding Shannon™s Entropy of the taxonomic profile and by counting the total number of species identified in each sample richness Shannon™s entropy accounts for the relative size of each group in diversity estimation and is defined as H ˆ’ cid31 ai log2ai where ai is the relative abundance of taxa i in the sampleWe generated profiles of antimicrobial resistance genes using MegaRes v10119 To generate profiles from MegaRes we mapped nonhuman reads to the database using Bowtie2 v230 very sensitive presets Subsequently alignments were analysed using Resistome Analyzer commit 15a52dd and normalized by total reads per sample and gene length to give RPKMs MegaRes includes an ontology grouping resistance genes into gene classes AMR mechanisms and gene groupsSkin metabolome The skin was swabbed using two sterile salinemoistened culture swabs and immediately frozen at ˆ’ a0°C Swab heads were removed and placed in a0ml methanol water Following sonication a0min a0ml of isopropanol was added and the solution was spun at a0g for a0min The swab was removed and the samples were dried using a vacuum concentrator operating at a0°C Prior to UPLCMS analysis samples were reconstituted in a0μl of HPLCgrade water sonicated for a0min and transferred to vials for analysisA Waters 2777C sample manager Waters Corp Milford MA USA was used for sample handling This was equipped with a a0μl Hamilton syringe a a0μl loop used for fullloop injections of prepared sample and a 3drawer sample chamber maintained at a0°C with a constant flow of dry nitrogen gas to prevent the buildup of condensation The LC component was an ACQUITY UPLC Waters Corp Milford MA USA composed of a binary solvent manager and column heatercooler module Metabolic profiles were acquired using reversedphase chromatography Water and acetonitrile each supplemented with formic acid mobile phases A and B respectively were selected for the mobile phase A — a0mm HSS T3 column was used at a0°C with a mobile phase flow rate of a0mlmin This generated a maximum pressure of psi in a wateracetonitrile gradient After a a0min isocratic separation at initial conditions A a linear gradient elution A to A in a0min proceeded followed by a quicker gradient A to A in a0min to final conditions The mobile phase flow rate was simultaneously increased to a0mlmin in the latter stage to facilitate faster column washing The MS component comprised a Xevo G2S QToF MS Waters Corp Manchester UK coupled to the UPLC via a Zspray electrospray ionization ESI source The cone gas flow was set to a0lh to protect the cone from residue accumulation during operation Both positive and negative ion modes RPC and RPCˆ’ respectively were used Raw spectra were converted into mzML files using MSConvert20 and processed with XCMS in R21 Peak picking and peak grouping were performed using inhouse scripts in R and matrices were normalized using a median fold change approach Log transformation scaling and data analysis was performed in SIMCA Umetrics Umea SwedenSkin biopsy After the skin swabbing the abdominal site was cleansed with Chloraprep swabs chlorhexidine and isopropyl alcohol Becton Dickinson Canaan CT Using sterile technique local anaesthesia was induced by infiltration of the area with a0ml of lidocaine Hospira Inc Lake Forest IL mixed with a0ml sodium bicarbonate The skin biopsy was performed using a a0mm punch Miltex Instruments York PA Fat tissue was carefully removed from the skin core of the biopsy The dermis and epidermis were divided into two halves one half placed in a cryomold for OTC flash freezing Agar Scientific Essex UK and stored at ˆ’ a0°C and the other half was placed in RNAlater Stabilization Solution Thermo Fisher Scientific Fair Lawn NJ refrigerated for a0h and then frozen at ˆ’ a0°C The fat tissue was removed from the biopsy divided between RNAlater refrigerated for a0h then frozen at ˆ’ a0°C and a dry Sarstedt tube that was flash frozen in liquid nitrogen and placed in ˆ’ a0°C The biopsy site was sutured closed and a dry sterile dressing was applied Subjects were discharged and scheduled to return for suture removalGene‘array and quantitative real‘time PCR analysis RNA was extracted followed by hybridization to Affymetrix Human U133 Plus gene arrays Santa Clara CA or quantitative RTPCR as previously described2223All statistical analyses were carried out in R Limma Log 2transformed qRTPCR measurements hARP normalized and microarray expression values were assessed with a mixedeffect The fixed factors were condition obese vs nonobese race African American vs Caucasian with random intercept for each subject Quality control of microarray chips was carried out using standard QC metrics and R package microarray quality control Images were scrutinized for spatial artefacts using Harshlight24 Expression measures were obtained using the GCRMA algorithm25 A batch effect corresponding to the hybridization date was detected by PCA and adjusted using the ComBat function from the SVA package Probe sets with at least samples with expression values were kept for further analysis Fold changes for the comparisons of interest were estimated and hypothesis testing was conducted with contrasts under the general framework for linear models with the limma package P values from the moderated paired ttests were adjusted for multiple hypotheses using the Benjamini“Hochberg procedure Hierarchical clustering was performed with Euclidean distance and a McQuitty agglomeration scheme26Data was deposited into Gene Expression Omnibus GEO repository GSE151839All study methods and procedures were carried out in accordance with Good Clinical Practice Guidelines by trained practitioners The protocol and informed consent were evaluated and approved by the Institutional Scientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression between the skin of obese and nonobese subjects A Heat map of the most differentially expressed genes in the skin of obese and nonobese subjects with an FCH fold change fdr false discovery rate B PCA principal component analysis plot of differentially expressed genes in the skin of obese and nonobese subjects with an FCH fdr Review Board and the Advisory Committee for Clinical and Translational Science at Rockefeller University prior to initiation of the study and annually thereafter Protocol JWA0921ResultsThis study was performed in ten healthy obese and ten healthy nonobese postmenopausal women matched for age and ethnicity Obese subjects had a mean weight of a0kg BMI of a0kgm2 and waist circumference of a0cm Nonobese subjects had a mean weight of a0kg BMI of a0kgm2 and waist circumference of a0cm Supplemental Table a0S1 The skin thickness for subjects with obesity was not significantly different from that of nonobese subjects Supplemental Fig a0S1Gene expression analysis of the skin The most differentially expressed genes in the skin between obese and nonobese subjects are displayed in the heat map in Fig a02a Comparing gene expression in obese versus nonobese skin showed greater gene expression of S100A7A encoding a calcium binding protein involved in psoriasis and CORIN encoding a natriuretic peptide converting enzyme which is expressed in the dermis and is involved in specifying skin colour However the expression of CREB3LA encoding a cyclic AMP response element was lower in the skin of obese subjectsA PCA model constructed on of the most differentially expressed genes between obese and nonobese subjects showed partial separation of the groups This difference was seen in PC1 which accounted for of the variation in the included genes Fig a02bThe complete list of skin genes whose expression significantly differed between the two groups are shown with fold changes in Supplemental Tables a0S2 and S3 Again the gene expression of S100A7A was 344fold higher in the obese skin compared to the nonobese skin Similarly the expression of DEFB4A Defensin B4A which encodes an antimicrobial peptide part of the betadefensive system and SPRR2C which encodes a proline rich protein strongly induced during differentiation of human epidermal keratinocytes was also significantly higher in the obese skin being and 17fold higher respectively Genes with lower expression profiles in obese subjects than nonobese included AOP that encodes aquaporin involved in water channels present in the skin PROM1 prominin involved in cell differentiation and proliferation and Keratin and important for fibrogenesis in the epidermis Also of interest was the significantly higher expression 282fold of CFTR the cystic fibrosis transmembrane conductance receptor in nonobese subjects compared to the obese groupQTPCR analysis of genes selected from the total list of significantly differentially expressed genes in skin confirmed increased expression of the S100A 373fold DEFB4A defensin B4A 329fold and CORIN fold in the skin of obese subjects Fig a0 Significantly lower gene expression in the skin of obese subjects was found with CFTR 36fold PROM1 556fold and GABRP gamma aminobutyric acid receptor 29foldScientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cGene expression pathway analysis showed a broad downregulation of many pathways in obesity with only out of of the most highly differentially expressed pathways higher in the obese Supplemental Fig a0S2 The pathways most downregulated included cardiac beta adrenergic signalling which appears to function in skin cyclin dependent Kinase CDK5 a mutation which is important in melanoma formation and functions in skin healing and gonadotrophic releasing hormone GNRH signalling which has many extra pituitary functionsGene expression analysis in skin fat We next examined differences in gene expression between the groups of subjects in subdermal fat removed from immediately below the skin portion of the biopsy A heat map of gene expression shows a markedly different pattern between the two groups Fig a04a The expression of many of the genes upregulated in obese subdermal fat are involved with inflammation and immune function including platelet activating factor PLA2G7 ILIRN involved in IL1 activation SPPI a cytokine that can increase interferon gamma and IL12 activity and several serpins mediators involved in inflammation and immune functionThe PCA plot of genes whose expression differed significantly in subdermal fat of obese and nonobese subjects Fig a04b clearly shows separation between the two groups Most of the difference was seen in PC1 which includes of the genes whose expression was determinedSupplemental Tables a0S4 and S5 show a list of genes whose expression was relatively greater in the subdermal fat of obese subjects The expression of SPPI that encodes osteopontin which can act as a cytokine augmenting the action of interferon gamma and interleukin was approximately tenfold higher in the obese subjects EGFL6 expression which encodes an epidermal growth factor found to be enhanced in obesity and alters insulin action was increased by 85fold MMP9 which encodes metalloproteinase and ILTRN was increased by sevenfold in obesity Genes significantly downregulated in obese subdermal fat included SLC27A2 acetylCoAsynthase tenfold and C6complement fivefoldBy QTPCR in subdermal fat from obese subjects the increased expression of genes encoding proteins important in inflammation and immune function was confirmed Fig a0 This includes genes encoding proteins that determine accumulation of immune cells in adipose tissues such as CD52 the high affinity immunoglobulin gamma FC receptor FCGR1β CCL3 CZXCL8 interleukin and CLEC7A a pattern recognition receptor found in monocytes and other myeloid cells IL17F a member of the IL17 family also was specifically increased in subdermal fat from the obese as compared to nonobese individualsExpression pathway analysis Supplemental Fig a0S3 showed upregulation of several inflammatory immune pathways including the Thelper dendritic cell maturation and inflammatory signalling pathways further indicating profound effects of obesity on inflammation in subdermal fat Dramatically lower in the obese subdermal fat was the LXRRXR activation pathwayGene expression analysis by race Two subgroups were observed in the gene expression profiles of the skin based on the subject™s race Using selfreported data and skin colour as criteria the data from African Americans was analysed separately from the data from European Americans This analysis showed striking differences in this very small group of subjects A heatmap of the most differentially expressed genes in skin from obese subjects divided by ethincity is shown in Fig a06a No clear differences in gene expression in the skin by ethnicity were found in nonobese subjects In contrast gene expression clearly differed between obese African American and obese European American subjects Fig a06a and also is illustrated in the PCA plot Fig a06b with PC1 responsible for the greatest variation The pattern of differences between obese and non “obese skin is further illustrated in Fig a06cA list of genes whose expression significantly differed between the obese African Americans and the obese European Americans is shown in Supplemental Tables a0S6 and S7 The expression of SLC6A4 a serotonin transporter CORIN and COL8AI a collagen gene encoding a protein that is dysregulated in atopic eczema was higher in African Americans while the expression of SCCB2A2 the secretoglobin expressed in skin sweat glands and CFTR was expressed higher in European AmericansComparing the skin of obese African Americans to obese European Americans by QTPCR the former showed significantly lower expression of MYBCPI 516fold and PROM1 43fold and CFTR Fig a0 In contrast there was a small increase in the expression of CORIN 22fold a gene encoding the atrial naturalistic peptide converting enzyme and BMP2 fold also present in the skin compared to obese European AmericansPathway analysis found no racerelated differences in the nonobese samples However in the obese Supplemental Fig a0S4 there was markedly reduced expression of oestrogen mediated sphase entry pathway aryl hydrolase receptor signalling pathway and cell cycle regulation through cyclins pathways in the African Americans compared to the skin of the European American groupSubdermal fat in African Americans exhibited few differences from that found in European Americans Fig a08A Figure a08bc show the differences by weight and by ethnicity respectively illustrating the impact of obesity in the two racial groups The expression of numerous inflammatoryimmune genes was upregulated in the fat of both groups of obese subjects Fig a0 Tables a0S5 and S6 Microbiota analysis We next examined whether the microbiota collected from skin swabs around the biopsy site differed between the obese and nonobese subjects We generated taxonomic profiles for each sample using KrakenUniq and a database built from all available microbial species in RefSeq We measured the total number of AMR genes detected in each sample by aligning reads to MegaRESOverall differences between groups were minor No significant differences were noted in average taxonomic alpha diversity as measured by either Shannon™s entropy or richness between the groups A PCA plot of the taxonomic profiles showed slight separation between obese and lean samples and slightly higher beta diversity for obese samples however these differences were minor Fig a010a The number of antimicrobial resistant AMR Scientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RT PCR between the skin of obese and nonobese subjects LS means of gene expression by RTPCR showing significant differences as p p p Figure a0 Differences in gene expression between the subdermal fat of obese and nonobese subjects A Heat map of the most differentially expressed genes in subdermal fat of obese and nonobese subjects with an FCH fdr B PCA plot of differentially expressed genes in subdermal fat of obese and nonobese subjects with an FCH fdr Scientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RTPCR between the subdermal fat of obese and nonobese subjects LS means of gene expression by RTPCR showing significant differences as p p p Figure a0 Differences in gene expression between the skin of African American and European American subjects A Heat map of the most differentially expressed genes in skin of African American and European American subjects with an FCH fdr B PCA plot of differentially expressed genes in skin of obese and nonobese African American and European American subjects with an FCH Left side of plot indicates differences in gene expression by ethnicity in nonobese subjects Right side of plot indicates differences in gene expression by ethnicity in obese subjects C PCA plot of differentially expressed genes in skin of obese and nonobese subjects by ethnicity with an FCH Left side of plot indicates differences in gene expression between obese and nonobese African American subjects Right side of plot indicates differences in gene expression between obese and nonobese European American subjectsScientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RT PCR between the skin of obese and nonobese African American and European American subjects LS means of gene expression by RTPCR showing significant differences as p p p genes identified was higher on average from obese subjects but this difference did not reach significance p Wilcox test Fig a010bDifferentially abundant taxa At the given sample size n no taxa were identified as significantly differentially abundant after Benjamini“Hochberg correction Before correction five taxa were significantly differentially abundant at p Wilcox test These five taxa were Corynebacterium aurimucosum Corynebac‘terium jeikeium Corynebacterium urealyticum Streptococcus salivarius and Streptococcus sp A12 All five taxa were more abundant in samples from obese subjects on averageMetabolome analysis As highlighted by the PCA analysis no variation in the metabolites analysed by liquid chromatographymass spectrometry from the skin swabs was observed between the obese and nonobese individuals PCA Supplemental Fig a0S6 Similarly no ethnicityrelated metabolic variation was observed These results were further confirmed by the poor predictive ability of the orthogonal projection to latent structuresdiscriminant analysis OPLSDA models comparing the two different ethnic groupsDiscussionFew comprehensive reviews of skin changes occurring with obesity have been conducted despite over of the US population being obese27 A broad review of the physiologic and clinical consequences and associations was published by Hirt et a0al in The authors include a discussion of circulatory and lymphatic changes which may enhance the frequency and severity of skin ulceration and provide a comprehensive review of skin disorders that can be associated with obesity expanding on previous reviews29 and studies in rodents30In our study the thickness of

Thyroid_Cancer

range of diseases including malignancies and autoimmune disordersIts high eï¬ectivenessprice ratio also won extensive application in ophthalmology On the other hand although MTX has anexcellent pharmacological efficacy MTX associated side eï¬ects in clinical use which vary from patient to patient are nonnegligible ere is no comparatively systematic review on MTX associated side eï¬ects and its risk factors is review aimed toreveal novel clinical approaches of MTX and its adverse eï¬ects in order to provide a reference for ophthalmic scholars in clinicalapplication of MTX IntroductionMethotrexate MTX is an antifolate metabolite that inhibitsDNA synthesis repair and cellular replication It was firstlyused as one of the essential treatments of pediatric leukemia[ ] According to previous studies MTX has also beenused to treat rheumatoid arthritis RA and psoriasis as anti‚ammatory and immunomodulatory agent [] as MTXcould not only optimize the efficacy of biological diseasemodifying antirheumatic drugs DMARDs [ ] but alsomake the therapeutic goals via lower doses in comparisonwith other conventional synthetic DMARDs [] Figure shows the pathway of folate in DNA synthesis the cellularpathway of MTX and how MTX works inside the cell Whileimmediate and lowdose MTX is used to treat nonmalignantand immunemediated disorders highdose MTX HDMTX more than mgm2week is widely used to treatmalignancies Until now HDMTX with or without radiation therapy is still the backbone of most modern chemotherapy regimens [] as well as the prevention ofsystemiccentral nervous system CNS lymphoma recurrence at a dose of gm2 per week []MTX has also been widely applied in ophthalmic diseases systemically and locally Recently published spay more attention to new clinical applications routes ofadministration and newly discovered side eï¬ects which arefoci of this review Clinical Applications in OphthalmologyAs one of the known corticosteroidssparing agents MTXhas been widely used in the treatment of anterior intermediate posterior or pan uveitis scleritis and ocularmucous membrane pemphigoid [] as well as advancedproliferative diabetic retinopathy [] However followedresearches reveal that MTX works with a significant difference in eï¬ectiveness ratings by anatomic location of‚ammation [] with treatment success achieved mostcommonly in patients with anterior uveitis and scleritis []In the treatment of noninfectious intraocular ‚ammationoral and intravenous are the most common routes with ausual dose range of mg to mg weekly e typical doseobserved was mgweek [ ] which is in the range oflowdose MTX e median time to achieve the success of 0cJournal of OphthalmologyFigure e cellular pathway of folate and MTX Dietary folate enters the cells through RFC1 as well as MTX In lowdose MTX treatmentMTX inhibits enzymes of the folate pathway Ultimately MTX leads to an increase in intracellular adenosine level which would cause anti‚ammatory eï¬ects RFC1 � reduced folate carrier ABC family � adenosine triphosphatebinding cassette ABC family DHF � dihydrofolate THF � tetrahydrofolate GGH � cglutamyl hydrolase FPG � folylpolyglutamate synthase MTXPG � methotrexate polyglutamate DHFR � dihydrofolate reductase MTHFR � methylene tetrahydrofolate reductase AICAR � aminoimidazole carboxamideribonucleotide ATIC � AICAR transformylasetreatment defined as control of ‚ammation with theability to taper corticosteroids to mg or less daily rangesfrom months to months for MTX [ ]Intravitreal MTX injection with or without systemicchemotherapy and radiotherapy has already been used totreat primary intraocular lymphoma patients [ “]According to Larkin [] intravitreal MTX injectioncould achieve remission in a proportion of patients withprimary intraocular lymphoma What is particularly noteworthy is that although MTX has a slow rate of onset ofeï¬ect when it was used to treat intraocular lymphoma viaintravitreal injection it prolonged local remission of oculardisease even with an aggressively growing tumor []erefore it has been taken as a relatively firstline choice forthe treatment of recurrent intraocular lymphoma [] although the treatment for primary intraocular lymphoma islacking solid justification because of the limited retrospectiveand prospective case series [] Local treatment via intraocular injection provides a consistent therapeutic MTXconcentration to reduce the systemic MTX associated sideeï¬ects [] erefore intraocular MTX injection is worthtrying especially for unilateral ocular diseases New Approaches of Applications of MTX MTX Used against Epithelial Downgrowth Previousstudies have already demonstrated safety of intravitrealMTX [] It has been used to treat intraocular lymphomaand proliferative vitreoretinopathy because ofits antiproliferative properties [] ere is a novel use of intravitreal MTX for recurrent epithelial downgrowth which wasnot treated by surgical and medical methods Lambert et al[] administered intravitreal MTX to patients with refractory proliferative membrane after cataract surgery whilemembrane peel and endolaser treatment failed e injectionof MTX was administered alone based on previous protocols and the presumed halflife of drugs in vitreous cavityAfter injections totally there was no membrane recurrence is case suggests that intravitreal MTX plays a role intreatment against epithelial downgrowth MTX Used in Conventional erapyResistant DiseasesGenerally the antivascular endothelial growth factor antiVEGF therapy has dramatically improved the prognosis ofneovascular agerelated macular degeneration nAMDHowever there are still some patients who remain refractoryto antiVEGF therapy which is termed as treatmentresistant nAMD As there is evidence that MTX has eï¬ects ininterrupting the angiogenesis cascade at various levels []Kurup oï¬ered intravitreal MTX to patients who wererefractive to standard antiVEGF therapy [] Although itwas an oï¬label use the patients™ visual acuity improved atfollowup visit while ophthalmic imaging examinationsshowed significantly reduced cystoid macular edema usFolatefolic acidRFC 1Folatefolic acidDHFTHFDHFR5MTHFMTHFRPurine and pyridine synthesis DNA synthesisMTXMTXABC familyFPGGGHMTXFGAICAR5FormylAICARATICAdenosine †‘AdenosineCell membraneCell membrane“““ 0cJournal of Ophthalmologypatients who are refractory to traditional antiVEGF therapymight benefit from intravitreal injection of MTXis approach is not alone Khalil [] had μg01 ml of MTX intravitreal injection once monthlyadministrated to adult Behcet™s disease BD patientssuï¬ering from BDassociated ocular ‚ammation withposterior segment involvement eir results prove thatintravitreal MTX improves visual acuity reduces posteriorsegment manifestations associated with Behcet™s disease andallows the reduction of corticosteroids and immunosuppressive drugs [] ese results also supported Taylor andassociates who conducted trials on patients with unilateral uveitis andor cystoid macular edema [] eirclinical trials suggest that intravitreal MTX may help patients with uveitisassociated posterior segment involvementto regain normal anatomical structure and then allowed thereduction of immunosuppressive therapy The Pharmacogenetics of MTXWith molecular sequencing and highthroughput technology large numbers of genetic polymorphisms can now bedetected accurately and rapidly [] Researchers pay moreattention to pharmacogenetics the study of genetic polymorphisms in drugmetabolizing enzymes and the translation of inherited diï¬erences to diï¬erences in drug eï¬ects[] e genes encoding transporting proteins and metabolizing enzymes for MTX are also known to harborfunctionally significant SNPs e SNPs may ‚uence theefficacy of MTX and have been suggested as potential riskfactors for enhanced MTX toxicity even in lowdose regimens based on previous researches []e research of pharmacogenetics of MTX could bedivided into genetic polymorphisms aï¬ecting MTX transport and SNPs that‚uence enzymes in the cellularpathway of MTX []Once taken MTX enters the cell through an activetransport which is mediated by the reduced folate carrier RFC1 e loss of RFC1 gene expression might lead toeï¬ects of uptake and intracellular levels of MTX A G80ASNP of RFC1 was proposed [] making a decreasing [] orincreasing [ ] eï¬ect on intracellular level of MTXerefore a significant association between RFC1 SNPs andMTX toxicity should be considered Chango state thatthese SNPs strongly impact the overall MTX associated sideeï¬ects by resulting in altered cellular MTX concentrationbut with no ‚uence on MTX efficacy [] However someresearchers argue that these SNPs have no definite eï¬ect[] us it remains controversial whether SNPs of RFC1aï¬ect the transport of MTX Moreover Pglycoprotein amembrane transporter that has ‚uences on the dispositionand bioavailability of MTX [] was studied SNPs ofABCB1 including C3435T SNP and C1236T SNP werebelieved to have eï¬ects on the expression of Pglycoprotein[] Gervasini speculate the C1236T SNP of ABCB1aï¬ects the administered doses of MTX and the incidence ofhematological toxicity [] However just like G80A SNPthere are disputes about the ‚uences of these SNPs asdiï¬erent studies had diï¬erent outcomes []Metabolizing enzymes were also being analyzed giventhe critical role of transporters in disposition of MTX and itsactive products as well as the folate metabolism MTXpharmacogenetics mostly focused on the SNPs in theMTHFR gene e present study shows that genetic polymorphisms in the folate metabolic pathway and in MTXtransporters ‚uence the toxicity but not the efficacy of thelowdose MTX treatment in patients with autoimmunediseases [] For example C677T and A1298C are knownin MTHFR gene to result in a lower enzyme activity []Windsor and associates reported that MTHFR A1298C andC677T were associated with MTX related nephrotoxicityand anemia [] ese SNPs might be associated withdecreased activity of methylenetetrahydrofolate reductaseelevated plasma homocysteine levels and altered distribution of folate [] us patients with this genotype weremore vulnerable to potential MTX induced toxicity sincethese reactions above may lead to slower folate metabolismand slower cell repair [] Weisman used univariatelogistic regression to reveal that the MTHFR C677T alsoincreases the occurrence of side eï¬ects in central nervoussystem manifested as headache and lethargy [] HoweverLambrecht argued that MTHFR C667T was not apredictive factor for toxicity [] Berkani found noassociation between A1298C polymorphism and MTXtoxicity [] Interestingly Grabar claimed that thepatients with MTHFR 1298C genotype have a lower risk forMTX toxicity than the carriers of MTHFR 1298A allele []To date the study of pharmacogenetics of MTX continues An increasing number of SNPs have been found to bepossibly associated with the efficacy and toxicity of MTXe newly discovered genotypes include C347G in ATICand ²UTR 28bp repeat and ²UTR 6bp deletion inTYMS which may ‚uence both efficacy and toxicity ofMTX similarly factors that may aï¬ect MTX associatedtoxicity are for example A2756G in MS and A66G in MTRR[ ] e genes and their SNPs that might beassociated with the eï¬ects and side eï¬ects of MTX aresummarized in Table Growing evidences suggest that asingle genetic factor is unlikely to adequately predict theefficacy and toxicity of MTX in polygenic disease such as RAand autoimmune associated ocular disease Given the impactof MTX in several metabolic pathways a complex of multiple risk genotypes examination would help to predict theefficacy of MTX and to identify patients who may haveadverse eï¬ects from MTX administrationTaken together the efficacy and toxicity of MTX mayremain associated with the genetic markers in the patientserefore although this remains a controversial subject it isreasonable to believe that pharmacogenetics may be able topredict who is at risk of MTX associated adverse eï¬ects andmay help in maximizing the benefitrisk ratio of MTX The Side Effects of MTXe doselimiting toxicity of MTX mainly includes hepatotoxicity and nephrotoxicity [“] but mortality hasoften been reported due to either pneumonitis or secondaryinfections [] 0cJournal of OphthalmologyTable Summary of genes and their SNPs which might have possible clinical eï¬ects towards MTXTransportingproteinsABCfamilyGeneRFC1 [“]ABCB1 [ ]ABCC1 []ABCC2 []ABCC4 []MetabolizingenzymesMTHFR [“]ATIC [“]TYMS [ ]GGH [ ]DHFR [ ]MS [ ]MTRR [ ]SNPsG80AC3435TC1236Trs246240Srs3784862A2412GG1249AG1058AC934AC677TA1298CC347G²UTR 28bp²UTR 6bprepeatdeletionC452TC401TT721AC830TA2756GA66GSome experts divided MTX associated pulmonarycomplications into ‚ammatory infectious and lymphoproliferative [] In the authors™ opinion all MTX relatedside eï¬ects can be classified into these three categoriesaccording to the pharmacological eï¬ects of MTXMajor adverse events for MTX are related to the folateantagonism and primarily aï¬ect highly proliferative tissuessuch as bone marrow and gastrointestinal mucosa []Given the immunosuppression eï¬ect of MTX pancyt iawas one of the most frequent severe toxicities of methotrexate [] Meanwhile the risk of developing an infectiousprocess is increased all along the treatment and the severityof the infected disease would be worsen [ ] includingcommon bacterial infections herpes zoster eruptions andopportunistic infections According to previous studies therisk is larger than that with other disease modifying antirheumatic nonbiological drugs DMARDsSecondly the MTX acts as the hapten [] and is likely toreact directly with nucleophilic groups present in proteins ieto combine with endogenous protein [ ] e proteinadducts thus act as an antigenic signal to direct the eï¬ector armof the immune response [] e provoked immune responsesare most commonly type I immediate hypersensitivity andtype III immune complex reactions [] Hypersensitivitypneumonitis is the most common severe and unpredictablecomplication with a mortality of up to almost []Moreover a few studies have shown that longterm MTXuse can lead to lymphoproliferative disorders LPDs in bothnodal sites and extra nodal sites such as the skin lungsepipharynx thyroid gland nasal cavity spleen and kidneysespecially for patients who are positive for EBV infection[“] e reported frequency of EBV positive in MTXassociated LPDs patients is “ [] Although themechanism of onset is not fully understood it is believedPossible clinical eï¬ectsIncreasingdecreasing intracellular MTX levelAï¬ecting efficacy of MTXAï¬ecting the distribution of MTX and incidence of hematological toxicityAssociation with MTX related toxicityLeading to accumulation of MTX to nephrotoxic levelsAssociation with MTX related gastrointestinal toxicityAssociation with MTX related hepatotoxicityAssociation with MTX related hematological toxicityAï¬ecting the toxicity but not the efficacy by resulting in a lower enzymeactivity association with related nephrotoxicity anemia and neurologicside eï¬ectsAï¬ecting efficacy and toxicity of MTXAï¬ecting efficacy and toxicity of MTXAï¬ecting efficacy of MTXAï¬ecting efficacy of MTXAï¬ecting efficacy of MTXAssociation with MTX associated toxicityAssociation with MTX associated toxicitythat the combination of immunodeficiency and the immunosuppressive eï¬ect of MTX has been implicated in thepathogenesis of MTX associated LPDs e World HealthOrganization WHO has classified MTX associated LPDs aslymphoid neoplasms whether iatrogenic or immunodeficiency associated diseases [ ] MTX associated LPDsoften take a spontaneous remission which tends to completemostly within weeks after the discontinuation of MTX[] But there are a few reports showing that the lymphoidneoplasms occur even after stopping using MTX [] e Eï¬ects of Administration Routes Generally the sideeï¬ects of MTX depend on the route of administration Dosedependent [] gastrointestinal side eï¬ects are the mostfrequent side events with orally administered MTX as oraladministration is the most common delivery method[ ] More than of MTX is excreted by therenal system thus MTX associated nephrotoxicity is common among patients taking MTX Fortunately the resolution usually occurs after discontinuation of therapy andsalvage treatment with highdose corticosteroids[]erefore to achieve treatment with less side eï¬ects theappropriate route of administration and dose of MTX arenecessary During the treatment monitoring of patients™general condition mattersAdverse eï¬ects of intravitreal injections of MTX occuronly within the eyeincluding hyperemia keratopathycataract iridocyclitis vitreous hemorrhage retinal detachment maculopathy and endophthalmitis []Splitting doses of MTX rather than intravenous administration is a new attempt to avoid MTX associated sideeï¬ects MTX is split and given twice or thrice in a week toachieve higher bioavailability and better clinical response 0cJournal of Ophthalmology[ ] thus providing us with a novel method of oraladministration of MTX with less adverse eï¬ects Is LowDose MTX Safer Based on clinical cases observation side eï¬ects which can lead to discontinuation ofMTX are rare during the typical ophthalmology treatmentbecause of the lower dose of MTX required [] e application of lowdose MTX regimen has also become one ofthe main therapies of a variety of immunemediated diseasesbecause of its efficacy and an acceptable safety profile asmost lowdose MTX associated toxicity has been describedin case reports and relatively small case series []However although welltolerated and mostly reversibleeven a lowdose regimen of MTX can result in clinicallysignificant toxicity with substantial death rates about according to Kivity™s cohort study [] e lowdose MTXassociated severe adverse eï¬ects include major centralnervous system complications [] mucositis pulmonaryinvolvement hepatotoxicity [] and myelosuppression Is MTX Safe to the Pregnant and Fetuses As one of thelipidsoluble and low molecular weight drugs MTX could bereadily transferred across the placental membrane duringpregnancy and adversely aï¬ect the fetus [] In addition MTXmight take longer time for elimination in fetal tissues []Regarding pharmacogenetics mutations caused by MTXlead to severe decrease of the expression of folate and nucleobaseenzymes which are critical for cellular homeostasis [] Inpractice MTX aï¬ected formation of the blastocyst and causeddysmorphic features and neurologic defects in early pregnancyleading to malformations in some cases [] Multiple congenitalabnormalities have been observed after weekly MTX treatmentsat a mg dose during the first months of pregnancy [] evenfetal death [] Verberne had reviewed cases of congenitalanomalies after in utero exposure to MTX and proved that somecongenital anomalies such as microcephaly craniosynostosistetralogy of Fallot pulmonary valve atresia limb reductiondefects and syndactyly were truly part of the œfetal methotrexatesyndrome [] Administration of MTX in childhood mightalso cause manifestations including visual defect [] andSmith“Magenis syndrome [] among patients with specificmutations us special care should be taken with pregnantpatients and children in particular e Risk Factors of MTX Associated Side Eï¬ects e mostcommon risk facts of MTX induced adverse eï¬ects areadvanced age age years and underlying disease incaused by MTX administration with100 g alcohol concluding renal andor hepatic insufficiency and lung diseaseespecially patients with chronic hepatitis B and diabetesmellitus [“] Patients with a history of alcohol intakemight have a greater risk of liver fibrosis and hepatotoxicitysumption per week [] Also preexisting hypoalbuminemiaand past use of any of the DMARDs and protonpumpinhibitors have been described in studies to increase theincidence of MTX induced side eï¬ects [ ] Moreovertaking drugs that may interact with MTX at the same timemight also be dangerous these drugs include salicylatescotrimoxazole chloramphenicol sulfonamides cyclosporine and pyrimethamine [] Although no significantprotective eï¬ect of folate supplementation on MTX relatedtoxicity has been found [] the folate deficiency is anotherreason for the side eï¬ects based on clinical cases []Heidari found that MTX administration elevatedkidney ROS levels decreased tissue antioxidant capacityincreased lipid peroxidation and depleted renal glutathionestores eir research data indicate that MTX caused tissuedamage and organ dysfunction through oxidative stresserefore they proposed that patients with preexistingmitochondrial defects might be vulnerable to MTX inducedrenal injury []e use of highdose MTX HDMTX is also the riskfactor of adverse eï¬ects MTX induced liver fibrosis is morelikely to become morphologically evident with high cumulative doses possibly largely exceeding to mg[ ] and the side eï¬ects caused by omeprazole use inthe past were found in cancer patients receiving HDMTXtreatment []e distribution of MTX in vivo also plays a role in MTXrelated side eï¬ects As MTX tends to accumulate in theextravascular compartment patients with pleural eï¬usionascites and massive edema should get extra caution due tothe risk of toxicity from reabsorption of extravascular fluid[]Another noteworthy risk factor is UV UV recall phenomenon also known as MTX associated UV reactivationhas been reported [ ] It is reactivity of sunburn areaswithin to days of the treatment with MTX [ ]According to Adams and associates this phenomenon mightbe due to the immune response by uncontrolled sunburninduced ‚ammation released by MTX [] Patients whopreviously suï¬ered sunburns deserve more detailed monitoring when methotrexate is needed Is Folate Supplementation Necessary for OphthalmicPatients To prevent MTX associated side eï¬ectsit iscommon to take folate [as either folic acid FA or folinicacid FLA] in clinic [ ] However there is noconsistent and evidencebased guideline for folate supplementation in ophthalmic patientsFolate and folic acid play significant roles in the de novosynthesis of purines and thymidylate which are required forDNA replication and repair [] Funk and associates founda significant reduction of circulating folate concentrations in of patients receiving MTX treatment [] Patientstreated with highdose MTX HDMTX got routine folatesupplementation to reduce HDMTX associated side eï¬ects[“] After a systematic literature review of HDMTXtherapy and folate supplementation Van der Beek et al[] found lower incidence of MTX associated adverseeï¬ects in regimens with higher cumulative doses and earlieradministration of folate supplementation in similar HDMTX dosage studies Folate supplementation in patientswith lowdose methotrexate is also being studied Ortiz et al[] had proved the protectivefolateeï¬ectof 0cJournal of Ophthalmologysupplementation by conducting a Cochrane review including more than patients taking lowdose MTX Untilnow folate supplementation had been proved to prevent andimprove MTX associated eï¬ects including gastrointestinalrespiratory and neurologic side eï¬ects [ ] Mori et alsupported the protective eï¬ect by demonstrating that patients treated with lowdose MTX without folate supplements were significantly associated with the development ofmyelosuppression and pancyt ia []However Arabelovic and associates™ preliminary studyshowed a significant increase of MTX dose needed []since folic acid fortification enriched cereal grain productswere fully implemented in the USA and Canada [] isconveyed a message to us that high dose of folate supplementation might have ‚uence on the efficacy of MTXAlDabagh found that the reduction in efficacy ofMTX cannot be ignored while folate supplementation didmake a significant reduction in associated adverse eï¬ects[] Salim declared the decreasing ‚uence betweenthe anti‚ammatory eï¬ect of MTX and folate supplementation by carrying out a doubleblind clinical trial []Chladek had conducted an labeltwowaycrossover study supporting the opinion above [] Additionally because of the unequal distribution of folic acidand MTX in organs and tissues [] MTX discontinuationis more common for some MTX associated side eï¬ects inophthalmic clinic [] rather than higher dosage of folatesupplementationere are no ophthalmic studies to demonstrate theprotective eï¬ects of folic acid supplementation us although the folate supplementation is widely used amongpatients treated with lowdose MTX [ ] the necessityand standardized dosage of folate supplementation in specific patients [] as well as the MTXfolate interactionstill warrant further studies DiscussionMethotrexate as one of the alternative pharmacologicalsteroidsparing immunosuppressive agentsis becomingmore and more popular as the preferred treatment in severalautoimmune conditions requiring longterm immunosuppression [] Lowdose MTX has anti‚ammatory andimmunomodulatory properties by increasing levels of intracellular and extracellular adenosine [] which is thefoundation of ophthalmic MTX treatment e standardizedand recommended administration of ophthalmic MTXtreatment is once a week starting with a dose of mg andescalating every to weeks up to “ mgweek whennecessary [ ] In patients with insufficient response toMTX alone cyclosporin with or without azathioprine wasadded []To avoid side eï¬ects split doses of MTX administrationand folate supplementation are gradually being used inophthalmic clinic Prescription of to mg of folatesupplementation has a significant role in MTX safety []but the higher dosage is less applied even with higher dose ofMTX [] Prophylactic folate supplementation is notnecessary in most patients [] ere is also research toconvey that ml100 g or above dosage of fish oil is aseï¬ective as folinic acid in therapeutic potential in preventingbone loss during MTX chemotherapy [] For some resistant andor mortal adverse eï¬ects the discontinuation ofMTX will work instantlyWith the increasing longterm use of MTX it is importantto monitor patients™ blood examination results including bloodroutine and liver and renal functions As pancyt ia can be alate manifestation [] elevation of urea creatinine aminotransferases and albumin as well as electrolytes disturbancesmay result in MTX associated liver and renal side eï¬ects []Plasma MTX level is not a reliable predictor for adverse eventsin MTX therapy [] On the contrary circulating folate levelsand folate polyglutamate distribution change sensitively withMTX exposure and exogenous folate supply [] and could beused as a biomarker of MTX efficacy [] It should be notedthat as erythrocytes have a halflife of approximately daysthe results of blood examinations might reflect both pretreatment and posttreatment status which need to be analyzedcarefully []Numerous studies had been conducted to prove thatMTX could be used as a welltolerated safe and eï¬ectivefirstline treatment Hence the MTX administration shouldnot continue to be stigmatized as a œcancer drug or to bediscouraged because of associated adverse eï¬ects Contrarily the indication and the routes of administration areabout to gradually widenConflicts of Intereste authors declare that they have no conflicts of interestReferences[] R Q H Kloos R Pieters C van den Bos œe eï¬ect ofasparaginase therapy on methotrexate toxicity and efficacy inchildren with acute lymphoblastic leukemia Leukemia Lymphoma vol no pp “ [] R K Bath N K Brar F A Forouhar and G Y Wu œAreview of methotrexateassociated hepatotoxicity Journal ofDigestive Diseases vol no pp “ [] W Wang H Zhou and L Liu œSide eï¬ects of methotrexatetherapy for rheumatoid arthritis a systematic review European Journal of Medicinal Chemistry vol pp “[] J Smolen and R Landew´e œEULAR recommendations forthe management of rheumatoid arthritis with synthetic andbiological diseasemodifying antirheumatic drugs update Annals of the Rheumatic Diseases vol no pp “ [] J A Singh K G Saag S L Bridges œ Americancollege of rheumatology guideline for the treatment ofrheumatoid arthritis Arthritis Rheumatology vol no pp “ [] M Holdhoï¬ P Ambady A Abdelaziz œHighdosemethotrexate with or without rituximab in newly diagnosedprimary CNS lymphoma Neurology vol no pp “ [] J Pe™er J M Rowe S Frenkel and E J Dann œTesticularlymphoma intraocularvitreoretinal lymphoma and brainlymphoma involvement of three immunoprivileged sites in 0cJournal of Ophthalmologyone patient American Journal of Hematology vol no pp “ [] S Gangaputra C W Newcomb T L Liesegang et alœSystemic immunosuppressive therapy for eye diseases cohort study methotrexate for ocular ‚ammatory diseasesOphthalmology vol no pp “ [] P W Hardwig J S Pulido J C Erie K H Baratz andH Buettner œIntraocular methotrexate in ocular diseasesother than primary central nervous system lymphomaAmerican Journal of Ophthalmology vol no pp “ [] E Esterberg and N R Acharya œCorticosteroidsparingtherapy practice patterns among uveitis specialists Journalof Ophthalmic Inflammation and Infection vol no pp “ [] K Durrani F R Zakka M Ahmed M MemonS S Siddique and C S Foster œSystemic therapy withconventional and novel immunomodulatory agents for ocular ‚ammatory disease Survey of Ophthalmology vol no pp “ [] S S Gangaputra C W Newcomb M M Joï¬e et alœComparison between methotrexate and mycophenolatemofetil monotherapy for the control of noninfectious ocular‚ammatory diseases American Journal of Ophthalmologyvol pp “ [] V K Ayuso E L van de Winkel A Rothova and J Helenade Boer œRelapse rate of uveitis postmethotrexate treatmentin juvenile idiopathic arthritis American Journal of Ophthalmology vol no pp “ [] S R Rathinam M Babu R undikandy œA randomized clinical trial comparing methotrexate and mycophenolate mofetil for noninfectious uveitis Ophthalmologyvol no pp “ [] A Galor D A Jabs H A Leder œComparison ofantimetabolite drugs as corticosteroidsparing therapy for‚ammation Ophthalmologynoninfectiousvol no pp “ ocular[] M D de Smet V S Vancs D Kohler D Solomon andC C Chan œIntravitreal chemotherapy for the treatment ofrecurrent intraocular lymphoma British Journal of Ophthalmology vol no pp “ [] E Kim C Kim J Lee and Y Cho œA case of primaryintraocular lymphoma treated by intravitreal methotrexateKorean Journal of Ophthalmology vol no pp “[] J Smith J T Rosenbaum D J Wilson œRole ofintravitreal methotrexate in the management of primarycentral nervous system lymphoma with ocular involvementhistorical image Ophthalmology vol no pp “ [] CC Chan and D J Wallace œIntraocular lymphomaupdate on diagnosis and management Cancer Controlvol no pp “ [] K L Larkin U S Saboo G M Comer œUse ofintravitreal rituximab for treatment of vitreoretinallymphoma British Journal of Ophthalmology vol no pp “ [] C P Fox E H Phillips J Smith œGuidelines for thediagnosis and management of primary central nervoussystem diï¬use large Bcell lymphoma British Journal ofHaematology vol no pp “ [] A Sadaka R Sisk J Osher O Toygar M Duncan andinfusion forœIntravitreal methotrexateC Riemannproliferative vitreoretinopathy Clinical Ophthalmologyvol pp “ [] N G Lambert DJ Wilson D M Albert andW D Chamberlain œIntravitreal methotrexate for recurrentepithelial downgrowth JAMA Ophthalmology vol no p [] A M Joussen F E Kruse HE V¨olcker and B KirchhofœTopical application of methotrexate for inhibition of cornealangiogenesis Graefe™s Archive for Clinical and ExperimentalOphthalmology vol no pp “ [] S K Kurup C Gee and C M Greven œIntravitrealmethotrexate in therapeutically resistant exudative agerelated macular degeneration Acta Ophthalmologica vol no pp e145“e146 [] H E M Khalil H A Raafat N A Azab H E Haroun andH A Elgendi œe role of intraocular methotrexate in themanagement of uveitis and posterior segment involvementin Behçet™s disease patients e Egyptian Rheumatologistvol no pp “ [] S R J Taylor A Banker A Schlaen œIntraocularmethotrexate can induce extended remission in some patients in noninfectious uveitis Re

Thyroid_Cancer

"ligandactivated transcriptional factors that belong to the nuclear receptor superfamily Among them PPAR alpha andPPAR gamma are prone to exert an antiangiogenic eï¬ect whereas PPAR betadelta has an opposite eï¬ect in physiological andpathological conditions Angiogenesis has been known as a hallmark of cancer and our recent works also demonstrate thatvascularspecific PPAR betadelta overexpression promotes tumor angiogenesis and progression in vivo In this review we willmainly focus on the role of PPAR betadelta in tumor angiogenesis linked to the tumor microenvironment to further facilitatetumor progression and metastasis Moreover the crosstalk between PPAR betadelta and its downstream key signal moleculesinvolved in tumor angiogenesis will also be discussed and the network of interplay between them will further be established inthe review IntroductionPeroxisome proliferatoractivated receptorsPPARs asligandactivated transcription factors belong to the steroidreceptor superfamily which includes three isoforms PPARalpha PPAR betadelta and PPAR gamma [] PPARs formheterodimers with retinoic X receptors and regulate theexpression of various genes upon ligand binding PPARs alsointeract with corepressors or coactivators to modulate thetranscription of its downstream target genes PPARs asimportant transcriptional regulators have been suggested tobe involved in lipid metabolism and multiple cellular functions For instance PPAR alpha also functions in fatty acidbetaoxidation and vascular ‚ammation [] PPAR gammaacts as a regulator in adipocyte diï¬erentiation and type diabetes [] PPAR betadelta is a key player in cardiac energyproduction angiogenesis and particularly in cancer progression []PPAR alpha and PPAR gamma exert predominantly anantiangiogenic eï¬ect [“] but there still exist conflictingstudies showing opposite results [ ] On the contraryPPAR betadelta produces more obviously proangiogeniceï¬ects [“] In this review we will focus on the promotingrole of PPAR betadelta in angiogenesis especially in tumorangiogenesis The network of interplay between PPAR betadelta and its various downstream signal molecules and alsobetween those key molecules will be further discussed andestablished Remarkably diverse important signal moleculesinvolved in tumor angiogenesis and progression and cancercell metabolism have been identified as direct PPAR betadelta target genes AngiogenesisAngiogenesis is the physiological process through which anew capillary network forms from the preexisting vasculature[ ] whereas vasculogenesis denotes de novo bloodvessel formation mostly during embryogenesis in whichendothelial progenitor cells EPC migrate to sites of vascularization then diï¬erentiate into endothelial cells EC andcoalesce into the initial vascular plexus [ ] Besides theinteraction between proangiogenic factors and antiangiogenic factors angiogenesis is also a multiple step biologicalprocess during which a variety of molecules cooperateincluding cell adhesion molecules matrix metalloproteinases 0cPPAR ResearchMMPs extracellular matrix ECM and basement membrane componentsAngiogenesis is a physiological and vital process in development and growth An imbalance of proangiogenic andantiangiogenic factors causes angiogenesis in pathologicalconditions such as diabetic retinopathy and tumor growthThus when the imbalance comes to a point at which angiogenesis is triggered by tumor cells then an œangiogenicswitch of tumor cells is turned on during tumor progression the œangiogenic switch is often activated and remainson [“] Inducing angiogenesis is known as a hallmarkof cancer [] and angiogenesis is also a fundamental stepby which most benign tumors transition into malignant ones Tumor Angiogenesis Tumor needs to sprout new vesselsand further develop a vascular network in order to supplynutrients and oxygen remove waste products support a continually high proliferative rate and ultimately expand neoplastic growth [ ] Hence angiogenesis is essential forhelping sustain tumor growth and facilitate tumor progression Besides being a requirement for angiogenesis an abnormal vasculature also helps to promote tumor progression andmetastasis The tumor vascular wall is imperfect and prone toleakage so it is much easier for tumor cells to directly penetrate into the blood vessels or lymphatic vessels and then proliferate at another distant site to form metastasis []Due to intensive abnormal neovascularization in tumortissues most malignant tumors grow rapidly and acquirethe ability to spread to adjacent and distant ans whichmakes them more malignant and even life threateningTherefore angiogenesis indeed plays an important role intumor progression and metastasis and to intervene with thisprocess would obviously prevent tumor development andspread Thus this has been regarded as a critical target forantitumor therapy PPAR Alpha and AngiogenesisIt was reported firstly that a selective PPAR alpha agonistWY14643 did not show any eï¬ect on angiogenesis or EC proliferation [] But some subsequent studies showed that theactivation of PPAR alpha inhibited angiogenesis in vitro byusing fenofibrate a clinically used PPAR alpha agonist []Moreover fenofibrate suppressed EC proliferation migration and tube formation through inhibition of protein kinaseB Akt and disruption of the cytoskeleton [] Furthermore PPAR alpha activation was shown to inhibit vascularendothelial growth factor VEGF induced EC migrationand basic fibroblast growth factor bFGFFGF2 inducedcorneal angiogenesis in vitro and in vivo [] Especiallyin vivo reduced tumor growth and microvessel numberswere observed in mice implanted with melanoma Lewis lungcarcinoma LLC fibrosarcoma and glioblastoma due to asystemic treatment of PPAR alpha ligand and the antiangiogenic state induced through activation of PPAR alpha withelevated thrombospondin1 TSP1 and endostatin expression []Howeverit was demonstrated inanother observation that activation of PPAR alpha stimuin that same yearlated neovascularization in vivo with increased phosphorylation of endothelial nitric oxide synthase eNOS and Akt via aVEGFdependent manner [] Furthermore Zhang andWard also suggested that PPAR alpha activation inducedproangiogenic responses in human ocular cells [] Inanother study it was shown that a new PPAR alpha agonistRK13675 had no eï¬ect on angiogenesis [] RecentlyPPAR alpha activation is further shown to have antineovascularization eï¬ects with downregulation of VEGF and angiopoietin expression in a rat alkali burn model []In summary the role of PPAR alpha in angiogenesis isstill controversial Some observations showed that ligandactivation of PPAR alpha had antiangiogenic eï¬ects mediated either through upregulation of antiangiogenic factorssuch as TSP1 and endostatin or downregulation of proangiogenic factors including VEGF FGF2 AKT and angiopoietins Others also reported opposite results showing aproangiogenic role upon PPAR alpha activation Thus thespecific molecular mechanism is still unclear and needs tobe further studied PPAR Gamma and AngiogenesisLigand activation of PPAR gamma was previously shown toinhibit human umbilical vein endothelial cell HUVEC tubeformation in collagen gels [] and VEGFinduced choroidalneovascularization in vitro and in vivo [] Another studyalso demonstrated that EC apoptosis was induced throughtreatment with the PPAR gamma ligand 15dPGJ2 [] Furthermore rosiglitazone a potent PPAR gamma agonist wasshown to inhibit primary tumor growth and metastasisthrough both direct and indirect antiangiogenic eï¬ectsin vitro and bFGFinduced corneal neovascularizationin vivo [] Moreover a similar observation also displayedthe inhibition of VEGFinduced angiogenesis in a chickchorioallantonic membrane model [] In a mouse modelwith ischemiainduced retinopathy pioglitazone a PPARgamma agonist also showed a protective eï¬ect against pathological neoangiogenesis through upregulation of anti‚ammatory adipokine adiponectin [] Additionally thePPAR gamma antagonist GW9662 was shown to reverseOmega3 polyunsaturated fatty acidinduced reduction ofESelectin angiopoietin2 vascular cell adhesion molecule and intracellular adhesion molecule1 [] implicatingan antiangiogenic potential of PPAR gamma itself Howeveropposite results also showed that pioglitazone enhanced neovascularization and inhibited apoptosis of EPC in vitro andin vivo via a Phosphoinositide3Kinase PI3K dependentmanner []Nadra observed that PPAR gammanull embryosdisplayed a vascular structural defect at E95 Moreover disanized placental layers and an altered placental microvasculature were observed in pregnant wildtype mice treatedwith the PPAR gamma agonist rosiglitazone as well asreduced expression of proangiogenic factorsincludingVEGF proliferin and plateletendothelial cell adhesionmolecule1 PECAM1CD31 [] suggesting a crucial roleof PPAR gamma in placental vascular development The 0cPPAR Researchmajor antiangiogenic properties on PPAR gamma activationwere also reviewed here []Notablyin most cancersthe canonical Wntbetacatenin pathway is upregulated while on the contrary PPARgamma is downregulated Interestingly in numerous tissuesthe activation of PPAR gamma inhibits the betacateninpathway whereascanonicalWntbetacatenin signal cascade also inactivates PPARgamma [] implicating a negative regulatory role of PPARgamma in carcinogenesis where tumor angiogenesis mightbe a fundamental stepstimulation ofthetheIn summary PPAR gamma predominantly displays anantiangiogenic eï¬ect that may be mediated through the inhibition of VEGF or bFGFinduced neovascularization andreduction of the expression level of some proangiogenicfactors PPAR BetaDelta and AngiogenesisUnlike PPAR alpha and PPAR gamma on the contrarymany studies have explicitly shown the proangiogenic eï¬ectsof PPAR betadelta on physiological and pathological angiogenesis The first evidence provided in a study is that activation of PPAR betadelta with GW501516 a highly selectivePPAR betadelta agonistinduces HUVEC proliferationand an increased expression of VEGF and its receptorVEGFR1 FLT1 [] Besides inducing EC proliferationPPAR betadelta activation by itsligand prostacyclinPGI2 also stimulates upregulation of alpha expression an antiapoptotic and anti‚ammatory protein whichthereby protects ECs from H2O2induced apoptosis and oxidant injury [] Moreover a subsequent study further provides evidence that activation of PPAR betadelta withGW501516 induces angiogenesis during which VEGFrelease is considered as a major trigger factor [] firstlysuggesting the promotion for angiogenesis upon PPARbetadelta activationMüllerBrüsselbach show that PPAR betadelta mice implanted with LLC and B16 melanoma exhibit diminished blood flow and immature microvascular structurescompared with wildtype mice Moreover reexpression ofPPAR betadelta into the matrigelinvading cells triggersmicrovessel maturation and restores normal vascularization[] indicating a crucial role of PPAR betadelta in tumorvascularization Additionally another study also observedreduced levels of calcium intracellular channel protein CLIC4 but it observed enhanced expression of cellular retinol binding protein CRBP1 in migrating ECs from PPARbetadeltanull mice [] both of which play a role in tumorvascularization [ ] It was reported that PPAR betadeltawas required for placentation [] and most of the PPARbetadeltanull mutant embryos died at E95 to E105 due toabnormal celltocell communication atthe placentaldecidual interface [] However in these studies [“] adefect in angiogenesis was not observed during normal development in PPAR betadeltaknockout miceSome observations also show the important role of PPARbetadelta in physiological angiogenesis For instance skeletal musclespecific PPAR betadelta overexpression leads toPPAR betadeltaan increase in the number of oxidative muscle fibers andrunning endurance in adult mice [“] Moreover PPARbetadelta activation promotes a rapid muscle remodelingvia a calcineurindependent manner and induces muscleangiogenesis in highly selective PPAR betadelta agonistGW0742treated animals [] Furthermore in the heartpharmacologicalstimulation withGW0742 induces rapid cardiac growth and cardiac angiogenesis through direct transcriptional activation of calcineurin [] Interestingly the same cardiac phenotype wasalso observed after treatment with the PPAR betadelta agonist GW501516implicating a response specificity forPPAR betadelta stimulation [] Calcineurin activationfurther leads to the stimulation of nuclear factoractivatedT cell c3 NFATc3 and an enhanced expression of hypoxiainducible factor alpha HIF1alpha and cyclindependentkinase CDK9 [] Overall the remodeling in skeletalmuscle and heart is perfectly the same as the phenotypeobserved with exercise and both of them are mediatedthrough activation of calcineurinPPAR betadelta may act as a key regulator in mediatingpathological angiogenesis For instance PPAR betadelta wasshown to regulate retinal angiogenesis in vitro and in vivoand its inhibition reduced preretinal neovascularization possibly via an Angiopoietinlike protein Angptl4 dependent manner []implicating the potential of PPARbetadelta in modulating pathological ocular angiogenesisRecently an observation reported that PPAR betadeltaknockdown in both retinal pigment epithelial and choroidalendothelial cells caused an antiangiogenic phenotype andPPAR betadelta promoted laserinduced choroidal neovascular CNV lesions in PPAR betadelta mice [] Moreover pharmacological inhibition of PPAR betadelta with theantagonist GSK0660 also resulted in a significantly decreasedCNV lesion size in vivo suggesting a functional role of PPARbetadelta in the development of CNV lesions [] This indicates that PPAR betadelta has an important association withpathological angiogenesisAngiotensin II Ang II the biologically active peptide ofthe reninangiotensin system RAS is a major blood pressure and cardiovascular homeostasis regulator and is alsorecognized as a potent mitogen Angiotensinconvertingenzyme inhibitors were introduced approximately yearsago as antihypertensive agents and have since become asuccessful therapeutic approach for high blood pressurecongestive heart failure and postmyocardial infarction Inexperimental systemsthe antitumor eï¬ects of diverseACE inhibitors show that these inhibit cell proliferationand possessand anti‚ammatory eï¬ects [“] It has been shown recentlythat activation of PPAR betadelta inhibits Ang IIstimulated protein synthesis in a concentrationdependentmanner and suppresses Ang IIinduced generation of reactive oxygen species ROS in vascular smooth muscle cells[] PPAR betadelta was further shown to inhibit AngIImediated atherosclerosis [] However it is not clearuntil now if PPAR betadelta activation can be consideredis foras an ACE inhibitormimicking approach as itexample the case for PPAR gamma activators[]antiangiogenicantimetastatic 0cPPAR Researchthe relevance ofFurthermorethis hypothetical PPARbetadelta feature might be limited for tumor angiogenesiswhere vascular smooth muscle hypertrophy and atherosclerosis do not contribute to the major pathologyBesides inducing angiogenesis it has been demonstratedthat PPAR betadelta directly acts on early EPC through activation of the AKT pathway and induces an enhanced vasculogenesis [] Similarlythe PPAR betadeltamediatedprovasculogenic eï¬ects are also observed on late EPC []He showed that PPAR betadelta activation withGW501516 induced EPC proliferation and tube formationwhereas EPC treated with an inhibitor of cyclooxygenaseCOX or PGI2 synthase or with PPAR betadeltaspecificsiRNA also displayed an opposite eï¬ect [] Furthermoreit has been demonstrated that PPAR betadelta inducesangiogenesis and skeletal muscle regeneration throughmatrix metalloproteinase MMP 9mediated insulinlikegrowth factor1 paracrine networks upon EPC activation[] Han also observed that PPAR betadelta activationpromoted a rapid wound healing with enhanced angiogenesis in a mouse model with skin punch wound [] Overallin addition to EC PPAR betadelta is also a key regulator ofEPC or even may act as an initiator of activation of EPC tofurther induce vasculogenesis PPAR BetaDelta and Tumor AngiogenesisLinked to Tumor MicroenvironmentPPAR betadelta expression is often upregulated and promotes cancer progression in many major human cancerslung breast and gastric cancers [“]such as colonwhich suggests a crucial role of PPAR betadelta in cancercells even though there exist some conflicting studies indicating that the functional role of PPAR betadelta in tumorigenesis or carcinogenesis still remains highly controversial [“] and dependent on specific tumor or cancer cell typesThus here we discuss the promotion of PPAR betadelta intumor progression through facilitating tumor angiogenesisPPAR betadelta has been suggested as a critical œhubnode transcriptional factor which governs a tumor œangiogenic switch [ “] In the transcriptional networkanalysis it was reported that tumor growth and tumor angiogenesis were markedly inhibited in PPAR betadeltanullmice in comparison with wildtype mice [] Moreoverthe elevated PPAR betadelta expression level was also considered to be highly correlated to pathologically advancedtumor stage and increased cancer risk for recurrence and distant metastasis in patients with pancreatic cancer [] indicating the crucial association of PPAR betadelta withtumor angiogenesis progression and cancer invasivenessPPAR betadelta may indirectly facilitate tumor angiogenesis and progression through its function on the tumormicroenvironment TME where tumor angiogenesis is fostered Moreover a tumor also releases some extracellular signals to closely communicate and constantly collaborate withTME to facilitate tumor angiogenesis in order to furtherenable tumor growth and progression For instance it wasshown that colon cancer cells with PPAR betadelta knockoutfailed to stimulate EC vascularization in response to hypoxicstress whereas wildtype cells exposed to hypoxia were ableto induce angiogenesis [ ] suggesting that PPAR betadelta is required for the promotion of angiogenesis in hypoxic stressmediated TME Moreover in the TME tumorltrating myeloid cells are considered as the most important cells for fostering tumor angiogenesis among the multiple diï¬erent kinds of stromal cells [] Besides stimulatingtumor angiogenesis tumor myeloid cells also support tumrowth by suppressing tumor immunity and promotingtumor metastasis to distinct sites [] Interestingly it hasbeen demonstrated that PPAR betadelta activation intumorltrating myeloid cells stimulates cancer cell invasion and facilitates tumor angiogenesis via an Interleukin IL10 dependent manner [] Moreover impairedtumor growth and angiogenesis were observed in PPARbetadelta KO BMT mice due to PPAR betadelta deficiencyin tumor myeloid cells [] suggesting that PPAR betadeltaplays a key role in tumor angiogenesis and progression intumor myeloid cells of TMEFurthermore the endoplasmic reticulum ER an essential anelle involved in many cellular functions is implicated in TME In cancer stressors like hypoxia nutrientdeprivation and acidosis disrupt ER function and lead toaccumulation of unfolded proteins in ER a condition knownas ER stress Cells adapt to ER stress by activating an integrated signal transduction pathway called the unfolded protein response UPR UPR represents a survival response bythe cells to restore ER homeostasis and has both survivaland cell death eï¬ects The mechanisms that determine cellfate during ER stress are not well understood For instanceshort exposure to ER stress initially increases AKT signalingbut longterm ER stress suppresses AKT signaling []PPAR betadelta activation has been shown to reduce endoplasmic reticulum ER stressassociated ‚ammation inskeletal muscle through an AMPKdependent mechanism[] and to reduce ‚ammation in response to chronic ERstress in cardiac cells [] Furthermore it has been nicelyshown that PPAR betadelta can repress RASoncogeneinduced ER stress to promote senescence in tumors [] Thisis mediated through the decrease of pAKT activity promoting cellular senescence through upregulation of p53 and p27expression [] It would be interesting to investigate thedirect eï¬ects of PPAR betadelta on senescence of tumorendothelial cells in an in vivo setting We recently showedthat senescent endothelial cells are indispensable for ahealthy lifespan and that removal of senescent endotheliumdisrupts vascular function leading to diminished vessel densities and fibrotic lesions [] If PPAR betadelta mediatessenescence of tumor endothelium thereby protecting vesselintegrity this might explain the enhanced tumor growthand vascularization upon PPAR betadelta activationobserved by us and others [ ]Most recently Zuo demonstrated that PPARbetadelta in cancer cells regulates tumor angiogenesisin vivo and in vitro by promoting the secretion of proangiogenic factors including VEGF and Interleukin IL8[] Most importantly in our recent works it has beenshown that conditionalinducible vascular endotheliumspecific PPAR betadelta overexpression in vivo leads to 0cPPAR Researchenhanced tumor angiogenesis tumor growth and metastasis formationfurther indicating a vascular ECspecificPPAR betadelta action mechanism in tumor progressionindependent of some controversial observations of PPARbetadelta in specific tumor or cancer cell types [] Wagner also firstly reported the mouse model in whichrapid induction of cardiac angiogenesis and cardiac hypertrophy were observed [ ] Crosstalk between PPAR BetaDelta and SignalMolecules PPAR betadelta activation or overexpressionmay upregulate the expression of its various downstream signal molecules involved in tumor angiogenesis includingproangiogenic factors such as VEGF PDGF and FGFproinvasive matrixdegrading enzymes such as MMP9pro‚ammatory mediators such as COX2 and cytokinesand chemokines such as IL1 and CXCL8 even some ofwhich have been further identified as PPAR betadelta directtarget genes Besides a leading role of PPAR betadelta amongthe signal molecules PPAR betadelta may function in TMElinked to diverse kinds of cells through direct or indirectmodulation of its downstream molecules Interplay between PPAR BetaDelta and InflammatoryAngiogenesis Inflammatory angiogenesis is a crucial processin tumor progression For instance the pro‚ammatorymediator cyclooxygenase2 COX2 is considered as a keyregulator of angiogenesis and tumor growth through multiple downstream proangiogenic mechanisms such as production of VEGF and induction of MMPs Moreover selectiveinhibition of COX2 has also been shown to suppress angiogenesis in vivo and in vitro [] It is well known that VEGFAplays a critical role in both angiogenesis and vasculogenesis[] and it leads the directional migration of tip cells andstalk cell proliferation in microtubule branches [ ] Ithas also been demonstrated that MMP9 triggers the œangiogenic switch during carcinogenesis and enhances the availability of VEGF to its receptors [] Furthermore it hasbeen reported that ‚ammatory cell MMP9 initiates theonset of tumor neovascularization during which there existsfunctionalincludingMMP9 [] LEPTIN is shown to mediate angiogenesisin vivo and in vitro through induction of EC proliferationand expression of MMP2 and MMP9 [] and to furtherpromote EC diï¬erentiation and directional migrationthrough enhancement of COX2 activity [] LEPTIN couldalso induce angiogenesis via transactivation of VEGFR inECs [] Additionally besides inducing angiogenesisPPAR betadelta also functions in chronic ‚ammationfacilitating tumorigenesis through induction of COX2 andits product prostaglandin E2 PGE2 in vivo [ ]Interestingly COX2 VEGF MMP9 and LEPTIN have beenidentified as PPAR betadelta target genes via a direct transcriptional activation mechanism in hepatocellular carcinoma cells [] colorectal cancer cells [ ] EPCs[ ] and liposarcoma cells [] respectivelylinks between VEGF and MMPsIn TME tumorltrating ‚ammatory cells also helpto induce and sustain tumor angiogenesis and further tofacilitate tissue invasion and tumor metastatic spread byreleasing some signal molecules such as proinvasive MMP9and ‚ammatory chemokines [“] Chemotaxis is alsoa crucial process for inducing angiogenesis in tumors eitherdirectly by attracting ECs towards tumor cells to form newvessels or indirectly by mediating immune ‚ammatorycells to ltrate eventually promoting tumor angiogenesis[] Chemotaxis of tumor cells and stromal cells in TMEis also required for tumor dissemination during tumor progression and metastasis [ ]CXC chemokines such as CXCL8 encoding IL8 andCXCL5 are also involved in COX2associated angiogenesisto contribute to nonsmallcelllung cancer progression[ ] It is further shown that IL8 directly regulatesangiogenesis via recruitment of neutrophils [] whichfurther drives VEGF activation [] MoreoverIL8responding neutrophils are considered as the major sourceof angiogenesisinducing MMP9 [ ] Chemokine CC motif ligand CCL2 in addition to the promotionof angiogenesis [ ] also enhances tumor metastasis[] Furthermore myeloid monocytic cellssuch asmyeloidderivedtumorMDSCsassociated macrophages TAMs and dendritic cells arerecruited to the tumor site mainly by CCL2 and producemany proangiogenic factorssuch as VEGF CXCL8plateletderived growth factor PDGF and transforminggrowth factor beta TGF beta [“] In fact bothTGF beta and hypoxia are potentinducers of VEGFexpression in tumor cells and collaborate with TME toprovide the foundation of tumor angiogenesis and cancercell invasion [] Importantly IL8 has been reported asa key target gene of PPAR betadelta to promote angiogenesis in vivo and in vitro [] and CCL2 expression isalso significantly upregulated upon vascular PPAR betadelta overexpression in vivo []suppressorcellsCOX2 also mediates IL1 betainduced angiogenesisin vitro and in vivo [ ] IL1 beta supports neovascularization through the regulation of the expression of VEGFand its receptor VEGFR2 FLK1KDR on ECs [] IL1 actsas an upstream pro‚ammatory mediator that initiates anddisseminates the ‚ammatory state by inducing a localinteractive network and increasing adhesion moleculeexpression on ECs and leukocytes which facilitates tumorassociated angiogenesis [] In TME ‚ammatory IL1beta recruits myeloid cells from bone marrow and activatesthem to produce proangiogenic factors such as VEGF VEGFfurther activates ECs and myeloid cells promoting tumorinvasiveness and fostering tumor angiogenesis [] In addition IL6 also stimulates angiogenesis and vasculogenesis[ ] However Gopinathan observed an IL6induced newly forming vascular structure with defectivepericyte PC coverage ex vivo [] thus facilitating cancercell ltration and tumor metastasis through vascular leakage Interestingly IL1 and IL6 expression levels are significantly upregulated in the PPAR betadelta overexpressionmouse model reported recently []In summary PPAR betadelta seems to act as a key leaderin ‚ammatory mediatordriven tumor angiogenesis linkedto TME in which many pro‚ammatory mediators chemokines and proangiogenic factors closely communicate with 0cPPAR Researcheach other and also associate with tumorltrating myeloid cells such as neutrophils TAMs and MDSCs Other Key PPAR BetaDeltaMediated ProangiogenicFactors It has been demonstrated that Wilms™ tumor suppressor WT1 is a major regulator of tumor neovascularization andtumor progression [] E26 avian leukemia oncogene ETS1 also plays a key role in regulating vascular development and haemopoiesis particularly in angiogenesis []In addition ETS1 promotes cancer cell invasion throughupregulation of MMPs [] Consistent with this silencingof ETS1 in highly invasive breast cancer cells also reducesthe expression of MMP9 and MMP1 []ETS1 also acts as a key regulator of MMPs such asMMP1 MMP3 and MMP9 in human cancerassociatedfibroblasts CAFs [ ] CAFs support tumor growthby secreting growth factors such as VEGF FGF PDGF andchemokines to stimulate angiogenesis and thereby promotecancer cell invasion and metastasis formation [ ]CAFs as metastatic tumor stroma are a crucial componentin tumor progression through the remodeling of the ECMstructure thus helping a tumor to acquire an aggressive phenotype [ ] PPAR betadelta in CAFs also exhibits aprotumorigenic eï¬ect It was reported that ablation of PPARbetadelta in CAFs attenuated tumor growth by altering theredox balance in TME [] suggesting that PPAR betadeltain CAFs is also an important player in tumor developmentETS1 induces the expression of VEGF VEGFR1 andVEGFR2 in ECs [“] In turn VEGF is also a majorinducer of ETS1 in ECs through the activation of either thePI3KAKT pathway or the MEKERK12 signal cascade[ ] WT1 is also reported to regulate tumor angiogenesis via direct transactivation of ETS1 []SRYrelated HMGbox SOX18 has also beenreported previously to induce angiogenesis during tissuerepair and wound healing [] and cancer progression[] And most recently it was further shown that specificECderived endovascular progenitors initiated a vasculogenic process and diï¬erentiated into more mature endothelial phenotypes within the core of the growing tumorsthrough reactivation of SOX18 [] Interestingly theseimportant proangiogenic molecules including WT1 ETS1and SOX18 are also significantly upregulated in the vascularPPAR betadelta overexpression model in vivo [] AndWT1 is also identified as a target gene of PPAR betadeltain melanoma cells [] PPAR BetaDelta May Facilitate Cancer Progression atDiverse Cellular Levels in TME PPAR betadelta activationis shown to induce colonic cancer stem cell CSC expansionand to promote the liver metastasis of colorectal cancerin vivo via direct transactivation of the Nanog gene []NANOG as a key transcriptional factor governs the selfrenewal and pluripotency of stem cells [] and cancer cellsexpressing NANOG also often exhibit stem cell properties[] Protooncogene cKITCD117 is known as the maststem cell factor receptor and receptor tyrosine kinase andits activation in CSCs may regulate the stemness to controltumor progression and drug resistance to tyrosine kinaseinhibitors Moreover cKIT has been identified as a potentialmarker of the cancer stemlike cells [] In addition cKITnot only functions on ECs [ ] but also belongs to thetumor angiogenesispromoting molecule [“] Studiesalso suggested that activation of cKIT enhances the expression of VEGF that can be suppressed by imatinib an inhibitor of cKIT in gastrointestinal stromal tumor cells whichthereby has an impact on tumor angiogenesis [ ] cKIT is also involved in pathological ocular neovascularization [] and is regulated transcriptionally by WT1 []and PPAR betadelta []PDGFB and its receptor PDGFR beta also known asangiogenic factors are suggested to enhance angiogenesisand vasculogenesis via their function in ECs [“] andEPCs [] and to regulate vascular permeability and vesselmaturation through recruitment of pericytes PCs [] and smooth muscle cells SMCs [] in newly formingvessels Moreover PDGFB and PDGFR beta also interactwith other proangiogenic factors such as FGF2 [ ]VEGFA and its receptor VEGFR2 [] FurthermorePDGFB and PDGFR beta may also aï¬ect cancer growthand progression by directly acting on TME Besides thecrosstalk with CAFs [“] PDGFR beta in stromalfibroblasts may mediate PDGFBinduced TAM recruitment[] thus implicating a role of PDGFR beta in tumorstroma to facilitate tumor progression Most recently it wasfurther shown that specific targeting of PDGFR beta kinaseactivity in TME inhibited cancer growth and vascularizationin cancers with high PDGFB expression such as LLC []Therefore this indicates the diverse role of PDGFB andPDGFR beta in facilitating tumor angiogenesis and progression at diï¬erent cellular levels in TME PDGFR beta is demonstrated as a target of telomeric repeat binding factor TRF2 that is further activated transcriptionally by WT1[] PDGFB and PDGFR beta have further been identifiedas critical targets of PPAR betadelta via a direct transactivation mechanism

Thyroid_Cancer

Radial shock waves prevent growth retardation caused by the clinically used drug vismodegib in ex vivo cultured bonesSowmya Ramesh123 Lars Svendahl245 Vrisha Madhuri135 farasat Zaman2In childhood medulloblastoma patients the hedgehog antagonist vismodegib is an effective anticancer treatment but unfortunately induces irreversible growth arrests and growth impairment limiting its use in skeletally immature patients We hypothesized that radial shock wave treatment rSWT may protect druginduced growth impairment owing to its osteogenic effects Fetal rat metatarsal bones were exposed to vismodegib day “ nM andor rSWT single session other bones from day were continuously exposed to a Gli1 antagonist GANT61 µM andor rSWT single session Control bones were untreated The bone length was measured at intervals histomorphometric analysis and immunostaining for PCNA Gli1 and Ihh were performed on the sectioned bones Bones treated with vismodegib showed impaired bone growth reduced height of the restingproliferative zone and reduced hypertrophic cell size compared to control In vismodegib treated bones a single session of rSWT partially rescued bone growth increased the growth velocity hypertrophic cell size and restored growth plate morphology Bones exposed to GANT61 showed impaired bone growth and disanized growth plate while when combined with rSWT these effects were partially prevented Locally applied rSWT had a chondroprotective effect in rat metatarsal bones and suggest a novel strategy to prevent growth impairment caused by vismodegibHedgehog Hh proteins are wellknown to be overexpressed in paediatric medulloblastoma1 Mutations that occur in the family of Hhpathway genes such as patched1 suppressor of fuse and smoothened leads to an increased level of the gliomaassociated oncogene Gli1 a downstream transcription factor of Hh2 In the clinic hedgehog inhibitors are used to decrease the Hhactivity and thereby impede tumor progression3“ However stable expression of the Hhgene is essential to maintain chondrocyte proliferation and hypertrophy during bone growth6 A recent study reported that prolonged exposure to vismodegib a Hhantagonist in children with medulloblastoma resulted in irreversible growth plate fusion causing growth arrest of long bones78 Preclinical studies in young mice exposed to a Hhantagonist also showed growth arrests and bone growth defects9 Mechanistic studies revealed that even brief exposure to a Hhinhibitor was enough to damage the growth plates by diminishing the numbers of reserve and proliferative chondrocytes9 These findings further imply that it may not be possible to arrive at a dose that selectively targets tumor growth with no sideeffects on bone development Therefore in children a protective strategy for growth plate shielding without interfering with the desired anticancer effects of vismodegib in the neural tissue is highly desiredIn vitro studies using cultured rat metatarsal bones10 and in a0vivo studies in rabbits11 and humans12 have shown that radial shock wave treatment rSWT a noninvasive modality used in the clinic have stimulatory effects on bone growth10 Furthermore the observed stimulation of chondrocyte proliferation and hypertrophy induced by rSWT was partially linked to local upregulation of Gli1 in cultured metatarsal bones10 Interestingly previous in a0vitro studies revealed that highenergy shock wave treatment increased the uptake of chemotherapy agents13 1Department of Paediatric Orthopaedics Christian Medical College Vellore India 2Division of Paediatric Endocrinology Department of Women™s and Children™s Health Karolinska Institutet Solna Sweden 3Centre for Stem Cell Research A Unit of inStem Bengaluru Christian Medical College Bagayam Vellore India 4Paediatric Endocrinology and Metabolism Astrid Lindgren Children²s Hospital Karolinska University Hospital Solna Sweden 5These authors contributed equally Lars Svendahl and Vrisha Madhuri email sowmyarameshkiseScientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Rescuing effects of rSWT on vismodegib treated bones a Fetal rat metatarsal bones cultured exvivo were treated with the Hhinhibitor vismodegib a0nM for a0days n dotted line a single session of highenergy rSWT a0Hz a0mJ n or both n and thereafter followed for a0days The graph shows increases in bone length over time a0from day All error bars indicate SD Twoway ANOVA was applied b Graph shows the increase in the growth velocity on day of control vismodegib rSWT and vismodegib rSWT treated bones All error bars indicate SD Representative images of metatarsal bones stained with Alcian blue c untreated control d vismodegib e rSWT and f vismodegib rSWT Magnification 10x g height measurements of R P zone and h hypertrophic cell size Quantification of immunostaining for i Gli1 and j Ihh using the ImageJ software p p p thereby lowering the dose of the drug when applied to cell lines derived from human osteosarcoma14 human colorectal adenocarcinoma15 and anaplastic thyroid cancer16 The effect was mediated by a transient increase in cell membrane permeability allowing passage of a higher concentration of the drug16A recent report suggested that locally applied rSWT can promote longitudinal bone growth of rat metatarsal bones cultured exvivo in the absence of serumgrowth factors10 Based on these findings we hypothesized that rSWT may also have the capacity to prevent growth failure caused by Hhinhibitors We aimed to investigate the potential for rSWT to prevent growth retardation caused by two different Hhantagonists vismodegib and the Gli1 antagonist GANT61 in a wellestablished model of cultured fetal rat metatarsal bonesResultsEffect of vismodegib on bone growth and rescuing effects of rSWT To allow transient inhibition of Hhactivity cultured fetal rat metatarsal bones were treated with vismodegib for a0days whereafter growth was monitored for another a0days without any treatment Bones treated with vismodegib grew less than untreated controls and the difference was significant when measured on day ± vs ± bone length increase from day respectively p Fig a01aTo address the primary objective of this study if rSWT can prevent growth failure caused by hedgehog inhibition we first studied if a single exposure to rSWT can rescue metatarsal bone growth after transient exposure to a0days treatment with vismodegib Indeed bones treated with rSWT single exposure on day in combination with vismodegib day “ grew significantly better than bones treated with vismodegib alone when measured Scientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Rescuing effects of rSWT on GANT61 treated bones a Fetal rat metatarsal bones cultured exvivo treated with the Hhinhibitor GANT61 a0µM n a single session of rSWT a0Hz a0mJ n or both n and followed for a0days The graph shows increases in bone length a0from day All error bars indicate SD Twoway ANOVA was applied Representative images of metatarsal growth plates stained with Alcian blue b untreated control c GANT61 d rSWT and e GANT61 rSWT Magnification 10x f Height measurements of R P zone and g hypertrophic cell size Quantification of immunostaining for h PCNA and i Gli1 using the ImageJ software p p on day ± vs ± respectively p Fig a01a Also the growth velocity tended to be higher in rSWT vismodegib compared to vismodegib alone Fig a01bGrowth plate morphology Histological evaluation revealed severe disruption in the growth plate morphology in the bones treated with vismodegib compared to control Fig a01c“d The disrupted growth plate morphology in vismodegib treated bones was found to be more normal in bones receiving rSWT alone Fig a01e and rSWT vismodegib Fig a01f When comparing vismodegib treated bones to controls we found reduced height of the resting proliferative R P zone ± a0µm vs ± a0µm respectively p Fig a01g The height of the R P zone tended to be increased in bones exposed to rSWT vismodegib when compared to vismodegib alone ± a0µm vs ± a0µm respectively p ns Fig a01g Histomorphometric analysis showed decreased size of hypertrophic chondrocytes in the bones treated with vismodegib compared to control ± a0µm vs ± a0µm respectively p Fig a01h In bones treated with rSWT vismodegib the hypertrophic chondrocytes were significantly larger compared to vismodegib alone and similar in size as in untreated control bones ± a0µm ± a0µm and ± a0µm respectively p vs vismodegib p ns vs control Fig a01h Immunostaining for Gli Fig a01i and Ihh Fig a01j did not show any significant effects of vismodegib andor rSWTShock wave treatment prevents bone growth retardation caused by GANT61 To study the effects of continuous inhibition of Hhactivity fetal rat metatarsal bones were treated with the Gli1 antagonist GANT61 from day of culture until the termination of the experiment on day Already on day GANT61 induced a pronounced suppression of the bone growth ± ± p vs control which remained at endpoint on day ± ± p vs control Fig a02aNext we asked if a single exposure to rSWT can rescue from growth retardation caused by continuous Hhinhibition induced by GANT61 Indeed metatarsal bones exposed to rSWT on day were rescued from GANT61induced growth retardation already from day of culture ± ± p vs GANT61 alone which remained until endpoint ± ± p vs GANT61 alone Fig a02aGrowth plate morphology Compared to control in the bones continuously treated with GANT61 growth plate morphology was found to be disturbed with disanized chondrocyte columns on day Fig a02b c Bones treated with rSWT alone showed anized growth plate morphology Fig a02d The growth rescuing Scientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0ceffect of rSWT in GANT61 treated bones was accompanied by a normalization of growth plate morphology including anized chondrocyte columns Fig a02eHistomorphometric analyses showed no significant differences in the height of the R P zone between GANT61 treated bones with or without rSWT ± a0µm ± a0µm p ns Fig a02f There was an increased size of hypertrophic chondrocytes in rSWT GANT61 group when compared to GANT61 alone ± a0µm ± a0µm p Fig a02g Furthermore the number of proliferative chondrocytes tended to be lower in GANT61 treated bones when compared to control ± cells ± cells p ns Fig a02h The number of proliferative PCNA positive cells tended to be higher in the rSWT GANT61 group compared to GANT61 alone ± cells ± cells p ns Fig a02h Immunoexpression of Gli1 tended to be reduced in GANT61 treated bones p ns vs control whereas bones treated with rSWT GANT61 tended to have higher Gli1 expression compared to GANT61 alone p ns Fig a02iDiscussionWe aimed to investigate the potential for locally applied rSWT to prevent bone growth impairment caused by the hedgehog inhibitor vismodegib a therapeutic investigational drug using a wellestablished model of ex a0vivo cultured fetal rat metatarsal bones Herein we report that a single session of rSWT partially prevented growth retardation caused by both transient and continuous Hhinhibition induced by vismodegib and GANT61 respectively The growth rescuing effects by rSWT were accompanied by preservation of growth plate morphology disrupted by the Hhinhibitors Altogether our data suggest that rSWT has the potential to noninvasively protect bones from growth retardation caused by vismodegibBone growth is majorly dependent on the preservation of a unique anization of chondrocytes in the growth plate17 Recent reports have demonstrated that long term exposure to vismodegib the first Hhantagonist approved in the US by FDA led to permanent growth impairment in children with medulloblastoma78 To date no successful strategy that targets tumor cells with no adverse effect on longitudinal bone growth has been described Previous reports have shown that rSWT a treatment modality that is already used in children for musculoskeletal indications18 can stimulate longitudinal bone growth locally in exvivo cultured metatarsal bones even in the absence of any systemic growth factors10 Proinflammatory cytokines are also known to impair bone growth19 and interestingly shockwave treatment has been shown to reduce inflammation and apoptosis while stimulating the regeneration of various tissues2021 These findings encouraged us to expand this knowledge and further investigate the potential for rSWT to prevent bone growth impairment caused by HhinhibitorsVismodegib at a0nM concentration has shown to impair bone growth in exvivo cultured metatarsal bones22 and decrease proliferation of the precursors of a0cerebellar granule neurons23 while in a0vivo studies in a model of medulloblastoma have also shown that vismodegib inhibits Gli1 at a IC50 of a0nM24 a similar range of concentration as used in the present study In young mice transient inhibition of the Hh pathway has been reported to cause permanent defects in bone and growth plate structure9 Similar to the previous in a0vivo observations in young mice9 our histomorphometric growth plate data suggest that partial loss of Hhactivity may result in the breakdown of chondrocyte columnar anization and reduced size of hypertrophic chondrocytes Also the disrupted growth plate ultrastructure caused by Hhinhibition explains the observed growth deficit in our study model system Besides undesired apoptosis of stemlike cells within the growth plate is another wellknown contributing factor linked to growth retardation caused by anticancer drugs2526Our key finding is that a single administration of rSWT not only prevented bone growth retardation caused by transient exposure to the Hhinhibitor vismodegib but also rescued bone growth under a condition of continuous Hhinhibition induced by another Hhinhibitor GANT61 Furthermore rSWT also improved growth velocity and restored growth plate morphology in bones exposed to vismodegib or GANT61 Thus our findings highlight the potential for shock wave technology to be developed as a new and safe treatment strategy to minimize deleterious effects of Hhinhibitors selectively in the growth plates of treated childrenHedgehog signaling drives chondrocyte proliferation and hypertrophy in the growth plate cartilage6 From in a0vitro studies we know that Hhinhibitors decrease the expression of Gli1 and induce cell cycle arrest in prostate cancer cells27 Despite rSWT rescued bones from Hhinhibitor impaired bone growth we did not see significant alterations in the expression of Gli1 and Ihh suggesting a crosstalk between hedgehog signaling and other pathways28 We speculate that the bone rescuing effect of rSWT is more evident if there is any ongoing disturbance within growth plate chondrocytes Indeed it was interesting to note that despite continuous exposure to a Hhinhibitor a single session of rSWT could partially rescue the bone growthOur study has several limitations Firstly the bone growth rescuing effects of rSWT were documented in an exvivo bone culture model and we do not know if this will be applicable under in a0vivo conditions Nevertheless in a0vivo studies in rats or mice are of limited value when it comes to exploring the potential for rSWT to rescue from vismodegib induced bone growth impairment as their growth plates do not normally fuse29 Secondly we only applied a single dose of rSWT while multiple sessions could potentially be even more efficient when it comes to preventing growth impairment caused by Hhinhibitors Thirdly the concentration of vismodegib used in the present study is different from the plasma concentration a0µM achieved in children30 Nevertheless mimicking a gradual decline in bone growth in order to test the rescuing effect of rSWT is more important in our experimental setting We therefore claim a protective effect and not a clinical effect which will require more rigorous testing Consequently our proof of concept finding s up a window of opportunity to explore the potential for locally applied rSWT to prevent bone growth impairment caused by vismodegib as it may not be possible to extrapolate the doses used for preclinical studies to a clinical setting3132In summary we here present a novel treatment strategy based on clinically used rSWT to locally prevent bone growth impairment caused by vismodegib a promising anticancer drug used in children with medulloblastoma Scientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A hypothetical schematic diagram showing how extracorporeal radial shockwave treatment rSWT may be administered to lowhigh growth velocity areas a0encircled selectively to restore vismodegibinduced bone growth impairment in children with medulloblastomaFig a0 Before any clinical studies our promising ex a0vivo findings need to be validated in an in a0vivo animal model like the rabbit where growth plate fusion normally occurs just like in humansMethodsThe experiments were approved by the local institutional review board Min No and the institutional animal ethics committee Min No at Christian Medical College Vellore Animal care compiled with the Guide for the Care and Use of Laboratory Animals A a0radial shock wave machine from Radialspec Medispec Gaithersburg MD USA was used for the studyBone an culture system Metatarsal bones were microdissected from the hind limbs of Sprague Dawley rat fetuses sacrificed on day of gestation Ex a0vivo cultures were performed as previously reported33 Briefly each bone was transferred to a 24well plate and cultured in medium containing DMEMF12 a0mM betaglycerophosphate ascorbic acid a0µgml and gentamycin The medium was replenished every two days Figure a0 shows the experimental overviewExposure to Vismodegib Vismodegib was purchased from Selleck Chemicals Houston Texas USA The experimental setup consisted of four groups Metatarsal bones were cultured for a0days and were either a treated with vismodegib a0nM n from day to b a single exposure to rSWT impulses a0Hz a0mJ n on day c vismodegib rSWT n or d were left untreated control n The bone length was measured on days and Exposure to GANT61 GANT61 was purchased from Sigma Aldrich St Louis Missouri USA Metatarsal bones were treated with the a Hhinhibitor GANT61 a0µM from day to n b a single exposure Scientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Experimental overview Middlethree metatarsals from the hind limb of embryonic rat at a 1920th day of gestation were dissected and b cultured exvivo c Bones were either exposed to vismodegibGANT61 a single session of highenergy radial shock wave treatment rSWT vismodegibGANT61 with rSWT or left untreated During this time d total bone length measurements at different intervals are performedto rSWT on day impulses a0Hz a0mJ n c GANT61 rSWT or d were left untreated control n The bone length was measured on days and Bone length measurement Digital images were captured for bone length measurements using an inverted microscope Leica Microsystems All measurements were performed by one of the investigators blinded to the nature of the group using an inbuilt ˜measurement tool™ Bone growth is expressed as percent bone length increase from day Bowed bones were measured in two parts added togetherQuantitative histology and immunostaining After termination of the culture the metatarsal bones were fixed with paraformaldehyde embedded in paraffin and fivemicrometer sections were cut along the longitudinal axis proximal to distal followed by staining with SafraninO and Alcian blue The microscopic description of the growth plate morphology included an assessment of the anization of the chondrocyte column Histomorphometric analysis was performed to measure the height of the restingproliferating zone at five different regions of the growth plate and the size of hypertrophic chondrocytes Hypertrophic cells were defined by a height along the longitudinal axis greater than a0µm Eight hypertrophic chondrocytes from the proximal and distal growth plate were measuredImmunostaining was performed as previously described34 Antigen retrieval was performed in citrate buffer at ° Celsius and endogenous peroxidase activity was quenched with H2O2 in methanol for a0min followed by a wash with PBS For immunostaining sections were blocked with bovine serum albumin for a0h incubated with primary antibodies dilution Gli1 Abcam Cambridge MA USA and Ihh mouse monoclonal antibody Santa Cruz Biotechnology Dallas TX USA overnight at ° Celcius After incubation for a0h with secondary polyclonal antimouse or antirabbit biotinylated antibody DakoCytomation Glostrup Denmark dilution sections were incubated with ABC solution and developed with diaminobenzidine Sections were counterstained with Alcian blue Nonimmune immunoglobin G IgG of the same species as the primary antibodies were used as negative controls Three to five bones per group were analyzed To quantify immunostaining the ImageJ software National Institutes of Mental Health Bethesda MD USA was used and the percentage of DAB positivity was calculated digitally using a plugin IHC profilerStatistical analysis All statistics were carried out using GraphPad Prism GraphPad Software Inc La Jolla CA USA Data were summarized using means ± SD for the bone length measurements and histomorphometric assessments A twoway ANOVA with Dunnett multiple comparisons test35 was performed to examine the change in bone length in terms of treatment and days Pairwise comparisons were done corrected for the alpha levels Margin plots with SD were presented to visualize the change in bone length Kruskal“Wallis and Man“Whitney U test were performed when the data were not normally disturbed A p value of was considered to indicate a significant differenceData availabilityAll data generated or analysed during this study are included in this manuscriptScientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cReceived March Accepted July References Gerber N et al Recent developments and current concepts in medulloblastoma Cancer Treat Rev “ Gorlin R J Neurocutaneous Disorders Phakomatoses and Hamartoneoplastic Syndromes “ Springer Berlin Gajjar A J Robinson G W Medulloblastoma”translating discoveries from the bench to the bedside Nat Rev Clin Oncol Kool M et al Molecular subgroups of medulloblastoma an international metaanalysis of transcriptome genetic aberrations and clinical data of WNT SHH Group and Group medulloblastomas Acta Neuropathol “ Kieran M W Targeted treatment for sonic hedgehogdependent medulloblastoma Neurooncology “ Ohba S Hedgehog signaling in endochondral ossification J Dev Biol Robinson G W et al Irreversible growth plate fusions in children with medulloblastoma treated with a targeted hedgehog pathway inhibitor Oncotarget Kieran M W et al Phase I study of oral sonidegib LDE225 in pediatric brain and solid tumors and a phase II study in children and adults with relapsed medulloblastoma Neurooncology “ Kimura H Ng J M Curran T Transient inhibition of the Hedgehog pathway in young mice causes permanent defects in bone structure Cancer Cell “ Ramesh S Zaman F Madhuri V Svendahl L Radial extracorporeal shock wave treatment promotes bone growth and chondrogenesis in cultured fetal rat metatarsal bones Clin Orthop RelatRes “ Gollwitzer H et al Radial extracorporeal shock wave therapy rESWT induces new bone formation in a0vivo results of an animal study in rabbits Ultrasound Med Biol “ Kertzman P Cs¡sz¡r N B Furia J P Schmitz C Radial extracorporeal shock wave therapy is efficient and safe in the treatment of fracture nonunions of superficial bones a retrospective case series J Orthop Surg Res Sansone V et al Shockwave Medicine vol “ Karger Publishers Basel Palmero A et al High energy shock waves enhance the cytotoxic effect of doxorubicin and methotrexate to human osteosarcoma cell lines Oncol Rep “ Anticancer Res “ Canaparo R et al High energy shock waves HESW for sonodynamic therapy effects on HT29 human colon cancer cells Gambihler S Delius M In a0vitro interaction of lithotripter shock waves and cytotoxic drugs Br J Cancer “ Pines M Hurwitz S The role of the growth plate in longitudinal bone growth Poult Sci “ Speed C A systematic review of shockwave therapies in soft tissue conditions focusing on the evidence Br J Sports Med FernandezVojvodich P Zaman F Svendahl L Interleukin6 acts locally on the growth plate to impair bone growth Ann Ciampa A R et al Nitric oxide mediates antiinflammatory action of extracorporeal shock waves FEBS Lett “ “ Rheum Dis e24“e24 Chen YL et al Extracorporeal shock wave therapy effectively prevented diabetic neuropathy Am J Transl Res Zaman F et al Humanin is a novel regulator of Hedgehog signaling and prevents glucocorticoidinduced bone growth impairment FASEB J “ Sharpe H J et al Genomic analysis of smoothened inhibitor resistance in basal cell carcinoma Cancer Cell “ Wong H et al Pharmacokinetic“pharmacodynamic analysis of vismodegib in preclinical models of mutational and liganddependent Hedgehog pathway activation Clin Cancer Res “ Eriksson E et al Bortezomib is cytotoxic to the human growth plate and permanently impairs bone growth in young mice PLoS Zaman F Fadeel B Svendahl L Proteasome inhibition therapies in childhood cancer Leukemia Gonnissen A et al The hedgehog inhibitor GANT61 sensitizes prostate cancer cells to ionizing radiation both in a0vitro and in a0vivo ONE e50523 Oncotarget Zhong L Huang X Karperien M Post J N The regulatory role of signaling crosstalk in hypertrophy of MSCs and human articular chondrocytes Int J Mol Sci “ Roach H I Mehta G Oreffo R O Clarke N M Cooper C Temporal analysis of rat growth plates cessation of growth with age despite presence of a physis J Histochem Cytochem “ Gajjar A et al Phase I study of vismodegib in children with recurrent or refractory medulloblastoma a pediatric brain tumor consortium study Clin Cancer Res “ Singer T Appropriate Dose Selection”How to Optimize Clinical Drug Development “ Springer Berlin ReaganShaw S Nihal M Ahmad N Dose translation from animal to human studies revisited FASEB J “ Chagin A S Chrysis D Takigawa M Ritzen E Svendahl L Locally produced estrogen promotes fetal rat metatarsal bone growth an effect mediated through increased chondrocyte proliferation and decreased apoptosis J Endocrinol “ Bov©e J V van den Broek L J CletonJansen AM Hogendoorn P C Upregulation of PTHrP and Bcl2 expression characterizes the progression of osteochondroma towards peripheral chondrosarcoma and is a late event in central chondrosarcoma Lab Investig “ Vincent K et al Aging of mouse intervertebral disc and association with back pain Bone “ AcknowledgementSR would like to personally acknowledge Dr Sumith K Mathew Assistant Professor Clinical Pharmacology Christian Medical College for his input on calculating for clinically relevant dose This study was supported by internal grants received from Christian Medical College Vellore Centre for Stem Cell Research Swedish Research Council Swedish Childhood Cancer Foundation HKH Kronprinsessan Lovisas f¶rening Ake Wibergs Stiftelse and Karolinska Institutet Sweden access funding provided by Karolinska InstituteAuthor contributionsSR LS VM FZ designed the study SR carried out data collection and statistical analysis SR LS VM FZ analysed data SR wrote the initial draft of the manuscript FZ VM and LS contributed to the revision of the manuscript All authors contributed to the interpretation of resultsScientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0ccompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to SRReprints and permissions information is available at wwwnaturecomreprintsPublisher™s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Access This article is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate if changes were made The images or other third party material in this article are included in the article™s Creative Commons license unless indicated otherwise in a credit line to the material If material is not included in the article™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this license visit httpcreat iveco mmons licen sesby40 The Authors Scientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0c'

Thyroid_Cancer

"Oral squamous cell carcinoma is one of the most common causes of malignancy‘associated death Early diag‘nosis of oral squamous cell carcinoma OSCC is important in patient treatment and prognostic evaluation Due to the lack of significant therapeutic benefit the ‘year survival rate has not improved Therefore effective novel markers are needed to improve diagnosis To determine novel promising diagnostic biomarkers for OSCC upregulated and downregulated differentially expressed genes were screened from OSCC tissues using an RNA microarray The results suggested that minichro‘mosome maintenance protein MCM5 mRNA was significantly overexpressed in OSCC tissues compared with that in adjacent normal tissues Moreover silencing of MCM5 expression an OSCC cell line SCC‘ significantly impaired proliferation and colony formation Furthermore negative regulation of the mRNA and protein expression of MCM5 and demonstrated that MCM5 served as a cancer‘promoting gene modulating OSCC cell proliferation through induced G2M phase arrest In this process the mRNA expression of cyclin E and cyclin‘dependent kinase was downregulated while p21 expression was upregu‘lated These results suggested that MCM5 may be an important pathogenic factor of OSCC High expression levels of MCM5 may serve as a marker for the early diagnosis of OSCC IntroductionOral squamous cell carcinoma OSCC affects individuals worldwide annually Presently the clinical treatment of OSCC is primarily surgery radiotherapy or chemotherapy Over the past decades the overall survival Correspondence to Dr Xiaofeng Wang Department of Stomatology China‘Japan Union Hospital of Jilin University Xiantai Changchun Jilin PR ChinaE‘mail wangxiaofengjlueducnContributed equallyKey words minichromosome maintenance protein cell cycle oral squamous cell carcinoma biomarker microarrayOS rate of patients with OSCC has not significantly improved with a ‘year survival rate of ‘ Insufficient sensitive and specific biomarkers may lead to the diagnosis of OSCC at advanced stages Therefore it is necessary to identify novel biomarkers for the early diagnosis and treatment of OSCCRecently with the continuous development of sequencing technology researchers can efficiently distinguish differentially expressed genes DEGs by transcriptome sequencing which allows screening of potential tumor markers or therapeutic drug targets For example a number of new potential tumor markers have been found in human malignancies such as breast cancer epithelial ovarian cancer and glioma Minichromosome maintenance protein MCM5 a member of mini‘chromosome maintenance family of proteins plays an important role in cell proliferation and DNA replica‘tion Some studies have confirmed that MCM5 is highly expressed in numerous human malignancies such as renal cell carcinoma pancreatic ductal adenocarcinoma cervical cancer and skin cancer Further studies have found that high expression of MCM5 is closely associ‘ated with the clinicopathological features of specific cancer types For example overexpression of MCM5 is significantly associated with overall survival rate OS in hepatocellular carcinoma Moreover increased expression of MCM5 is positively correlated with larger tumor size positive lymph node metastasis more advanced clinical stage higher histo‘logical grade deeper invasion depth and perineural invasion of OSCC However thus far the expression function and potential mechanisms of MCM5 in OSCC are still unclear Therefore the present study aimed to analyze the DEGs in OSCC using a microarray screen for MCM5 and further eval‘uate the possible functions of MCM5 in OSCC The present results may provide evidence to support the value of MCM5 as a biomarker or a therapeutic target of OSCC Materials and methodsTissue sampling Pairs of OSCC tissues and adjacent normal tissues were obtained from patients undergoing resec‘tion operations at the China‘Japan Union Hospital of Jilin University Changchun China Clinicopathological data were also collected No patient received preoperative treatment including radiotherapy or chemotherapy No other inclu‘sionexclusion criteria were used Matched normal OSCC 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAtissues were obtained from a segment of the resected specimens ‘cm away from the tumor Pathological analysis was used to identify surgically resected specimens Pathological analysis was performed by our group with no specific diagnostic guide‘lines Three paired samples were obtained for transcriptome sequencing Then to confirm the reliability of sequencing data the samples size was increased using the remaining paired tissues and analyzed using quantitative qPCR All compari‘sons between OSCC tissues and adjacent normal tissues were performed simultaneously The Kaplan‘Meier analysis of OS and survival curves were from the Cancer Genome Atlas database TCGA wwwcancergovThe study was approved by the Ethics Committee of China‘Japan Union Hospital of Jilin University Written informed consent was obtained from all patients who participated in this studyTranscriptome sequencing and functional annotation analysis Total RNA extraction RNA library construction and transcriptome sequencing were performed at Sangon Biotech Co Ltd The biological relevance of unique genes in expression profiles of DEGs were screened according to the threshold values of log2fold‘change‰¥ and P005 Then the differentially expressed mRNAs were analyzed by Gene Ontology GO whose annotations were downloaded from Gene Ontology httpgeneontology UniProt sparqluniprot and NCBI wwwncbinlmnihgov Significant GO categories were identified using Fisher's exact test with a P005 which indicated that signifi‘cantly upregulated genes in the set of DEGs were assigned to a specific functional category more often than expected by chance Significant pathways of the DEGs were then analyzed and identified according to the Kyoto Encyclopedia of Genes and Genomes KEGG database wwwkeggjpCell lines The human tongue squamous cell carcinoma SCC‘ and CAL‘ were obtained from the American Type Culture Collection CAL‘ cells were cultured in DMEM medium with fetal bovine serum FBS Gibco Thermo Fisher Scientific Inc Uml penicillin and streptomycin at ˚C in a humidified atmosphere containing CO2 SCC‘ cells were cultured in MEM medium with FBS NEAA Uml penicillin and streptomycin at ˚C in a humidified atmosphere containing CO2MCM5‘specific siRNA and transfection Three MCM5 siRNA sequences were synthesized by Suzhou GenePharma Co Ltd The sequences were as follows '‘' siRNA‘ Forward CCG ACU ACU UGU ACA AGC ATT and reverse UGC UUG UAC AAG UAG UCG GTT siRNA‘ forward CCA AAU GUC UAU GAG GUC ATT and reverse UGA CCU CAU AGA CAU UUG GTT siRNA‘ forward GUC GUC UGU AUU GAC GAG UTT and reverse ACU CGU CAA UAC AGA CGA CTT and scrambled forward UUC UCC GAA CGU GUC ACG UTT and reverse ACG UGA CAC GUU CGG AGA ATT The mock was an untransfected empty vector serving as the controlSCC‘ cells 45x104 cellswell were cultured in ‘well plates overnight at ˚C Then cells were transfected with nM negative control siRNA or MCM5 siRNA using Lipofectamine® Transfection Reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's protocol After h transfection cells were collected and then RNA was extracted by TRIzol® regent Invitrogen Thermo Fisher Scientific Inc for further experiments as indicatedReverse transcription RTqPCR RNA extracted from tissues samples were reverse transcribed into cDNA using a GoScript Reverse Transcription System kit Monad httpwwwmonad‘biotechcom according to the manufacturer's instructions Relative mRNA expressions were quantified by qPCR using the QuantiTect SYBR Green PCR kit Roche Diagnostics and normalized to GAPDH using primers listed in Table I The cycling parameters were cycles of ˚C for sec ˚C for sec and ˚C for sec Relative mRNA levels were assessed by the comparative ‘ΔΔCq method All analyses of the samples were conducted in triplicateFor association of MCM5 expression levels with clinico‘pathologic features of OSCC the relative expression levels of MCM5 were evaluated using qPCR as aforementioned Relative mRNA levels of paired samples adjacent vs cancer tissues were assessed by the comparative ‘ΔΔCq method A ratio was considered to have high MCM5 expression whereas a ratio ‰¤ was considered to have low MCM5 expression Cell proliferation assay To analyze cell proliferation a Cell Counting Kit CCK‘ Dojindo Molecular Technologies Inc was used according to the manufacturer's instructions In total cells were cultured to each well of ‘well plates After h post siRNA transfection cells were incubated for and h CCK‘ reagent was added h prior to detec‘tion The OD was measured at nm using a microplate reader Bio‘Tek The experiment was performed three timesColony formation assay This assay was performed according to a previous study Briefly cells were cultured in ‘well plates at cellswell followed by culture in complete medium DMEM supplemented with FBS Uml peni‘cillin and streptomycin for weeks The colonies were fixed with methanol for min at room temperature and washed with PBS and stained with crystal violet at room tempera‘ture Beyotime Institute of Biotechnology solution for min A cell colony was defied as a group formation of at least cells Finally formed colonies were observed and images were captured under a light microscope at magnification x200 Cell migration analysis using scratching assays SCC‘ cells were cultured in a ‘well plate at 5x105 cellswell overnight at ˚C Then the cells were scratched and scraped with fresh DMEM Cells were observed and images were captured under a light microscope magnification x200 at h The width of the scratch was measured and referred to as Wbefore Then the cells were starved with no FBS and returned to the incubator for h at ˚C The width of the same scratch was measured and referred to as Wafter Migrating distance was calculated by subtracting Wafter from Wbefore The migration of the control was set as Western blot analysis The protein extractions from the cells were isolated using RIPA Lysis Buffer P0013B Beyotime Institute of Biotechnology Then ‘ µg protein was loaded per lane on a gel resolved using SDS‘PAGE and electroblotted onto 0cONCOLOGY LETTERS Table I Primers used for reverse transcription quantitative‘PCRmRNA MCM5 P21 CyclinE CDK2 GAPDH MCM5 minichromosome maintenance protein cyclin‘dependent kinase Forward primer '‘' GATCCTGGCATTTTCTACAG GGAGACTCTCAGGGTCGAAA TTCTTGAGCAACACCCTCTTCTGCAGCC GCTAGCAGACTTTGGACTAGCCAG AGAAGGCTGGGGCTCATTTG Reverse primer '‘'CCCTGTATTTGAAGGTGAAGGGATTAGGGCTTCCTCTTGGTCGCCATATACCGGTCAAAGAAATCTTGTGCCAGCTCGGTACCACAGGGTCAAGGGGCCATCCACAGTCTTCFigure Volcano plots and KEGG pathway analysis of differentially expressed genes between OSCC cancer tissue group and adjacent normal tissue group A Differences in gene expression profiles between OSCC cancer tissue group and adjacent normal tissue group The horizontal line corresponds to a ‘fold log2 scaled change up or down and the vertical line represents P005 The red points on the plot represent the differentially expressed genes with a ‘fold change upregulation while the green points represent downregulation with P005 B Top KEGG enrichment terms of DEG in OSCC The vertical axis represents the pathway category and the horizontal axis represents the enrichment score [‘lgP‘value] of the pathway LgP was the logarithm of P‘value and P005 was considered significant KEGG Kyoto Encyclopedia of Genes and Genomes OSCC oral squamous cell carcinoma DEG different expressed genes OSCC oral squamous cell carcinomaPVDF membranes Roche Diagnostics After blocking at ˚C for h non‘fat milk in PBS plus Tween the blots were incubated with primary antibodies against anti‘MCM5 D220960‘ BBI Life Sciences anti‘p21 Proteintech anti‘cyclin E ProteinTech Group Inc and anti‘β‘actin D16H11 CST Biological Reagents Co Ltd at ˚C for h Western blots were probed with secondary antibodies and detected using the Odyssey infrared imaging system LI‘COR BiosciencesCell cycle analysis SCC‘ cells were harvested and fixed with ethanol on ice for min and then washed with PBS to decant the ethanol solution Then the cells were suspended and stained by PI and RNase A treatment Cell cycle analysis was performed using a flow cytometer FACSARIA…¡ BD Biosciences The data was performed using CXP Analysis software Beckman Coulter Inc Statistical analysis All the data analysis was performed using SPSS version SPSS Inc The results are presented as mean SD Associations between MCM5 mRNA expres‘sion and clinicopathological factors were analyzed using the Pearson's χ2 test or Fisher's exact test The differences in MCM5 mRNA expression between carcinoma and adjacent normal tissues were evaluated by a paired t‘test One‘way ANOVA followed by Tukey's post hoc test was used to determine the differences between groups and unpaired t‘tests for the rest of the data The survival rate was calculated by the Kaplan‘Meier method and compared using the log‘rank test P005 was considered to indicate a statistically significant difference All experiments were performed in triplicateResultsRNA sequencing and functional annotation analysis To explore novel biomarkers for OSCC the RNAs derived from tissue samples by sequencing were detected Three matched primary OSCC tissues and adjacent normal tissues were randomly selected As shown in Fig 1A the aberrant expression of genes was detected in tissue samples To screen 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMATable II Twenty randomly selected differentially expressed genes between oral squamous cell carcinoma and adjacent tissue samples The genes selected randomly instead of listing based on rank or fold‘change in expressionGene ID ENSG00000160182 ENSG00000205592 ENSG00000171195 ENSG00000126549 ENSG00000090382 ENSG00000161798 ENSG00000161055 ENSG00000107562 ENSG00000214711 ENSG00000106066 ENSG00000184330 ENSG00000137745 ENSG00000243207 ENSG00000107159 ENSG00000183072 ENSG00000196611 ENSG00000171217 ENSG00000178445 ENSG00000100297 ENSG00000127564 Log2 fold‘change ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ Gene nameTFF1MUC19MUC7STATHLYZAQP5SCGB3A1CXCL12CAPN14CPVLS100A7AMMP13PPAN‘P2RY11CA9NKX2‘MMP1CLDN20GLDCMCM5PKMYT1P‘value 881x10‘ 468x10‘ 144x10‘ 140x10‘ 209x10‘ 528x10‘ 959x10‘ 604x10‘ 601x10‘ 846x10‘ 234x10‘ 753x10‘ 222x10‘ 161x10‘ 380x10‘ 154x10‘ 705x10‘ 751x10‘ 565x10‘ 240x10‘ Result Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation the candidate biomarkers for diagnosing OSCC the DEGs were selected when changes in RNA expression were fold‘changes As shown in Fig 1A aberrant RNAs were significantly downregulated while RNAs were upregu‘lated In order to represent all differentially expressed genes twenty randomly selected dysregulated genes between OSCC and adjacent tissue samples are summarized in Table II The P‘value and log2 fold‘changes of all aberrant expression genes are shown in Table SI The DEGs were selected randomly instead of listing based on rank or fold‘change in expression To explore the role of differentially expressed RNAs in OSCC KEGG pathway analysis was performed Depending on the P‘value and enrichment signal pathways associ‘ated with OSCC were identified Table SII The top KEGG enrichment terms of DEGs are shown in Fig 1B including ˜cell cycle™ ˜pathways in cancer™ ˜cell cycle‘yeast™ ˜meiosis‘yeast™ and ˜cytokine‘cytokine receptor interac‘tion™ Among these it was reported that some genes such as MCM5 cell division cycle ‘related protein kinase and Cyclin‘dependent kinase homolog were primarily enriched in the ˜cell cycle™ pathway Some genes such as lysozyme C statherin and aquaporin‘ were enriched in the ˜saliva secretion™ pathway MCM5 which participated in cell cycle regulation and had high expression in OSCC was selected for further study and it was hypothesized that MCM5 might be a candidate tumor marker for OSCCValidation of MCM5 using RT‘qPCR To further verify the aforementioned expression profile data MCM5 expression levels were investigated using RT‘qPCR in tumor and Figure Relative expression levels of MCM5 mRNA in paired adjacent normal tissues and oral squamous cell carcinoma tissues using quantita‘tive PCR The relative expression data were analyzed by the ‘ΔΔCq method GAPDH was used as an internal control P005 P001 vs adjacent normal tissue MCM5 minichromosome maintenance proteinadjacent normal tissues As shown in Fig MCM5 mRNA was significantly upregulated in of tumor tissues compared with that in matched normal tissues These results showed that MCM5 was highly expressed in OSCC tissues which was in line with the sequencing data Association of MCM5 expression levels with clinicopathologic features of OSCC and survival analysis The results of the potential association between MCM5 expression and clinicopathological features in patients with OSCC are presented in Table III No significant association with MCM5 expression was found for age sex histological differentiation metastasisrecurrence and survival status P05 0cONCOLOGY LETTERS Value n MCM5 expression‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘Low n3 High n12 Table III Association between expression of MCM5 and clinicopathologic features of patients with oral squamous cell carcinoma Characteristic Age years ‰¥ Sex Male Female Histological differentiation Well and Moderate Poor MetastasisRecurrence Yes No Survival status Death Survival P‘valueA Kaplan‘Meier analysis of OS is shown in Fig Analysis of clinical data from TCGA showed that high MCM5 expres‘sion was no associated with a shorter OS in patients with OSCC log‘rank P062 These results suggested that MCM5 might not be a prognostic biomarker for OSCC Inhibitory effect of MCM5 in OSCC cell lines To determine the functional role of MCM5 first MCM5 expression was analyzed using RT‘qPCR in two OSCC cell lines Notably SCC‘ cells expressed significantly higher levels of MCM5 compared with Cal‘ cells P001 Fig 4A Considering that knockdown of MCM5 in the cell line with high MCM5 expres‘sion may bring about more significant changes the SCC‘ cell line was selected for further investigation of the functional role of MCM5 Three specific siRNA sequences were designed to inhibit MCM5 expression and transfected in SCC‘ cells and the impact on MCM5 expression was determined using RT‘qPCR As shown in Fig 4B siRNA1 siRNA2 and siRNA3 transfection decreased MCM5 expression by 651P001 P001 and P001 respectively compared with the negative control Then the efficiencies were confirmed using western blotting Fig 4C The inhibitory effect of siRNA1 and siRNA2 was significant but not found in siRNA3 The results were consistent with those of RNA expression siRNA1 trans‘fection reduced MCM5 expression significantly in SCC‘ cells Therefore siRNA1 was used for subsequent experiments Effect of MCM5 inhibition on proliferation colony formation and migration To determine whether MCM5 regulates cell cycle and modulates cell proliferation in OSCC the effect of inhibiting MCM5 expression on SCC‘ cell proliferation was investigated As shown in Fig 5A the results showed that downregulation of MCM5 had significant anti‘proliferative Figure Survival curves from The Cancer Genome Atlas datasets n518 containing high and low MCM5 expression levels MCM5 minichromosome maintenance protein HR hazard ratio TPM transcripts per millioneffect compared with the negative control P001 Colony formation assays were performed and the results revealed that compared with the number of colonies in the control group downregulation of MCM5 significantly reduced colony formation P001 Fig 5B and C To estimate the impact of MCM5 on OSCC migration scratching assays were conducted and inhibition of MCM5 showed no significant impact on the migration of SCC‘ cells P005 Fig 5D These results suggested that inhibiting MCM5 expression inhibited cell proliferation and colony formation but had no effect on migration in SCC‘ cells\x0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAFigure Inhibition of MCM5 expression in oral squamous cell lines A MCM5 expression in Cal‘ and SCC‘ cells B Efficiency of siRNA‘MCM5 was confirmed by reverse transcription quantitative‘PCR in SCC‘ cells C The efficiency of siRNA‘MCM5 was confirmed by western blot in SCC‘ cells MCM5 minichromosome maintenance protein si small interfering NC negative controlFigure Effects of MCM5 inhibition on antiproliferation colony formation and migration capacity in SCC‘ cells A Downregulation of MCM5 expression suppressed SCC‘ cell proliferation compared with the corresponding control at different time points B Downregulation of MCM5 expression inhibited SCC‘ cells colony formation compared with the corresponding control C Quantification of MCM5 inhibition on colony formation compared with the corresponding control D Cells scratching wounds observed by microscopy E Downregulation of MCM5 had no effect on the migration capacity of SCC‘ cells P001 vs NC MCM5 minichromosome maintenance protein si small interfering NC negative controlEffect of MCM5 inhibition on cell cycle To determine the potential mechanism for the observed proliferation inhibition of SCC‘ cells by MCM5 inhibition cell cycle analysis was performed using flow cytometry As shown in Fig 6A and B after MCM5 inhibition the number of cells were decreased in the G0G1 phase and the S phase but significantly increased in the G2M phase compared with the negative control These results indicated that MCM5 inhibi‘tion significantly induced G2M phase arrest compared with that in the control group\x0cONCOLOGY LETTERS Figure Effects of MCM5 inhibition on cell cycle regulation in SCC‘ cells A Flow cytometry assays were performed to analysis the cell cycle progres‘sion when SCC‘ cells transfected with siRNA‘MCM5 B The bar chart represented the percentage of cells in G0G1 S or G2M phase as indicated C Expression levels of cell cycle‘regulated genes detected by quantitative PCR and normalized to GAPDH D Expression levels of cell cycle‘regulated proteins determined using western blotting β‘actin was used as the loading control Data are presented as mean ± SD P005 P001 vs control group MCM5 minichromosome maintenance protein si small interfering NC negative control CDK2 cyclin‘dependent kinase To elucidate the mechanism underlying this effect the expressions levels of cyclin E cyclin‘dependent kinase CDK2 and p21 related to cell cycle arrest were deter‘mined using RT‘qPCR As shown in Fig 6D MCM5 inhibition decreased both cyclin E and CDK2 mRNA levels but increased the mRNA expression of p21 significantly Then cyclin E and p21 were selected to detect the protein levels using western blotting As shown in Fig 6C cyclin E levels decreased while p21 levels increased in MCM5‘downregulated SCC‘ cells which was consistent with the RT‘qPCR results DiscussionDespite notable progress in cancer research and treatment the survival rate of patients with OSCC has not significantly improved in the past few decades To date there are no effective tumor‘specific biomarkers for the early detection and prognosis prediction of OSCC Several studies have shown that DEGs serve an important role in the development of tumors in different cancer types ‘ However few studies have reported differentially expressed genes in OSCC The present screened genes that regulate the progression of oral cancer by gene expression profiling and found that genes were dysregulated of which DEGs were upregulated and were downregulated DEGs significantly affected GO terms and KEGG pathways MCM5 did not have one of the highest log2 fold‘change values and log10 qValues however MCM5 is one of the differentially expressed genes in cell cycle signaling pathway which was the most signifi‘cant enrichment of differentially expressed genes Therefore MCM5 which regulates the cell cycle was selected for further investigation However previously published studies on biomarkers in OSCC mainly focused on pathological studies The present study not only verified the over‘expression of MCM5 in OSCC but also confirmed using cell experiments that MCM5 affects cell proliferation by regulating the cell cycle MCM5 is a member of the MCM family of proteins and is a component of the starting complex for DNA synthesis MCM5 has been identified as a cell cycle biomarker of aber‘rant proliferation which is associated with the progression of various cancer types Previously MCM5 has been found to be overexpressed in numerous human malignancies such as esophageal thyroid and ovarian cancer For example increased MCM5 levels in urine sediment cells predicts the presence of bladder cancer Inhibition of transcription factor SOX‘ can inhibit the proliferation of skin melanocytes and MCM5 expression is significantly decreased following downregulation of SOX‘ Moreover high expression of MCM5 is associated with poor prog‘nosis and poor malignant status in patients with cervical adenocarcinoma It is well known that immunohistochemistry and western blot‘ting are necessary methods to evaluate protein expression 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAHowever due to a limited number of tissue samples the present study did not have enough samples for simultaneous qPCR western blotting and immunohistochemistry detection Therefore this is a limitation of the present study However the study did perform qPCR to evaluate the expression of MCM5 at the mRNA level The results demonstrated that of patients with OSCC have high MCM5 expression which is consistent with the results of the aforementioned studies indicating that high MCM5 expression may play an important role in the pathogenesis of OSCC In addition other members of the MCM family of proteins have also served as biological markers of dysplasia and malignancy such as glioma cervical colorectal breast prostate and lung cancer ‘ Therefore some researchers even suggested that changes in MCM5 expression may be a sign of cell cycle disorders It is worth noting that some researchers reported that the high expression of MCM family members may be closely related to tumorigenesis and prognosis For example MCM2 MCM4 and MCM6 are overexpressed in breast cancer of high histological grades MCM7 expression is a potent prognostic marker in non‘small cell lung cancer while MCM5 may be an independent adverse prognostic marker in lung squamous cell carcinoma It is well known that the cell cycle is related to cell proliferation signaling path‘ways In most tumors an increase in the expression level of genes encoding proteins that regulate cell proliferation is observed The abnormal expression of cell‘cycle‘related genes is associated with infinite proliferation of tumors and poor prognosis Thus far only Yu reported the relationship between MCM5 and OSCC The study reported that overexpression of MCM5 in patients with OSCC was significantly associated with tumor site tumor size positive lymph node metastasis later clinical stage higher histological grade deeper infiltration depth and peripheral nerve infiltration However in the present study association between high expression of MCM5 with survival metastasis and poor histologic differentiation was not observed A Kaplan‘Meier analysis of the overall survival rate was not significantly changed in patients with high MCM5 expression compared with patients with low MCM5 expression It was speculated that due to the small number of samples that there were large differences between individuals Therefore in future research a larger sample size should be used to clarify the relationship between high expression of MCM5 and prognosis of OSCC Little is known about the role and potential function of MCM5 in OSCC In the present study a loss‘of‘function analysis was conducted and it was demonstrated that MCM5 participated in regulating cell cycle and cell proliferation in OSCC cells In fact inhibiting the expression of MCM5 in SCC‘ cells resulted in the downregulation of cyclin E and CDK expression and upregulation of p21 expression which ultimately led to G2M phase arrest in oral cancer cells These results further verified that MCM5 is highly expressed in patients with OSCC which promotes the proliferation of OSCC cells and regulates cell cycle In addition it was observed that MCM5 was not only expressed in SCC‘ cells but also expressed in CAL‘ cells Fig 4A Notably according to the ATCC the MCM5 gene had no mutations in either of the two cell lines indicating that the two cell lines selected in this study have similar genetic backgrounds and could be used for the study of MCM5 cell functions Considering MCM5‘knockdown experiments in SCC‘ cells with high MCM5 expression received more significant results SCC‘ cells were selected for follow‘up studies However analyzing the functional effects of MCM5‘knockdown in CAL27 cell lines may provide more information In addition both SCC‘ and CAL‘ cells are transformed cell lines In future investigations untransformed cell lines for multiple comparisons should be used to clarify the role of MCM5 in OSCC Surgical resection is currently the main method to treat OSCC However considering the particularity of the oral struc‘ture surgical resection will lead to a huge impact on patients' quality of life Therefore it is important to find effective diagnostic biomarkers for early detection or to develop targeted drugs for OSCC In the present study it was reported that MCM5 is overexpressed in OSCC and that MCM5 can affect cell proliferation by regulating cell cycle Therefore the results suggested that MCM5 might be one of the important patho‘genic factors of OSCC and is expected to be used as a potential tumor marker for OSCC target drugs The specific mechanism of action of MCM5 is still worthy of further investigationOverall the present study evaluated differentially expressed genes using sequencing patterns in OSCC tumor tissues and further validated MCM5 upregulated expression in OSCC tissues By knocking down MCM5 expression in SCC‘ cells it was revealed that cell proliferation and colony formation was significantly inhibited by inducing G2M phase arrest The results also suggested that during this process cyclin E and cell cycle‘related gene expression levels were decreased while p21 was significantly upregulated Therefore MCM5 may modulate OSCC cell proliferation by regulating the cell cycle MCM5 is an important pathogenic factor and might have important role as a potential diagnostic marker or drug target for OSCCAcknowledgementsNot applicableFunding This study was funded by the National Natural Science Foundation of China grant no the Bethune Project of Jilin University of China grant no 2018B02 the Education Department of Jilin Province grant no JJKH20190074KJ and Department of Science and Technology of Jilin Province grant no 20190103086JH and 20200201398JC Availability of data and materials The datasets used andor analyzed during the present study are available from the corresponding author on reasonable request The other datasets generated andor analyzed during the current study are available in The Cancer Genome Atlas wwwcancergov Gene Ontology httpgeneon‘tology UniProt sparqluniprot NCBI wwwncbinlmnihgov and Kyoto Encyclopedia of Genes and Genomes wwwkeggjp databases\x0cONCOLOGY LETTERS Authors' contributions HW CZ and CL performed the experiments HW MH and XW analyzed the data MH and HW wrote the manuscript MH and XW designed and supervised

Thyroid_Cancer

"Microwave ablation MWA is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body This study aimed to investigate the effects of MWA on cytokines inpatients who underwent MWA for a hepatic malignancyMethods Patients admitted to the Oncology Department in the First Affiliated Hospital of Soochow Universitybetween June and February were selected Peripheral blood was collected from patients with a hepaticmalignancy treated with MWA The levels of cytokines IL2 IFNÎ TNFα IL12 p40 IL12 p70 IL4 IL6 IL8 IL10and vascular endothelial growth factor VEGF were detected with a Milliplex® MAP Kit The comparison times wereas follows before ablation h after ablation days after ablation and days after ablation Data were analyzedusing a paired sample ttests and Spearman™s correlation analysisResults A total of patients with hepatic malignancies were assessed There were significant differences in IL2IL12 p40 IL12 p70 IL1 IL8 and TNFα at h after MWA Significant increases 2fold vs before ablation wereobserved in IL2 IL1 IL6 IL8 IL10 and TNFα after MWA Elevated IL2 and IL6 levels after ablation werepositively correlated with energy output during the MWA procedureConclusions WA treatment for hepatic malignancies can alter the serum levels of several cytokines such as IL2 and IL6Keywords Microwave ablation Hepatic malignancy Cytokines IL2 IL6 ImmunoregulationBackgroundPrimary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwideIn China hepatocellular carcinoma HCC has the secondhighest mortality rate of malignancies [] The treatmentof primary and secondary hepatic malignancies via Correspondence lengbengsudaeducn Jing Zhao Qiang Li and Merlin Muktiali contributed equally to this work2Department of Oncology the First Affiliated Hospital of Soochow UniversitySuzhou China5Division of Neurosurgery City of Hope Beckman Research Institute DuarteCalifornia USAFull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] Recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for HCC and its fiveyear survival rateis similar to that of resection [] Microwave ablationMWA is widely used to treat unresectable HCC and recurrent HCC and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy Immune checkpoint inhibitors ICIs The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhao BMC Cancer Page of such as PD1PDL1 and CTLA4 antibodies have beenwidely applied in several cancers and studies have indicated that ICI treatment could enhance the effect of ablation [] Evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process [] Cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer Several cytokineswhich can arise from either tumor cells or immunocytes[] such as tumor necrosis factor TNFα interleukinIL1 IL6 IL8 IL10 and vascular endothelial growthfactor VEGF have been linked with cancers and can either promote or inhibit tumor development The serumlevels of cytokines differ during cancer development Although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after MWA It is still unknown whether the above cytokines changed before andor after MWA in patientswith hepatic malignancies In this study we investigatedthe effects of MWA on the serum levels of cytokines inpatients with hepatic malignanciesMethodsPatients and samplesThe patient population examined in this study was derivedfrom the First Affiliated Hospital of Soochow UniversityPatients were admitted to the Oncology Department between June and February The total number ofpatients was with liver metastases and primaryliver cancers The inclusion criterion was a tumor locatedat a hepatic site either primary or metastases All patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease SD or partial responsePR for more than days Informed consent for blooddraw and the relevant therapy was obtained from all patients The protocol was approved by the Human EthicsCommittee of the First Affiliated Hospital of SoochowUniversity and was conducted in accordance with theDeclaration of Helsinki All written informed consent wasobtained from all participants and clearly stated Wholeblood mL was drawn into EDTA anticoagulant tubeson days ˆ’ to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesAblation procedureThe ablation procedure used in this research was MWAThe puncture site and pathway were determined underthe guidance of a computed tomography CT scanLocal infiltration anesthesia was achieved by using lidocaine The placement of microwave ablation probeswas guided by a CT scan or ultrasonic device and allprobes were placed at the maximum diameter layerDouble probes were employed when the maximumdiameter of the tumor was up to cm The power andtime of ablation were designed for each patient in therange of W and min respectively basedon the size number and position of the tumor Theboundaries of ablation zones were designed as extended cm upon the tumor siteCytokine detectionA Milliplex MAP Kit with human cytokinechemokinepanels that measured IFNÎ IL2 IL6 IL8 IL10 IL12p40 IL12 p70 IL1 TNFα and VEGF was utilized according to the manufacturer™s instructions Briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °C washedand then incubated with a biotinylated detection antibodyAfter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a Luminex analyzer Luminex Corporation All samples were measured in duplicate Standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgmL The minimum detectable concentrations were as follows IFNÎ pgmL IL2 pgmLIL12 p40 pgmL IL12 p70 pgmL IL1 pgmL IL6 pgmL IL8 pgmL IL10 pgmL TNFα pgmL and VEGF pgmL All resultsbelow the minimum concentrations were processed as theminimum concentrationsStatistical analysisIBM SPSS Statistics software was used for the statistical analysis along with GraphPad Prism for figurecreations Normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval CI Cytokinesat different times were compared using a onetailedpaired ttest Spearman™s correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceResultsClinical characteristics of the enrolled patientsAs shown in Table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed The patients™ cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0cZhao BMC Cancer Page of Table Clinical characteristics of the patients enrolled n CharacteristicSexmalefemaleAgePathogenesisprimarysecondaryPrimary site For metastatic hepatic malignancesColon rectalPancreasStomacheBreastOthersMaximum tumor length mmAblation probe usedAblation time minAverage power per probe W ± ± ± ± Average energy time × power time × power–¼–¼ Time and power indicate the time and power respectively ofdifferent probes used during the operation ± IFNÎ IL12 p40 and IL12 p70 were slightly increasedafter MWA treatmentAs shown in Table and Fig the median level ofIFNÎ before the MWA treatment was pgmL CI “ pgmL at days and days after theMWA treatment there was a slight increase comparedto that preMWA with median levels of pgmL CI “ pgmL and pgmL CI“ pgmL respectively The median level of IL p40 before the MWA treatment was pgmL CI “ pgmL There was a slight increase to pgmL CI “ pgmL days postMWAThe median IL12 p70 level before the MWA treatmentwas pgmL CI “ pgmL and increasedto pgmL CI “ pgmL days afterthe MWA treatment and to pgmL CI “ pgmL days postMWA No significant alteration in the VEGF median level was detected after theMWA treatmentIL2 IL1 IL6 IL8 and IL10 were elevated over 2foldafter the MWA treatmentAs shown in Table Fig and Fig the median levelof IL2 before the MWA treatment was pgmL CI “ pgmL There was a significant increase at h postMWA with a median level of pgmL CI “ pgmL The median level ofIL1 before the MWA treatment was pgmL CI “ pgmL and a significantincrease wasnoted days after the MWA treatment pgmL CI “ pgmL The median level of IL6before the MWA treatment was pgmL CI“ pgmL and significantly increased daysafter the MWA treatment pgmL CI “ pgmL The median level ofIL8 before theMWA treatment was pgmL CI “ pgmL and increased significantly to pgmL CI“ pgmL days after the MWA treatmentThe median level of IL10 before the MWA treatmentwas pgmL CI “ pgmL and increasedsignificantly days after the MWA treatment pgmL CI “ pgmL The median level ofTNFα before the MWA treatment was pgmL CI “ pgmL and increased significantlyto pgmL CI “ pgmL days afterthe MWA treatmentlevelsElevated IL2 and IL6 levels after ablation were positivelycorrelated with energy output during MWATo further evaluate the relationship between the increased cytokineand MWA treatment weemployed the concept of œenergy time × power time × power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the MWA procedure As shown in Table and Fig the IL2 levels at h postMWA and the IL levels at days postMWA illustrated significant correlations with energy the relative indexes were and respectivelyDiscussionAs technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used MWA for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed [] A consensus guideline was recently developed to address indications for MWA in these patientsThermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °C Terminal treatment requiresthat a necrotic area surrounds the target site with anadditional “10mm margins [] However in the liverhigh tissue perfusion and large blood vessels can cause aœheat sink effect around the ablation zone making itdifficult to achieve terminal ablation [] The heat sink 0cZhao BMC Cancer Page of Table Median levels of cytokines before and after MWACytokineIFNÎIL2preMWA pgmL CI “ CI “ CI “ CI “ CI “ CI “ CI “ CI “ CI “ CI “ h postMWA pgmL CI “ CI “ –¼ CI “ CI “ CI “ CI “ CI “ CI “ CI “ CI “IL12 p40IL12 p70IL1IL6IL8IL10TNFαVEGF p vs preMWA –¼ 2fold vs preMWA days postMWA pgmL CI “ CI “ CI “ CI “ CI “ –¼ CI “ –¼ CI “ –¼ CI “ –¼ CI “ –¼ CI “ days postMWA pgmL CI “ CI “ CI “ CI “ CI “ CI “ CI “ CI “ CI “ CI “effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression LTP [] however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsIn our study significant increases in the secretion ofchemokines IL8 proinflammatory cytokines IL1IL12 IFNÎ and TNFα and antiinflammatory cytokines IL10 were observed after MWA IL8 is mainlyproduced by macrophages the classical biological activity of IL8 is to attract and activate neutrophils whichcan lead to a local inflammatory response However recent studies have indicated that IL8 both macrophageand cancer cellderived can recruit Myeloidderivedsuppressor cells MDSCs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] IL1 is mainlyproduced by macrophages B cells and NK cells couldproduce IL1 under certain circumstances Generallycells can only synthesize and secrete IL1 after beingstimulated by foreign antigens or mitogens IL1 couldpromote the Th1 response promoting the activation ofDendritic cells DCs and Cytotoxic T lymphocytesCTLs IL12 is mainly produced by B cells and macrophages Human IL12 is a heterodimer with two subunits p40 kD and p35 kD which areinactivated in isolated form In general IL12 functionsas a combination of two subunits IL12 p70 while p40alone possesses partial functions of IL12 p70 It™s mentionable that IL12 p40 and p35 are not expressed inequal proportions so the amounts of IL12 p40 and IL p70 are different in one cell IL12 can stimulate theproliferation of activated T cells and promote the differentiation of Th0 cells into Th1 cells Moreover IL12could induce the cytotoxic activity of CTLs and NK cellsand promote the secretion of several cytokines such asIFNÎ [] and TNFα [] Previous research indicatedthat TNFα may play a crucial role in MWA in combination with immunotherapy [] Notably our data illustrated that the IL12 results were consistent with thoseof IFNÎ after the ablation operation but not with thoseof TNFα This result indicated that upregulation ofIFNÎ may be a major effect of the IL12 increase afterMWA On the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after MWA IL10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses IL10 was reported to increaseafter thermal ablation in the literature [ ] Strategiesto inhibit IL10induced immunosuppression after thermal ablation treatment would be of interestIL10asAblation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of œin situ vaccination [] Moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] Many immunoregulatory cytokineswere released or expressed after thermal ablation Notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle Previously researchers demonstrated that proinflammatory cytokines such as IL1 IL6 IL8 IL18 andTNFα were increased several hours or days after thermalablation [ ] To our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction [] This systemic reaction would becaused by different mechanisms First interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines Second heat injurycould cause acute thermal necrosis in liver and tumor 0cZhao BMC Cancer Page of Fig Levels of cytokines before and after MWA treatment Slightly increased IFNÎ IL12 p40 and IL12 p70 levels after MWA treatment Over fold enhancement of IL2 h postMWA and of IL1 IL6 IL8 IL10 and TNFα d postMWA p 0cZhao BMC Cancer Page of Fig Trends in cytokines significantly altered after MWA treatment The levels of IL2 at h postMWA IL1 at d postMWA IL6 at dpostMWA IL8 at d postMWA and IL10 at d postMWA were elevated over 2fold compared to the levels preMWATable Correlation between the ablation energy and significantly elevated cytokinesEnergyvsIL2 h postMWAEnergyvsIL1 d postMWAˆ’EnergyvsIL6 d postMWAEnergyvsIL8 d postMWAEnergyvsIL10 d postMWAEnergyvsTNFα d postMWASpearman™s rP value onetailed p 0cZhao BMC Cancer Page of Fig Correlation between the ablation energy and the serum levels of IL2 and IL6 The serum levels of IL2 at h postMWA and IL6 at dpostMWA were positively correlated with energy output during the MWA procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions Generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions Ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions Theseexplanations may be the reason why the cytokine changeslasted different durations Moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the œenergyindex In our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time Terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction This reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor Our findings indicated that IL2 andIL6 were significantly altered after the ablation procedureand positively correlated with MWA energy IL2 is commonly derived from activated T cells primarily Th1 cellsIL2 can stimulate T cells to proliferate and differentiateactivate natural killer NK cells and macrophages and enhance the functions of cytotoxic T lymphocytes CTLs[] Our data illustrated that IL2 is significantly increased at h after MWA indicating that IL2 may induce a nonspecific immune response after MWA But IL decreased after h postMWA in our study suggesting that the IL2induced immune response may not belong lasting Mentionable many cytokines detected IL8IL1 IL12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellThis result support the theory that MWA could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction Additional cytokines alterationsuch as IL6 after ablation may be no anspecific inliver Evidences indicate that increase of IL6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum IL8 IL1 IL6 IL10 IL12and TNFα were significantly raised after radiofrequencythermal ablation [] Moreover Joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of IL6 and IL10 []Another question remain unveiled was if our result wasœcancerspecific We checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules[] and adenomyosis [] According to these literatureIL6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of IL6 may be caused by tumour antigenreleased by ablation treatment However the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection To the research about adenomyosis on the other hand experiment design was determined to followup the IL6 at months afterHIFU ablation As our data demonstrated mostly cytokines were return to preMWA level after monthdetection after months may miss the modulation ofIL6 Overall few evidences support that some of thecytokines were altered in a œcancerspecific mannerwhile no solid results could confirm that Further animal experiments were required to make a clarifieddata and answer this question 0cZhao BMC Cancer Page of thetumorassociated immuneIn recent years ablationinduced systemic effects suchasresponse haveattracted increased attention [] de Baere T first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis [] Although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled œabscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management In it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity [] however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors ICIs [ ] ICIs suchas PD1PDL1 and CTLA4 antibodies are widely applied in several cancers and studies have indicated thatICI treatment could enhance the effect of ablation []Evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process [] However opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders PD1 immunotherapy [] suggesting that ablation treatment may promote tumorprogression Our data demonstrated that IL6 was significantly increased after MWA treatment IL6 is derived from monocytes macrophages DCs Th2 cells andsometimes cancer cells and it plays a key role in T cellproliferation and survival [] The role of IL6 appearsto be rather complex Korn classified IL6 as œdifferentiation factor which could involve in differentiation ofTh17 cells [] However IL6 does not direct the commitment to the Th1 or Th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as TGF TNF or IL21 []For instance IL6 activated STAT3 pathway in naiveCD4 T cells in the presence of the morphogen TGFbpromotes the population expansion of Th17 cells [] Recent evidence indicates that IL6 plays an indispensable role in T cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy [] Incontrast IL6 can increase MDSCs [] inhibit the development and maturation of dendritic cells DCs []and inhibit the polarization of Th1 cells [] eventuallyresulting in negative immunomodulatory effects According to Muneeb Ahmed™s work the adjuvant uses ofa nanop smallinterfering RNA siRNA can besuccessfully used to target the IL6mediated locoregional and systemic effects of thermal ablation IL6 knockout via a nanop antiIL6 siRNA in mice coulddecrease the local VEGF level at the ablation site []Therefore how to utilize the positive effect of IL6 whileavoiding the negative effect after MWA needs further investigation Preclinical research indicated that IL6 andPDL1 blockade combination therapy reduced tumorprogression in animal models [ ] Thus an antiIL strategy after ablation should be considered whencombined with ICI therapy Previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation This resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapyConclusionsOur results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionThis reaction could lead to a detectable alteration of cytokine levels Further investigation is required to revealwhether the cytokines altered by MWA treatment couldaffect cancer progression whether positive or negativeAbbreviationsMWA Microwave ablation HCC Hepatocellular carcinoma ICIs Immunecheckpoint inhibitors TNF Tumor necrosis factor IL InterleukinVEGF Vascular endothelial growth factor SD Stable disease PR Partialresponse CT Computed tomography CI Confidence interval LTP Likelihoodof local tumor progression MDSCs Myeloidderived suppressor cellsCTLs Cytotoxic T lymphocytes NK Natural killer siRNA Small interfering RNAAcknowledgementsNot applicableAuthors™ contributionsJZ Conceptualization data curation writing“original draft and writing“review and editing QL Conceptualization and writing“review and editingMM Conceptualization and writing“review and editing BRConceptualization and writing“review and editing and collect samples YHExecute Milliplex assay and collect data DPL Patient enrollment executeMWA ablation and collect samples ZL Execute MWA ablation and collectsamples DML Patient enrollment execute MWA ablation and collectsamples YX Execute Milliplex assay and collect data MT Conceptualizationand writing“review and editing RL Conceptualization data curation formalanalysis visualization writing“original draft and writing“review and editingAll authors have read and approved the manuscriptFundingThis work was supported by the National Natural Science Foundation ofChina the Natural Science Foundation ofJiangsu Province of China BK20140295 the Jiangsu GovernmentScholarship for Oversea Studies JS2018179 and the œSix one projects forhighlevel health personnel in Jiangsu Province LGY2018077Availability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThe protocol was approved by the Human Ethics Committee of the FirstAffiliated Hospital of Soochow University and was conducted in accordancewith the Declaration of Helsinki Patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0cZhao BMC Cancer Page of research and that their privacy would be maintained All written informedconsent was obtained from all participants and clearly statedConsent for publicationNot applicableCompeting interestsThere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workAuthor details1Department of Radiation Oncology the First Affiliated Hospital of SoochowUniversity Suzhou China 2Department of Oncology the First AffiliatedHospital of Soochow University Suzhou China 3Department of LymphaticHematologic Oncology Jiangxi Cancer Hospital Nanchang China4Department of Interventional Radiology the First Affiliated Hospital ofSoochow University Suzhou China 5Division of Neurosurgery City of HopeBeckman Research Institute Duarte California USAReceived January Accepted August ReferencesFu J Wang H Precision diagnosis and treatment of liver cancer in ChinaCancer Lett “Bruix J Han KH Gores G Llovet JM Mazzaferro V Liver cancer approachinga personalized care J Hepatol SupplS144“Rognoni C Ciani O Sommariva S Bargellini I Bhoori S Cioni R FacciorussoA Golfieri R Gramenzi A Mazzaferro V Transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis BMC Cancer Nault JC Sutter O Nahon P GanneCarrie N Seror O Percutaneoustreatment of hepatocellular carcinoma state of the art and innovations JHepatol “Yin J Dong J Gao W Wang Y A case report of remarkable response toassociation of radiofrequency ablation with subsequent Atezolizumab instage IV nonsmall cell lung cancer Medicine Baltimore 20189744e13112Shi L Chen L Wu C Zhu Y Xu B Zheng X Sun M Wen W Dai X Yang M PD1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor Clin Cancer Res “Lippitz BE Cytokine patterns in patients with cancer a systematic reviewLancet Oncol 2013146e218“Jin YB Zhang GY Lin KR Chen XP Cui JH Wang YJ Luo W Changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy IMRT PLoS One 2017122e0172264Kim MJ Jang JW Oh BS Kwon JH Chung KW Jung HS Jekarl DW Lee SChange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma Cytokine “ Gillams A Goldberg N Ahmed M Bale R Breen D Callstrom M Chen MHChoi BI de Baere T Dupuy D Thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans Frontieres meeting Eur Radiol “ Ahmed M Solbiati L Brace CL Breen DJ Callstrom MR Charboneau JWChen MH Choi BI de Baere T Dodd GD 3rd Imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate Radiology “ Chiang J Hynes K Brace CL Flowdependent vascular heat transfer duringmicrowave thermal ablation Conf Proc IEEE Eng Med Biol Soc “ Huang HW Influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors Med Phys Najjar YG Rayman P Jia X Pavicic PG Jr Rini BI Tannenbaum C Ko JHaywood S Cohen P Hamilton T Myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withIntratumoral expression of IL1beta IL8 CXCL5 and Mip1alpha Clin CancerRes “ Alfaro C Teijeira A Onate C Perez G Sanmamed MF Andueza MP AlignaniD Labiano S Azpilikueta A RodriguezPaulete A TumorproducedInterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps NETs Clin Cancer Res “Kundu M Roy A Pahan K Selective neutralization of IL12 p40 monomerinduces death in prostate cancer cells via IL12IFNgamma Proc Natl AcadSci U S A “ Onishi H Kuroki H Matsumoto K Baba E Sasaki N Kuga H Tanaka MKatano M Morisaki T Monocytederived dendritic cells that capture deadtumor cells secrete IL12 and TNFalpha through IL12TNFalphaNFkappaBautocrine loop Cancer Immunol Immunother “ Yu Z Geng J Zhang M Zhou Y Fan Q Chen J Treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathOncotarget “ Yang W Wang W Liu B Zhu B Li J Xu D Ni Y Bai L Liu GImmunomodulation characteristics by thermal ablation therapy in cancerpatients Asia Pac J Clin Oncol 2018145e490“Erinjeri JP Thomas CT Samoilia A Fleisher M Gonen M Sofocleous CTThornton RH Siegelbaum RH Covey AM Brody LA Imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 J Vasc Interv Radiol “ den Brok MH Sutmuller RP van der Voort R Bennink EJ Figdor CG RuersTJ Adema GJ In situ tumor ablation creates an antigen source for thegeneration of antitumor immunity Cancer Res “ Zerbini A Pilli M Laccabue D Pelosi G Molinari A Negri E Cerioni SFagnoni F Soliani P Ferrari C Radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous NKcell responseGastroenterology “

Thyroid_Cancer

Neuropilin1 regulated by miR320a participates in the progression of cholangiocarcinoma by serving as a coreceptorthat activates multiple signaling pathways The present study sought to investigate upstream lncRNAs that control theexpression of miR320aneuropilin1 axis and dissect some of the underlying mechanisms Here we report lncRNATTNAS1 titinantisense RNA1 acts as a sponging ceRNA to downregulate miR320a and is highly expressed in humancholangiocarcinoma tissues and cells The expression of the above three molecules is correlated with theclinicopathologic parameters of cholangiocarcinoma patients In this study multiple bioinformatics tools anddatabases were employed to seek potential lncRNAs that have binding sites with miR320a and TTNAS1 wasidentified because it exhibited the largest folds of alteration between cholangiocarcinoma and normal bile ductepithelial cells The regulatory role of TTNAS1 on miR320a was further evaluated by luciferase reporter and RNApulldown assays coupled with in situ hybridization and RNA immunoprecipitation analyses which showed that TTNAS1 bound to miR320a through an argonaute2dependent RNA interference pathway in the cytoplasm ofcholangiocarcinoma cells Knockdown and overexpression assays showed that the regulatory effect between TTNAS1and miR320 was in a oneway manner TTNAS1 promoted the proliferation and migration of cholangiocarcinomacells via the miR320a neuropilin1 axis The function of TTNAS1 on tumor growth and its interaction with miR320awere confirmed in animal models Further mechanistic studies revealed that TTAAS1 through downregulating miR320a promoted cell cycle progression epithelial“mesenchymal transition and tumor angiogenesis by upregulatingneuropilin1 which cointeracted with the hepatocyte growth factorcMet and transforming growth factor TGFTGF receptor I pathways In the present results demonstrate that lncRNA TTAAS1 is a sponging ceRNAfor miR320a which in turn downregulates neuropilin1 in cholangiocarcinoma cells indicating these three moleculesrepresent potential biomarkers and therapeutic targets in the management of cholangiocarcinomaIntroductionCholangiocarcinoma CCA arises from the epithelialcells facing the lumen of the biliary trees and is the secondmost frequent primary hepatic tumor after hepatocellularCorrespondence Jun Lu lujunsd126com orXueying Sun sunxueyinghrbmueducn1Department of Hepatobiliary Surgery Shandong Provincial Hospital Affiliatedto Shandong First Medical University Jinan China2The Hepatosplenic Surgery Center the First Affiliated Hospital of HarbinMedical University Harbin ChinaFull list of author information is available at the end of the Edited by A Stephanoucarcinoma globally12 CCA is usually diagnosed atadvanced incurable stages due to the absence of priorrecognizable clinical manifestations coupled with thecurrent unavailability of specific tumor biomarkers3Despite the latest progress in the development of molecular targeted therapies the prognosis for this devastatingcancer remains grim3 Pemigatinib has recently beenapproved for “ of CCA patients harboring a fusionor rearrangement of growth factor receptor gene4 andivosidenib has been shown to significantly improve theprogressionfree survival of patients with isocitrate The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of dehydrogenase1 mutant advanced CCA in a phase clinical trial5 However CCA is a heterogeneous malignancy and bears a high mutation burden6 thus thepotential druggable genome alterations in a small proportion of CCAs are not ideal therapeutic targets owing tosignaling pathways7the anticipated redundancy ofTherefore there is great urgency in further elucidating themolecular mechanisms and pathways underpinning thisdisease so that the clinical outcome of CCA patients couldbe improvedNeuropilin1 NRP1 is a nontyrosine kinase transmembrane molecule overexpressed in gastrointestinalcancers89 and serves as a coreceptor for several cellularsignaling pathways involved in cancer progression10“We have recently demonstrated that human CCA tissuesexpressed higher levels of NRP1 which coactivates thevascular endothelial growth factor VEGF epidermalgrowth factorEGF and hepatocyte growth factorHGFmediated pathways involved in the progression ofCCA14 It is known that microRNAs miRNAs regulatemultiple cellular functions and have emerged as potentialtargetsIn exploring themiRNAmediated mechanisms that lead to the overexpression of NRP1 we have shown that miR320anegatively regulates NRP1 by binding to the ²UTR of itspromoter and is expressed at low levels in CAA tissuesand cells14 MiR320a is regarded as a tumorsuppressivemiRNA16 and inhibits the proliferation and metastasis ofCCA cells in vitro and in vivo through downregulatingNRP114 However its upstream regulatory mechanismsremain unknownin anticancer campaign15Long noncoding RNAs lncRNAs are a group of noncoding RNAs ncRNAs with over nucleotides inlength and comprise of ncRNAs Emerging studiesprovide strong evidence that lncRNAs exert pivotal rolesin regulating gene expression in many diseases17 One ofthe main regulatory functions of lncRNAs is to act ascompeting endogenous RNAs ceRNAs to sponge miRNAs leading to the loss of the ability to degrade silenceor hamper translation of their downstream genes17 ManylncRNAs have been shown to regulate key factorsinvolved in cancer cells18 and some of them representpotential diagnostic markers and therapeutic targets forCCA1920 Therefore we carried out the present study toexplore potential upstream lncRNAs that can regulate themiR320aNRP1 axis in CCAResultsIdentification of lncRNA TTNAS1 as a potential targetin CCAThe overexpression of NRP1 in clinical CCA tissueswas confirmed by using immunohistochemistry of tissuemicroarrays Supplementary Fig S1 A panel of CCA celllines expressed different levels of NRP1 where the orderOfficial journal of the Cell Death Differentiation Associationof cell lines with the highest to lowest expression was RBEHCCC9810 QBC939 CC262 and FRH0201 but allexpressed higher levels of NRP1 than normal humanbiliary epithelial HIBEC cells Supplementary Fig S2A BRBE cells expressed the highest levels of NRP1 proteinand mRNA which were and fold higher thanHIBEC cells respectively and expressed the lowest level ofmiR320a which was of that of HIBEC cells FigS2C A negative correlation was found between expression levels of miR320a and NRP1 mRNA Fig S2DLncRNAs that have binding sites with miR320a werescreened by using multiple bioinformatics tools anddatabasestarbasesysueducn DIANATarBasewwwlncrnadb LncBase Experimental v2 lncactdb20omictoolscom and httpbioinfolifehusteducnand potential candidates were selected based on thecriteria of free energy ‰ kcalmol and score Supplementary Table S1 We then detected their expressionlevels in RBE and HIBEC cells by quantitative reversetranscription polymerase chain reaction qRTPCR withspecific primers Supplementary Table S2 Among the candidates TTNAS1 titinantisense RNA1 was shownto have the largest folds of alteration between RBE andHIBEC cells Supplementary Fig S3A B Notably TTNAS1 is a novel lncRNA derived from the opposite strandoftitin TTN gene and has partial sequence complementarity with TTN gene21 LncRNA TTNAS1 hasbeen shown to promote the progression of several cancertypes including esophageal squamous cell carcinomaESCC21 lung adenocarcinoma22 and papillary thyroidcancer23 The expression of TTNAS1 was also detected inall the available CCA cell lines and showed a positivecorrelation with NRP1 but a negative correlation withmiR320a Fig S3CEAssociation of TTNAS1 expression with clinicopathologicparameters of CCA patientstissuesThe qRTPCR analyses revealed that CCA tumor tissuesexpressed significantly higher levels of TTNAS1 Fig 1aand NRP1 mRNA Fig 1b and significantly lower levelsof miR320a Fig 1c compared with adjacent normal bileductIn CCA tissues an inverse correlationbetween expression levels of TTNAS1 and miR320aFig 1d and between miR320a and NRP1 mRNAFig 1e and a positive correlation between TTNAS1 andNRP1 mRNA Fig 1f were found by using Pearsoncorrelation analyses Based on the expression levels ofTTNAS1 we divided CCA cases into the high meanand low ‰mean groups and analyzed the associationbetween TTNAS1 expression and clinicopathologicparameters The results showed that the expression ofTTNAS1 was significantly correlated with tumor differentiation and lymph node metastasis and marginallycorrelated with portal vein invasion while not with gender 0cZhu Cell Death and Disease Page of Fig The expression of lncRNA TTNAS1 miR320a and NRP1 and their correlation in CCA tissues The expression of TTNAS1 a NRP1mRNA b and mature miR320a c in pairs of human CCA tissues and corresponding adjacent normal biliary tissues was detected by qRTPCRn number of samples examined Statistical analyses were performed by a Student™s t test d“f The correlation between miR320aTTNAS1NRP1mRNAmiR320a and TTNAS1NRP1 mRNA expression was analyzed with a Pearson testage tumor location or TNM staging Table NamelyCCA patients with poor tumor cell differentiation positivelymph metastasis and portal vein invasion had higherexpression of TTNAS1 Table By using the sameanalyses based on expression levels of NRP1 mRNA andmiR320a we found that both were correlated with tumordifferentiationlymph node metastasis and portal veininvasion Further NRP1 mRNA expression levels alsocorrelated with TNM staging Table TTNAS1 functions as a ceRNA to sponge miR320aFor examining the regulatory effects between TTNAS1and miR320a we first showed that transfection of miR320a mimics had little effect on TTNAS1 expression butdepletion of TTNAS1 significantlyincreased theexpression of miR320a in RBE and HCCC9810 cellsSupplementary Fig S4A Bimplying that TTNAS1might negatively regulate miR320 in CCA cells Based onthe putative binding sites between TTNAS1 and miR320a luciferase reporter and RNA pulldown assays wereemployed to examine their direct binding SupplementaryFig S5 The luciferase intensity was decreased by cotransfected miR320a mimics and wildtype TTNAS1reporter vector but not the mutant reporter vector lackingthe miR320a binding site Consistently miR320a wasprecipitated by wildtype TTNAS1 but not TTNAS1mutant and TTNAS1 was pulled down by biotinlabeledwildtype miR320a but not miR320a mutant Fig S5TTNAS1 regulates miR320a in an argonaute2dependentmannerThe above results indicate that miR320a binds tolncRNATTNAS1 without causing TTNAS1 degradation TTNAS1 and miR320a were both located in thecytoplasm of CCA cells as detected by In situ hybridization Fig 2a“c suggesting that TTNAS1 may bind tomiR320a through the argonaute2 Ago2dependentRNA interference pathway24 As expected RNA immunoprecipitation RIP assay showed levels of miR320aand TTNAS1 precipitated by an antiAgo2 Ab weremarkedly increasedresulting in a and 3foldenrichment compared with controlIgG respectivelyFig 2d e Meanwhile endogenous TTNAS1 pulldownby the antiAgo2 Ab was specifically enriched uponectopic overexpression of miR320a Fig 2f These datasuggest that TTNAS1 binds to miR320a in the cytoplasm in an Ago2dependent mannerIn addition the expression of miR320a was downregulated by TTNAS1 overexpression and upregulatedby TTNAS1 knockdown and these effects could beabolished by miR320a mimics and antagomiR320arespectively Supplementary Fig S6A B However nosignificant difference in TTNAS1 expression was detected by transfection of miR320a mimics or antagomiR320a Fig S6C D These results indicate that the regulatory effects between TTNAS1 and miR320a are in aoneway mannerOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Table Correlations of the expression of TTNAS1 NRP1 mRNA or miR320a with clinicopathological parameters ofCCA patientsParametersTotaln TTNAS1P value NRP1 mRNAP value miR320aP valueLown Highn Lown Highn Lown Highn GenderMaleFemaleAge years‰¥Tumor differentiationWellmoderatePoorTumor locationIntrahepaticPerihilarTumor size mm‰¥ mmTNM stagebIIIIIIIVLymph metastasisNegativePositivePortal vein invasionNoYes00042a00182a001823a001543a002709a003307aaindicates a significant differencebAccording to the 8th UICC Union for International Cancer ControlTNM staging system P value was estimated by a χ2 testCCA cholangiocarcinoma TTNAS1 lncRNA titinantisense RNA1 NRP1 neuropilin10009076a00194a004106aTTNAS1 promotes the proliferation of CCA cellsvia miR320aNRP1We have previously reported that NRP1 depletion andectopic expression of miR320a inhibited the proliferationof CCA cells14 In accord we confirmed that depletion ofNRP1 significantly reduced cell viability while miR320amimics showed a similar effect by downregulating NRP1expression Supplementary Fig S7 We could furthershow that knockdown of TTNAS1 significantly reducedcell viability while antagomiR320a partially restored cellviability Supplementary Fig S8A Mechanistically TTNAS1 knockdown led to a significant downregulation ofNRP1 cyclindependent kinase CDK2 and cyclin E asignificant upregulation of p27 but had little effect on theexpression of cyclin D1 and p21 The above molecules arekey factors involved in cell proliferation and cycle progression25 AntagomiR320a counteracted the effect ofTTNAS1 knockdown Fig S8B Cell cycle distributionassays showed that knockdown of TTNAS1 led to morecells arrested at the G0G1 phase while antagomiR320apartially abolished this effect of TTNAS1 knockdownSupplementary Fig S9On the other hand exogenous overexpression of TTNAS1 increased the viability of FRH0201 cells while miROfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig TTNAS1 regulates the expression of miR320a in the cytoplasm of CCA cells in an Ago2dependent manner a RBE cells weresubjected to in situ hybridizations of miR320a ²DIG tagged probe identified with Cy3conjugated Ab in red and TTNAS1 ²DIG tagged probeidentified with FITCconjugated Ab in green and stained with DAPI blue Three images from the same cells were merged Scale bar μmb c Total RNA was extracted from nuclear NU and cytoplasmic CY fractions of RBE cells and the expression of TTNAS1 b and miR320a c wasmeasured by qRTPCR and normalized U1 and U6 were used as internal nuclear controls for TTNAS1 and miR320a respectively and GAPDH as aninternal cytoplasmic control d e RBE cells were subjected to RNA immunoprecipitation RIP assays The fold enrichment of miR320a d andTTNAS1 e by an antiAgo2 Ab was normalized to a nonspecific IgG acting as a negative control f RBE cells transfected with negative control NC ormiR320a mimics were subjected to RIP to measure relative enrichment of TTNAS1 by the antiAgo2 Ab P indicates a significant differencefrom respective controls320a mimics partially abolished this effect Fig S8CTTNAS1 overexpression resulted in the upregulation ofNRP1 cyclin E and CDK2 and downregulation of p27while miR320a mimics could neutralize the effect ofTTNAS1 overexpression Fig S8DTTNAS1 promotes the migration of CCA cells via the miR320aNRP1 axisKnockdown of TTNAS1 significantly reduced theability of RBE cells to migrate while antagomiR320aFig 3a“d CCA cellspartially abolished this effectacquire the migratory and invasive properties through acritical process known as epithelialmesenchymal transition EMT26 Therefore we examined the effects ofTTNAS1 knockdown on the expression of decisive facinvolved in the process of EMT27 TTNAS1torsknockdown significantly downregulated the expressionof NRP1 Snail Ncadherin matrix metalloproteinaseMMP2 and MMP9 and upregulated the expression ofEcadherin Fig 3e The results were supported bygelatin zymography assays which showed that TTNAS1knockdown significantly reduced activities of MMP2 andOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig Knockdown of TTNAS1 inhibits cell migration via regulating miR320a RBE cells were transfected with scrambled shRNA controlshRNATTNAS1 or shRNATTNAS1 antagomiR320a for h Cells were subjected to transwell migration a b and scratch c d assays a Migratedcells were visualized using Giemsa staining Scale bar μm a and µm c b Numbers of migrating cells were counted c Scratch areas wererecorded d Scratch distances were quantified at indicated time points e f Cells were immunoblotted for detecting key EMT proteins and the densityof each band was normalized to actin g Cells were subjected to gelatin zymography assays for analyzing the gelatinolytic activity of MMP9 andMMP2 P vs controls and P and P vs shRNATTNAS1MMP9 while antagomiR320a partially counteracted thiseffect Fig 3f On the other hand overexpression ofTTNAS1ofFRH0201 cells while miR320a mimics partially abolishedthis effect Supplementary Fig S10the migratoryincreasedabilityTTNAS1 contributes to the growth of CCA tumors inanimal modelsThe functional role of TTNAS1 was also confirmed inCCA tumors in vivo Subcutaneous RBE tumors wereestablished in mice which were randomly assigned todifferent treatments when tumors reached mm3Tumors treated with shRNATTNAS1 were significantlysmaller ± mm3 than control tumors ± mm3 however cotreatment of antagomiR320aOfficial journal of the Cell Death Differentiation Associationcould partially restore the growth of tumors ± mm3 as measured days after treatment commencement Fig 4a The results of tumor volume correlatedwith the weight of tumors Fig 4b Treatment of shRNATTNAS1 led to TTNAS1 downregulation and miR320aupregulation in tumors harvested days after treatmentsby in situ hybridization and downregulation of NRP1 byimmunohistochemistryofantagomiR320a partially abolished the effects of shRNATTNAS1 on miR320a upregulation and NRP1 downregulation but had little effect on TTNAS1 expressionFig 4c Treatment of shRNATTNAS1 significantlyinhibited cell proliferation in situ Fig 4d e and reducedtumor vasculature while antagomiR320a neutralized theeffects of shRNATTNAS1 Fig 4d f In agreement with4c CotreatmentFig 0cZhu Cell Death and Disease Page of Fig Knockdown of TTNAS1 inhibits the growth and angiogenesis of CCA tumors in vivo Subcutaneous CCA tumors were established inmice by inoculation of RBE cells and received respective treatments as described in Supplementary Information a The growth curve of RBE tumorswas recorded b RBE tumors were resected weighed and photographed at the end of experiments c Two mice were killed from each group toharvest tumors days after treatments and the expression of TTNAS1 and miR320a was examined by in situ hybridization magnification × Scalebar μm and NRP1 expression by immunohistochemistry Magnification × Scale bar μm Tumors harvested at the end of experimentsd Illustrated are representative tumor sections immunostained by Abs against Ki67 and CD31 respectively Magnification × Scale bar μm Insitu cell proliferation index e and tumoral microvessel density f were quantified g Tumor tissue homogenates were immunoblotted for detectingthe expression of key proliferation proteins P vs controls P and P vs shRNATTNAS1the in vitro results Fig S8A B immunoblotting analysisof tumor homogenates showed that shRNATTNAS1treatment led to downregulation of NRP1 cyclin E andCDK2 and upregulation of p27 while antagomiR320acounteracted the effects of shRNATTNAS1 Fig 4gOn the other hand by adopting another subcutaneousCCA tumor mouse model with FRH0201 cells whichwere shown to express a lower level of TTNAS1Fig S2A we demonstrated that exogenous overexpression of TTNAS1 promoted tumor growth bypromoting in situ cell proliferation and tumor angiogenesis while miR320a mimics partially abolished theseeffects Supplementary Fig S11TTNAS1 regulates the cMet and TGF pathways via NRP1We have previously demonstrated that NRP1 coactivates the HGFcMet pathway in CCA cells14 Controland shRNATTNAS1transfected RBE cells were incubated with recombinant human HGF protein in the presence or absence of tivantinib a cMet inhibitor and anOfficial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig TTNAS1 regulates the cMet and TGF pathways via NRP1 RBE and cells were transfected with negative control or shRNATTNAS1 andthen incubated for h in the presence or absence of recombinant HGF protein ngml and tivantinib μgml a or TGF protein ngmland LY2157299 μgml b Cell lysates were immunoblotted to determine the expression of key proteins involved in the above pathways asindicated The density of each band was normalized to actin P and P indicate a significant difference P indicates asignificant difference from negative control cells treated with vehicleanticancer drug used in CCA clinical trial28 TTNAS1knockdown led to downregulation of NRP1 expressionresulting in downregulation of phosphorylated cMetpcMet and sequential downregulation of phosphorylated Akt pAkt and upregulation of p27 Fig 5aIncubation of HGF protein did not affect NRP1 expression but could activate the cMet pathway evidenced byupregulation of pcMet and pAkt and downregulationof p27 and incubation of tivantinib showed the oppositeeffects to HGF ligand Fig 5aNRP1 cointeracts transforming growth factor TGFpathway29 which is crucial for EMT of cancer cells30Therefore we examined the effect of TTNAS1 knockdown on this pathway in CCA cells Control and shRNATTNAS1transfected RBE cells were incubated withrecombinant human TGF protein orand LY2157299 aspecific TGF receptor TGFR inhibitor31 Incubationof TGF protein or LY2157299 did not affecttheexpression of NRP1 or TGFRI Fig 5b HoweverTGF induced the upregulation while LY2157299expression of pTGFRI TTNAS1reduced theknockdown had little effect on TGFRI expression butsignificantly inhibited its phosphorylation Fig 5b TheOfficial journal of the Cell Death Differentiation Associationactivation of TGF pathway by TGF protein increasedthe sequential expression of pSmad23 and Snail whileLY2157299 and TTNAS1 knockdown demonstratedopposite effects and abolished the activating effects ofTGF protein Fig 5bDiscussioninotherrole wasconfirmedLncRNA TTNAS1 was initially reported to participatein the progression and metastasis of ESCC21 Later itsfunctionalcancertypes22233233 Importantly TTNAS1 exerts regulatoryeffects via acting as a ceRNA to sponge different miRNAsin different cancers For instance TTNAS1 regulated themiR133bactinbinding protein fascin homolog axis inESCC cells21 while promoted the migration and EMT oflung adenocarcinoma cells by sponging miR1425p toregulate CDK522 As schematically summarized in Fig we have in the present study found that TTNAS1 servesas a ceRNA to sponge miR320a through complementarybinding sites in an Ago2dependent manner in CCA cellsLncRNAs exhibit different functions depending on theirsubcellular localization This study showed that TTNAS1was mainly localized in the cytoplasm of CCA cells while 0cZhu Cell Death and Disease Page of dependent way and in a oneway manner in CCA cells Inaccord it has been reported that TTNAS1 was locatedmainly in the cytoplasm and acted as a ceRNA spongingmiRNAs in ESCC and papillary thyroid cancer cells2123MiR320a is one of the two most highly downregulatedmiRNAs in clinical CCA tissues and is closely associatedwith the progression and severity of CCA35 MiR320aalso represents a critical suppressor component of theprogression of other cancers163637 We have previouslyreported that miR320a negatively regulated the expression of NRP1 by binding to the ²UTR of NRP1 promoter and inhibited cell proliferation and migration ofCCA cells14NRP1 functions as versatile coreceptors that bind to anumber of growth factors and couple with cognatereceptor tyrosine kinase signaling pathways involved incancer progression111438 In the present study we havefurther demonstrated that NRP1 acts as a coreceptor forthe activation of HGFcMet pathway which induces thephosphorylation of Akt39 a downstream of cMet signaling40 Akt activation leads to the sequential downregulation of p2741 which inactivates the CDK2cyclinE complex resulting in cell cycle arrest41 Howevertivantinib a specific cMet inhibitor can block NRP1induced activation of the HGFcMet pathway Fig Onthe other hand NRP1 cointeracts with TGF29 leadingto the activation of the TGFTGFRI pathway whichin turn increases the expression of phosphorylated Smad2and Smad3 The latter two combine with Smad4 to form atrimeric SMAD complex that upregulates the expressionof Snail which conveys TGFinduced repression ofEcadherin and stimulation of Ncadherin42 thus promoting EMT of CCA cells However LY2157299 a specific TGFR inhibitor31 can block NRP1inducedactivation of the TGFTGFRI pathway Fig Asdemonstrated previously14 but not investigated in thisstudy the above signaling pathways may also crosstalkwith each other and contribute to the proliferation andmetastasis of cancer cells43In summary to the best of our knowledge this is thefirst study that reports the functional role of TTNAS1 asa sponging ceRNA for miR320a its high expression inCCA tissues and a significant association with clinicopathologic parameters of CCA TTNAS1 displays itsregulatory activity by binding to miR320a through theAgo2dependent RNA interference pathway and in a oneway manner in the cytoplasm of CCA cells Throughdownregulating miR320a TTAAS1 promotes cell cycleprogression EMT and angiogenesis via NRP1 which cointeracts HGFcMet and TGFTGFRI pathways inCCA cells Taken together the present study has unveileda novel axis consisting of TTNAS1miR320aNRP1which may also represent a therapeutic target and biomarkers in the management of CCAFig Schematic diagram of the TTNAS1miR320aNRP1 axiscontributing to the progression of CCA LncRNA TTNAS1 serves asa ceRNA to sponge miR320a through complementary binding sites inan Ago2dependent manner in CCA cells On the other hand miR320a downregulates the expression of NRP1 by binding to its ²UTRAn NRP1 protein molecule is composed of five extracellular domainsa1 a2 b1 b2 and c one transmembrane domain and a shortcytosolic tail and acts as a coreceptor for ligands HGF and TGF tostimulate the activation of respective cMet and TGF signalingpathways œ†’ indicates promotion positive regulation or activationœŠ¥ indicates inhibition negative regulation or blockade œp indicatesphosphorylation of proteins Ago2 argonaute2 CCAcholangiocarcinoma CDK2 cyclindependent kinase HGFhepatocyte growth factor NRP1 neuropilin1 ORF readingframe TGF transforming growth factor TGFR TGF receptorUTR untranslated regionmiR320a was located in both nuclear and cytoplasmsubcellular compartments It is well established that thematuration of miRNAs occurs in the cytoplasm wherethey execute posttranscriptional gene silencing via anRNAinduced silencing complex pathway34 Intriguinglythe ectopic expression of miR320a reduced the luciferaseactivities of the wildtype TTNAS1 reporter but theexpression of TTNAS1 remained unchanged uponoverexpression of miR320a Moreover endogenousTTNAS1 and miR320a could be pulled down by an antiAgo2 Ab These data suggest that miR320a recognizesand binds with TTNAS1 without triggering the degradation of TTNAS1 which plays a posttranscriptionalregulatory role in downregulating miR320a via an Ago2Official journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Materials and methodsClinical CCA tissuesA total of pairs of CCA and matched adjacent normalbile duct tissues were collected at the Department ofHepatobiliary Surgery Shandong Provincial HospitalAmong them pairs have been described previously14while new pairs of tissues were collected between April and September Of the cases were perihilar CCA and were intrahepatic CCA The criteria ofthe included specimens were consistent with our previousstudy14 The current study has been approved by the EthicsCommittee of Shandong Provincial Hospital Jinan Chinaand informed consent was obtained from all subjectsCells antibodies and reagentsHuman CCA celllines HCCC9810 RBE QBC939CC262 and FRH0201 and normal human biliary epithelialHIBEC cells were obtained from the Cell Bank of theChinese Academy of Sciences Shanghai China1444 Cellswere routinely cultured at °C in RPMI1640 mediumsupplemented with vv fetal bovine serum in ahumidified atmosphere of CO2 Cell lines were confirmed to be negative for mycoplasma infection by using aPCRbased Universal Mycoplasma Detection kit AmericanType Culture Collection Manassas VA USA Relevantinformation regarding antibodies Abs reagents and kitsare described in detail in Supplementary InformationAnimal experimentsThe experimental protocol has been described previously1444 and approved permit SYXK20020009 by theInstitute Animal Ethics CommitteeImmunodeficientnude BALBc mice H2b were housed in the AnimalResearch Center the First Affiliated Hospital of HarbinMedical University China Two sets of experiments weredesigned to examine the effects of TTNAS1 knockdownand overexpression on tumor growth Detailed information for animal experiments is included in SupplementaryInformation Briefly cells were injected subcutaneouslyinto mice and palpable tumors were monitored Around“ weeks later mice bearing tumors with a volume ofˆ¼ mm3 were randomly assigned to different groupsn The TTNAS1 knockdown study had threegroups of animals which received intratumoral injectionsof control shRNATTNAS1 or shRNATTNAS1 antagomiR320a respectively while the TTNAS1 overexpression study comprised three groups of animalswhich received injections of either control TTNAS1 orTTNAS1 miR320a mimics respectively Two micefrom each group were killed days after injection fordetecting gene expression The remaining mice werefurther monitored and euthanized days after treatments commencedOfficial journal of the Cell Death Differentiation AssociationImmunohistochemistry Tissue microarrays Establishment of stable transfectants depleted of NRP1 Assays ofcell viability cell cycle Transwell migration Cell scratchqRTPCR western blot and Gelatin zymography Cellfraction isolation In situ hybridization RNA pulldownand RIP assays Transfection of miR320a mimicsantagomiR320a and TTNAS1 expression vectors Plasmid constructs and luciferase assay In situ Ki67 proliferation index and Assessment of tumor vascularityThe detailed description for these methods is includedin Supplementary Information and has also been described previously1114Statistical analysisGraphPad Prism GraphPad Software San DiegoCA USA was employed for performing statistical analyses Data are expressed as mean values ± standarddeviation Multiple comparisons were made with a oneway analysis of variance ANOVA followed by a Tukeyposthoc test Comparisons between two groups weremade by a ttest Correlations of TTNAS1 NRP1mRNA or miR320a with clinicopathological parameters were estimated by a χ2 test The relationshipbetween two variables was analyzed by using Pearson™scorrelation coefficient P was considered statistically significantAcknowledgementsThis study was supported in part by the grants from the National Key Researchand Development Program of China 2017YFC1308602 the Supportive Fundby Heilongjiang Provincial Department of Science and TechnologyGX18C010 Natural Scientific Foundation of Shandong ProvinceZR2019MH089 Natural Scientific Foundation of Heilongjiang ProvinceH2018028 and LH2019H018 and Research Projects from the Fourth AffiliatedHospital of Harbin Medical University HYDSYXH201904 an

Thyroid_Cancer

Lenvatinib inhibits tyrosine kinases including vascular endothelial growth factor VEGF receptor fibroblast growth factor receptor platelet derived growth factor receptor alpha RET proto oncogene and KIT proto oncogene receptor tyrosine kinase We assessed the efficacy and safety of lenvatinib in patients with metastatic colorectal cancer after failure of standard chemotherapiesPatients and methods This was an label single centre single arm phase study Eligible patients had unresectable metastatic colorectal adenocarcinoma refractory or intolerant to fluoropyrimidine irinotecan oxaliplatin trifluridinetipiracil anti VEGF therapy and anti epidermal growth factor receptor therapy for tumours with wild type RAS Patients were treated with oral lenvatinib at mg one time a day in day cycles until disease progression or unacceptable toxicity The primary endpoint was centrally assessed disease control rate Secondary endpoints included safety response rate progression free survival and overall survival The planned sample size was patients to expect a disease control rate of with a threshold disease control rate of one sided alpha of and power of Results Between October and January patients were enrolled and had received or ‰¥ lines of prior chemotherapy for metastatic disease respectively The median number of lenvatinib cycles was range “ The centrally assessed disease control rate was CI to one sided p00001 patients had a partial response and had a stable disease Median progression free survival was months CI to Median overall survival was months CI to The most common grade ‰¥ adverse events were hypertension thrombocyt ia increased alanine aminotransferase and anorexia eachConclusions Lenvatinib showed promising clinical activity and was tolerated in patients with metastatic colorectal cancer after failure of standard chemotherapiesTrial registration number UMIN CTR UMIN000023446 and JAMCCT CTR JMA IIA00261InTRoduCTIonThe combination of cytotoxic chemotherapy with a molecular targeted agent has significantly Key questionsWhat is already known about this subject –º No studies have previously reported the efficacy and safety of lenvatinib monotherapy in patients with metastatic colorectal cancer refractory to standard chemotherapiesWhat does this study add –º Lenvatinib showed promising antitumour activity with acceptable toxicity for heavily pretreated patients with metastatic colorectal cancer refractory to standard chemotherapies –º No unexpected safety signals were observed and toxicities were manageable with dose modification interruptions and supportive medicationsHow might this impact on clinical practice –º Further prospective randomised studies are warranted to evaluate the efficacy of lenvatinib in patients with metastatic colorectal cancer refractory to standard chemotherapiesimproved the survival of patients with unresectable metastatic colorectal cancer1“ From results of recent clinical trials trifluridinetipiracil and regorafenib are recognised as new treatment options for patients with metastatic colorectal cancer refractory or intolerant to standard therapies6 Nevertheless the prognosis of patients which are refractory or intolerant to standard chemotherapies is poor and there are still an unmet medical needs for these patients especially for those who are in a good performance status and eligible for further therapiesLenvatinib is an oral multitargeted tyrosine kinase inhibitor of the vascular endothelial growth factor receptor VEGFR “ fibroblast growth factor receptors “ platelet derived growth factor receptor alpha RET and KIT8 Preclinical studies have shown that Iwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776 0c accesslenvatinib not only interferes the interaction between cancer cells and endothelial cells but also inhibits tumour growth10 Several phase trials of patients with solid tumours in the USA11 Europe12 and Japan13 showed that the optimum dosage of lenvatinib was mg one time a day in a day cycleA total of patients were enrolled in four phase studies of lenvatinib monotherapy of whom had colorectal cancer Disease control rate DCR was achieved in out of patients including one with a partial response which continued for weeks mg two times a day for weeks of a week cycle Grade palmar plantar erythrodysesthesia was reportedly much lower in of patients treated with lenvatinib for thyroid cancer in a Japanese population of the SELECT trial than that of reported in a Japanese population of CORRECT trial using regorafenib for metastatic colorectal cancer15 These results suggested that lenvatinib may have a potential for improving the outcomes of patients with unresectable metastatic colorectal cancer who have already received conventional chemotherapy with a fluoropyrimidine irinotecan and oxaliplatinWe conducted a single centre phase study to evaluate efficacy and safety in patients with metastatic colorectal cancer failing to standard therapiesPaTIenTs and meTHodsstudy design and patientsThis study was a single arm phase study conducted at National Cancer Center Hospital Tokyo Japan The inclusion criteria were histological diagnosis of colorectal adenocarcinoma excluding carcinoma of the appendix and the anal canal unresectable metastatic disease an Eastern Cooperative Oncology Group performance status of or an age of “ years no previous treatment with regorafenib or lenvatinib sufficient oral intake adequate an and bone marrow function at least one measurable lesion in accordance with the Response Evaluation Criteria in Solid Tumors RECIST version refractory or intolerant to fluoropyrimidine irinotecan oxaliplatin therapy and antiepidermal growth factor receptor therapy for tumours with wild type RAS and no systemic therapy for at least weeks weeks if any investigational drug had been administered before study enrolment The exclusion criteria were provided in the online supplementary materialtrifluridinetipiracil anti VEGF All patients provided written informed consentProceduresPatients received lenvatinib at mg one time a day in day cycles orally until disease progression or unacceptable toxicity The dose was reduced to mg mg mg mg and mg if a patient had an intolerable grade or grade adverse event Treatment was discontinued if a dose interruption was required for more than consecutive daysTumour response was assessed by the independent radiological review committee based on the CT or MRI performed at baseline every weeks for weeks and every weeks thereafter until confirmed objective disease progression Safety assessments including laboratory tests were done at screening days and of cycle and days and of the subsequent cycles Urinalysis thyroid function prothrombin time international normalized ratio PT INR and tumour markers both carcinoembryonic antigen and carbohydrate antigen “ were measured at screening and on day of each treatment cycle Adverse events were recorded from the first day of the protocol treatment to days after the last dose of study medication and graded using the National Cancer Institute Common Terminology Criteria for Adverse Events version Blood sampling for biomarker analyses was done at baseline on days and and at the end of treatment Plasma levels of angiopoietin2 were measured by the Human Angiopoietin2 Quantikine ELISA Kit RD Systems Minneapolis USAoutcomesThe primary endpoint was centrally assessed DCR which was defined as the proportion of patients with a complete response partial response or stable disease persisting for more than weeks from the initiation of study treatment according to RECIST version A complete response and partial response were needed to be confirmedThe secondary endpoints were the objective response rate ORR proportion of patients who had a complete response or partial response progression free survival PFS time from the enrolment until investigator assessed disease progression or death overall survival OS time from the enrolment until death due to any cause and adverse events The incidence of adverse events was calculated based on the information of the worst grade of each adverse event experienced in each patient Relative dose intensity which is unprespecified outcome was calculated as the proportion of the actual cumulative dose divided by planned cumulative dose mg times treatment daysstatistical analysisFor this single arm study the required sample size of patients provided power to reject the null hypothesis of DCR ‰¤ with expectation that of patients would have a disease control one sided α of Considering the possibility of a few ineligible patients we planned to recruit patientsThe final analysis was planned approximately months after enrolment of the last patient We included all eligible patients in the efficacy analysis and all patients receiving a least one dose of lenvatinib in the safety analyses For the primary analysis binomial test was performed and the centrally assessed DCR was estimated with CI using the Clopper and Pearson method which corresponds to one sided α of We also estimated the investigator assessed DCR a supplementary analysis of the primary Iwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776 0cTable Baseline patient characteristicsCharacteristicsTable ContinuedOverall N CharacteristicsOverall N access Median range   Continued Intolerant Wild type Mutant RAS mutational status BRAF mutational status Wild type Mutant UnknownMSI status MSS Unkown There is an overlapping This number includes patients with the RAS wild type and patient with mutant RASECOG Eastern Cooperative Oncology Group EGFR epidermal growth factor receptor MSI Microsatellite instability MSS Microsatellite stableendpoint and ORR with CIs using the same method We estimated the median time and month and year probability of OS and PFS with the Kaplan Meier method The CIs for the median time were calculated using Brookmeyer and Crowley method The CIs of month and year survival probabilities were calculated based on the Greenwood™s formula HRs and CIs were estimated by Cox regression We did subgroup analyses divided by prespecified baseline patient and disease characteristic variables including RAS status for DCR PFS and OS We also did a prespecified exploratory analysis of potential predictive biomarkers in blood samples We did all analyses with SAS V94ResulTsPatient characteristicsBetween October and January patients with unresectable metastatic colorectal cancer were enrolled All patients were eligible and received the study medication Table summarises the baseline characteristics of all enrolled patients The median number of previous lines of palliative chemotherapy was range “ and patients had received or ‰¥ prior lines of chemotherapy for metastatic disease respectively The data cut off date was January with median follow up of months IQR “efficacyThe centrally assessed DCR was CI to one sided p00001 two patients had a partial response and had a stable disease including unconfirmed PR table figure A total of patients had a reduction in target lesion size from baseline figure Time on treatment for all patients is ‰¥ ‰¥ Male Female months ‰¥ months Right sided colon Left sided colorectum Lung Liver Lymph node PeritoneumAge years Sex ECOG performance status Primary site Number of metastatic site Metastatic an Time from start of first line chemotherapy Number of previous palliative chemotherapy Previous chemotherapy and reason for discontinuation Fluoropyrimidine Refractory IntolerantOxaliplatin Irinotecan TAS102 trifluridinetipiracil Angiogenesis inhibitor Anti EGFR inhibitor Refractory Intolerant Refractory Intolerant Refractory Refractory Intolerant Refractory IntolerantIwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776 0c accessTable Best response to treatmentComplete responsePartial responseStable diseaseProgressive diseaseNot evaluableDisease control rate CIResponse rate CICentral assessmentn30 to to Investigator assessmentn30 to to shown in online supplementary figures and Events for PFS were recorded in all patients and median PFS was months CI to figure All deaths were recorded median OS was months CI to with a month and year OS of CI to and CI to figure safetyPatients received the study treatment for four cycles at median range “ The median relative dose intensity was IQR “ Dose interruptions and reductions were required in and patients respectively The major treatment related adverse events ‰¥ for dose reduction were proteinuria patients palmar plantar erythrodysesthesia patients diarrhoea patients hypertension patients fatigue patients and thrombocyt ia patients The reasons for treatment discontinuation of all patients were disease progression in patients and adverse events in patients gastrointestinal perforation and grade proteinuria in of each After treatment with lenvatinib patients received a subsequent treatment online supplementary table Most patients only had mild grades “ adverse events table The most common grade ‰¥ adverse events were hypertension patients thrombocyt ia patients increased alanine aminotransferase and anorexia patients each No clear relationship was found between the incidence of lenvatinib associated adverse event of any grade and baseline body surface area online supplementary table Serious adverse events occurred in four patients including Figure Waterfall plot analysis of maximum percentage change from baseline in measurable target lesions Response Evaluation Criteria in Solid Tumors version central reviewIwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776 0c accessFigure Kaplan Meier curves of A progression free survival PFS by investigator assessment and B overall survival OS in all patients n30five treatment associated events anorexia in two and gastrointestinal perforation central venous catheter related bloodstream infection caused by Staphylococcus aureus and nausea in each one in each of four patients all patients recovered from these adverse eventssubgroup analysisIn patients with wild type RAS the median PFS was months CI to and that was months CI to in patients with mutant RAS online supplementary figure In patients with wild type RAS the median OS was months CI CI to and months CI to in patients with mutant RAS online supplementary figure Plasma angiopoietin2 levels were decreased by lenvatinib treatment in almost all patients and increased at the Iwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776time of treatment discontinuation online supplementary table With a first quartile cut off point17 the eight patients with a first quartile or lower level of angiopoietin2 had a median OS of months CI to months compared with months CI to in the patients with higher than a first quartile level of angiopoietin2 HR CI to online supplementary figure Patients with a first quartile or less level of angiopoietin2 had a median PFS of months CI to compared with months CI to in the patients with more than a first quartile level of angiopoietin2 HR CI to online supplementary figure 0c accessTable Treatment related adverse events occurring in ‰¥ patients N30Any gradeGrade ‰¥Treatment related adverse eventHypertensionProteinuriaThrombocyt iaFatigueHypothyroidismWeight lossHoarsenessPalmar plantar erythrodysesthesia syndromeAnorexiaDiarrhoeaMucositis oralSerum AST increasedSerum creatinine increasedAST Aspartate transaminase dIsCussIonPatients with metastatic colorectal cancer with disease progression after three or more lines of therapy have limited treatment options In this label single arm phase study of patients with previously treated metastatic colorectal cancer lenvatinib demonstrated manageable toxic effects and promising antitumour activity A total of out of patients had disease control including with partial responses Moreover patients experienced reduction in measurable tumour size The overall toxicity profiles were similar to that reported for lenvatinib across a spectrum of advanced malignant neoplasmsTwo recent international phase studies reported that regorafenib or trifluridinetipiracil provided significant improvements in DCR PFS and OS compared with placebo in patients with metastatic colorectal cancer after failure of standard chemotherapies DCR median PFS months median OS months in the CORRECT study and DCR median PFS months median OS months in the RECOURSE study6 Interestingly the present single arm phase study of lenvatinib revealed favourable DCR and median PFS values in patients with metastatic colorectal cancer compared with those in the regorafenib or trifluridinetipiracil study Moreover about half of the patients received post study treatment which led to a favourable OSThe lenvatinib safety profile in this study was similar to the published safety profiles of lenvatinib for thyroid cancer and hepatocellular carcinoma in the Japanese population18 Moreover we found no unexpected or off target safety signals The most common adverse events were hypertension proteinuria thrombocyt ia and fatigue while the most case of grade or hypertension and proteinuria required treatment interruption and dose reduction While the target population for thyroid cancer or hepatocellular carcinoma that showed efficacy for lenvatinib was first line setting20 this study targeted patients receiving salvage line therapy Most patients with metastatic colorectal cancer in the salvage line setting had grade or proteinuria and hypertension at baseline because of the long term prior treatment with anti VEGFVEGFR treatment whereas the occurrence of grade hypertension was significantly higher compared with that of regorafenib in a similar study population in the CORRECT CONCUR and CONSIGN trials7 It was manageable by dose reduction or interruption but it may be necessary to consider the starting dose in the future Although palmar plantar erythrodysesthesia is a not life threatening toxicity these adverse events have a significant impact on treatment schedules and quality of life in treated patients Grade ‰¥ palmar plantar erythrodysesthesia has been observed in and of patients treated with lenvatinib in this study and the SELECT Japanese population15 respectively while in patients treated with regorafenib in the CORRECT Japanese population16 To date the clear mechanism of palmar plantar erythrodysesthesia by VEGF receptor tyrosine kinase inhibitors is not known but it has been reproduced that palmar plantar erythrodysesthesia by lenvatinib is well tolerated Overall it is suggested that lenvatinib might be a favourable treatment option in terms of toxicitiesSeveral preclinical studies demonstrated that VEGF targeted treatment affects immune suppression by promoting the expansion of suppressive immune cell populations such as regulatory T cells and myeloid derived suppressor cells24 Several clinical studies suggested that modulation of VEGF mediated immune suppression via angiogenesis inhibition could potentially augment the immunotherapeutic activity of anti programmed cell death PD1 antibody26 Regorafenib and nivolumab showed antitumour activity in patients with metastatic colorectal cancer including those with microsatellite stable tumours in a phase study28Angiopoietin2 a relatively novel regulator of angiogenesis that acts through the TEK tyrosine kinase endothelial Tie2 receptor has been identified as a potential prognostic biomarker for some types of cancer Although the baseline Ang2 level was a predictive biomarker in patients with thyroid cancer in the SELECT trial17 it did not become a reliable biomarker of lenvatinib response in this study Prior treatment with anti VEGFVEGFR antibodies probably had an effect on baseline angiopoietin2 levels because the study population was refractory to standard treatment in this study The decrease in angiopoietin2 levels was observed after treatment therefore it may be an indicator of treatment responseThe limitations of our study include its small size which could limit the interpretation of the subgroup analyses Iwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776 0cand the absence of a comparison group However the level of clinical benefit in the form of confirmed responses observed in this study was remarkable in the historical context of other clinical trials done in heavily pretreated patients with metastatic colorectal cancer Moreover most of the patients in our study had left sided tumours which were known to have a better prognosis compared with right sided tumoursIn conclusion lenvatinib provided promising activity with prolonged survival relative to the anticipated median PFS in heavily pretreated patients with metastatic colorectal cancer The safety profile of lenvatinib was similar to that in other tumour types with no new safety signals recorded Based on these findings further investigation of lenvatinib with anti PD1 antibody or other novel combinations with the potential to build on the benefit of lenvatinib is currently taking place NCT03797326 and NCT04008797acknowledgements The authors thank the patients and their families the members of the Clinical Research Support Office for their support with data collection and running the study and NAI incorporated for editing a draft of this manuscriptContributors All authors conceived and designed the study and drafted and revised the manuscript for publication SI NO HS YH AT KK TH NB and YY collected data AK GO MK and KN analysed the data and managed data and study progress All authors interpreted the data and approved the final version of the manuscriptFunding The study was supported by the Project Promoting Clinical Trials for Development of New Drugs and Medical Devices Japan Medical Association from the Japan Agency for Medical Research and Development Grant Number JP18lk0201037 and by Eisai CoCompeting interests SI has received research grants from Eisai and Merck Biopharma TH has received research grants from Eisai and honoraria from Merck Serono YY has received honoraria from EisaiPatient consent for publication Not requiredethics approval The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines The study protocol was approved by the National Cancer Center Institutional Review Board T4329Provenance and peer review Not commissioned externally peer revieweddata availability statement Data are available upon reasonable request Proposals should be directed to siwasa ncc go jp The data will be available for achieving aims in the approved proposal access This is an access article distributed in accordance with the Creative Commons Attribution Non Commercial CC BY NC license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited any changes made are indicated and the use is non commercial See a0http creativecommons licenses by nc oRCId idsSatoru a0Iwasa http orcid Yasuhide a0Yamada http orcid RefeRences Saltz LB Clarke S Díaz Rubio E et a0al Bevacizumab in combination with oxaliplatin based chemotherapy as first line therapy in metastatic colorectal cancer a randomized phase III study J Clin Oncol “ Tabernero J Yoshino T Cohn AL et a0al Ramucirumab versus placebo in combination with second line FOLFIRI in patients with metastatic colorectal carcinoma that progressed during or after first line therapy with bevacizumab oxaliplatin and a fluoropyrimidine raise a randomised double blind multicentre phase study Lancet Oncol “ access Van Cutsem E Tabernero J Lakomy R et a0al Addition of aflibercept to fluorouracil leucovorin and irinotecan improves survival in a phase III randomized trial in patients with metastatic colorectal cancer previously treated with an oxaliplatin based regimen J Clin Oncol “ Yamazaki K Nagase M Tamagawa H et a0al Randomized phase III study of bevacizumab plus FOLFIRI and bevacizumab plus mFOLFOX6 as first line treatment for patients with metastatic colorectal cancer WJOG4407G Ann Oncol “ Price TJ Peeters M Kim TW et a0al Panitumumab versus cetuximab in patients with chemotherapy refractory wild type KRAS exon metastatic colorectal cancer ASPECCT a randomised multicentre label non inferiority phase study Lancet Oncol “ Mayer RJ Van Cutsem E Falcone A et a0al Randomized trial of TAS102 for refractory metastatic colorectal cancer N Engl J Med “ Grothey A Van Cutsem E Sobrero A et a0al Regorafenib monotherapy for previously treated metastatic colorectal cancer correct an international multicentre randomised placebo controlled phase trial Lancet “ Matsui J Yamamoto Y Funahashi Y et a0al E7080 a novel inhibitor that targets multiple kinases has potent antitumor activities against stem cell factor producing human small cell lung cancer H146 based on angiogenesis inhibition Int J Cancer “ Matsui J Funahashi Y Uenaka T et a0al Multi Kinase inhibitor E7080 suppresses lymph node and lung metastases of human mammary breast tumor MDA MB231 via inhibition of vascular endothelial growth factor receptor VEGF R and VEGF R3 kinase Clin Cancer Res “ Wiegering A Korb D Thalheimer A et a0al E7080 lenvatinib a multi targeted tyrosine kinase inhibitor demonstrates antitumor activities against colorectal cancer xenografts Neoplasia “ Hong DS Kurzrock R Wheler JJ et a0al Phase I dose escalation study of the multikinase inhibitor lenvatinib in patients with advanced solid tumors and in an expanded cohort of patients with melanoma Clin Cancer Res “ Boss DS Glen H Beijnen JH et a0al A phase I study of E7080 a multitargeted tyrosine kinase inhibitor in patients with advanced solid tumours Br J Cancer “ Yamada K Yamamoto N Yamada Y et a0al Phase I dose escalation study and biomarker analysis of E7080 in patients with advanced solid tumors Clin Cancer Res “ Nakamichi S Nokihara H Yamamoto N et a0al A phase study of lenvatinib multiple receptor tyrosine kinase inhibitor in Japanese patients with advanced solid tumors Cancer Chemother Pharmacol “ Kiyota N Schlumberger M Muro K et a0al Subgroup analysis of Japanese patients in a phase study of lenvatinib in radioiodine refractory differentiated thyroid cancer Cancer Sci “ Yoshino T Komatsu Y Yamada Y et a0al Randomized phase III trial of regorafenib in metastatic colorectal cancer analysis of the correct Japanese and non Japanese subpopulations Invest New Drugs “ Tahara M Schlumberger M Elisei R et a0al Exploratory analysis of biomarkers associated with clinical outcomes from the study of lenvatinib in differentiated cancer of the thyroid Eur J Cancer “ Takahashi S Kiyota N Yamazaki T et a0al A Phase II study of the safety and efficacy of a0lenvatinib in patients with advanced thyroid a0cancer Future Oncol “ Yamashita T Kudo M Ikeda K et a0al REFLECT a phase trial comparing efficacy and safety of lenvatinib to sorafenib for the treatment of unresectable hepatocellular carcinoma an analysis of Japanese subset J Gastroenterol “ Kudo M Finn RS Qin S et a0al Lenvatinib versus sorafenib in first line treatment of patients with unresectable hepatocellular carcinoma a randomised phase non inferiority trial Lancet “ Schlumberger M Tahara M Wirth LJ et a0al Lenvatinib versus placebo in radioiodine refractory thyroid cancer N Engl J Med “ Li J Qin S Xu R et a0al Regorafenib plus best supportive care versus placebo plus best supportive care in Asian patients with previously treated metastatic colorectal cancer concur a randomised double blind placebo controlled phase trial Lancet Oncol “ Van Cutsem E Martinelli E Cascinu S et a0al Regorafenib for patients with metastatic colorectal cancer who progressed after standard therapy results of the large single arm label phase IIIB CONSIGN study Oncologist “Iwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776 0c access Ott PA Hodi FS Buchbinder EI Inhibition of immune checkpoints and vascular endothelial growth factor as combination therapy for metastatic melanoma an overview of rationale preclinical evidence and initial clinical data Front Oncol Kato Y Tabata K Kimura T et a0al Lenvatinib plus anti PD1 antibody combination treatment activates CD8 T cells through reduction of tumor associated macrophage and activation of the interferon pathway PLoS One 201914e0212513 Taylor MH Lee C H Makker V et a0al Phase IbII trial of lenvatinib plus pembrolizumab in patients with advanced renal cell carcinoma endometrial cancer and other selected advanced solid tumors J Clin Oncol “ Makker V Rasco D Vogelzang NJ et a0al Lenvatinib plus pembrolizumab in patients with advanced endometrial cancer an interim analysis of a multicentre label single arm phase trial Lancet Oncol “ Fukuoka S Hara H Takahashi N et a0al Regorafenib plus nivolumab in patients with advanced gastric GC or colorectal cancer CRC an label dose finding and dose expansion phase 1B trial REGONIVO EPOC1603 JCO Iwasa a0S et a0al ESMO 20205e000776 101136esmo 2020000776 0c'

Thyroid_Cancer

properly citedIntroduction Endogenously produced antiganglioside antibodies could aï¬ect the evolution of cutaneous melanoma Epidemiologicaland experimental evidence suggest œchronic ‚ammation to be one of the hallmarks in skin cancers The aim of the study was tocharacterize the relation between antiganglioside antibodies and ‚ammation in cutaneous melanoma focusing on gangliosidesGM1 GM2 GM3 GD1a GD1b GT1b GQ1b Material and Method We performed an observational study that included subjects subdivided into three groups patients with metastatic melanoma cases patients with primary melanoma casesand healthy subjects subjects The assessment of antiganglioside antibodies IgG and IgM classes against GM1 GM2GM3 GD1a GD1b GT1b GQ1b was performed using immunoblot technique EUROLine kit Results The presence of IgGand IgM antiganglioside antibodies in primary melanoma was as follows antiGM1 and GM2 and GM3 and GD1a and GD1b and GT1b and GQ1b and In metastaticmelanoma the level of antiganglioside antibodies was significantly lower compared with primary melanoma p while inthe control group they were absent Antiganglioside antibodies antiGM1 and GD1a were positively correlated while antiGM3GD1b and GT1b were negatively associated with the ‚ammatory markers interleukin IL8 and C reactive protein CRPConclusions Tumour ganglioside antigens generate an immune response in patients with primary melanomas The host™s ability toelaborate an early antiganglioside response could be considered as a defence mechanism directed toward eliminating a dangersignal from the tumour microenvironment Antiganglioside antibodies associated with ‚ammation markers could be used asdiagnostic monitoring and treatment tools in patients with cutaneous melanoma IntroductionGangliosides are a group of bioactive glycolipids located onthe outer face of cell membranes These glycolipids play amajor role in cell proliferation diï¬erentiation migrationapoptosis signal transduction cell adhesion modulatinggrowth factor or hormone receptor antigen recognition protein trafficking viral transformation and oncogenesis [“]Atypical expression of some ganglioside antigens associatedwith certain tumours neuroblastomas melanomas gliomaslymphomas small cell lung cancer and prostate cancer andfurthermore could play an importantrole in cancer 0cJournal of Immunology Researchimmunotherapy [“] Gangliosides that are released inextracellular spaces could have dual action antitumor andprotumour eï¬ect [“] Data regarding the endogenousimmune response directed toward tumour gangliosides andthe significance of this response are limited A series of studiesperformed in in vivo experimental models and in vitro inmurine and human cancer cells have shown that monoclonalantiganglioside antibodies have antitumor potential Theseantibodies exert numerous antitumor eï¬ects through variousmechanisms An important mechanism is the translocationof gangliosides from the plasmatic membrane into theintracellular spaces so binding of antibodies to the surfaceof the tumor cells and complement activation that leads to celllysis mediated by complementdependent cytotoxicity andantibodymediated cellular cytotoxicity [ ] Antiganglioside antibodies modulate ceramide synthesis [ ]reception and transduction of the cytotoxic signal [] theyare involved in suppression or induction of cell death throughdiï¬erent pathways apoptosis necrosis oncogeneslike structural and functional changes of mitochondria accumulationof reactive oxygen species acetylation of gangliosides accumulation of sphingosine sphingamine ceramides [ ]Proteomic studies showed that antiganglioside antibodiescould induce changes like the disruption of signalling systemsP38MAPK PARP JNK123 METc ERK12 P13KAKTand FAK modulation of the level and function of transcription factors P53 SP1 MYCN and HSF1 regulating the balance between apoptosisinducing and apoptosissuppressingfactors cysteineaspartylproteases Bax Bcl2 [ “]These antibodies stimulate the cytotoxicity of chemotherapeutic drugs and small molecule inhibitors [ ] As a result antiganglioside antibodies could be used as diagnostic monitoringand treatment tools in cancer patients [ ]Ganglioside levels are increased in malignant melanocytes and represent an important topic of research [ ]Several researchers have emphasized the role of glycolipidsas markers of melanoma A study analysing the expressionof gangliosides in melanocyte lines and melanoma cell linesfound out an increased expression of GD3 synthase genesin melanoma cells but not in melanocytes The same resultswere obtained for GM2GD2 synthase [] It seems thatgangliosides induce cell proliferation and invasion throughp130Cas and paxillin in melanoma cells []Inflammatory mechanisms play an important role inmelanoma Multiple studies have shown that plasma levelsof C reactive protein CRP increase during tumor proliferation and several relations have been evaluated CRPsurvivalrelationship CRPresponse therapy CRP‚ammationNowadays CRP is considered a true marker for assessing‚ammation in melanoma as well as a marker for responseto treatment Prospective studies have provided consistentresults in the predictive value of CRP in neoplastic diseaseproving high sensitivity and specificity [] In addition inmelanoma elevated levels of CRP may reflect the amountand activity of circulating pro‚ammatory cytokines eginterleukin IL8 IL8 plays a crucial role in regulating cellfunction for host defence and for developing natural immunity [ ] Moreover IL8 is released by various cell typesincluding polymorphonuclear neutrophils PMNs monoSerum dilution Incubation with fixed on strip antigens degrees min with balanceWashing with universal tampon times min each with balanceIncubation with conjugated enzyme degrees min with balanceWashing with universal tampon times min each with balanceIncubation with fixed on strip antigens degrees min with balanceWashing with universal tampon times min each with balanceEUROLine scan evaluationFigure Antiganglioside detectioncytes T lymphocytes and endothelial cells upon exposureto ‚ammatory stimuli Melanoma cells have been reportedto express IL8 and this ‚uences their oncogenic properties[ ] IL8 follows the evolution of melanoma progression and regression under treatment reflecting the stage ofthe disease [“]Based on these accumulating data we have investigatedantiganglioside antibodies in correlation with other ‚ammatory markers IL8 CRP and the clinical evolution of the melanoma patients Clarifying these relations could significantlyimprove the prediction of clinical outcomes Furthermore itcan lead to the development of appropriate therapeutic strategies in patients with cutaneous melanoma Material and Method Patients We performed an observational prospectivestudy during years in Clinical Hospital for Infectious andTropical Diseases œVictor Babes”Dermatology Department Bucharest The study was approved by the Ethics Committee of the Hospital All participants agreed to be includedin research studies without prejudice of the diagnosis orpersonal image and signed the informed consent accordingto the Declaration of HelsinkiThe study included adult patients with cutaneous melanoma with no other pathologies and no treatment for theprimary disease Exclusion criteria were age under yearspregnancy alcohol use melanoma under treatmentWe performed an observational study that included subjects subdivided in three groups patients with metastaticmelanoma cases patients with primary melanoma cases and healthy subjects with matching sex and age subjects Patients were selected and examined accordingto ESMO Clinical Practice Guidelines for melanoma 0cJournal of Immunology ResearchTable Test strips coated with parallel lines of purified antigensAntigenGM1GM2GM3GD1aGD1bGT1bGanglioside typeSourceMonosialoganglioside GM1Monosialoganglioside GM2Monosialoganglioside GM3 Dog erythrocytesBovine brainBovine brainDisialoganglioside GD1aDisialoganglioside GD1bTrisialoganglioside GT1bBovine brainBovine brainBovine brainGQ1bTetrasialoganglioside GQ1bBovine brainStructureGal3GalNAc4[Neu5Ac3]Gal4GlcCerGalNAc4[Neu5Ac3]Gal4GlcCerNeu5Ac3Gal4GlcCerNeu5Ac3Gal3GalNAc4[Neu5Ac3]Gal4GlcCerGal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCerNeu5Ac3Gal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCerNeu5Ac8Neu5Ac3Gal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCer Glcglucose Gal galactose GalNAc Nacetylgalactosaminediagnosis based on clinical histopathological immunohistochemical and imagistic data All the events related to theprogression of the disease were recorded relapse metastasisneurotoxicity hyper reactivation of the immune system upontreatment The group characteristics were similar for age andsex the primary melanoma group included women and men with a mean age of ± years the metastaticmelanoma group included women and men with a meanage of ± years and the control group included women and men with mean age of ± years Materials and Reagents In this work the assessment ofantiganglioside antibodies was made by the immunoblottechnique using EUROLine kits Figure This methodallows the evaluation of antibodies IgG and IgM classesagainst GM1 GM2 GM3 GD1a GD1b GT1b and GQ1bfrom serumplasma The kit contains strips marked withpurified antigens Table Theevaluation ofantigangliosideantibodies wasperformed using the EUROLine Scan software After readingthe signal intensity on the strips marked with ganglioside antigens the results were evaluated and the results are presented asoptical sensibility The assessment of IL8 was performed bythe ELISA method using Enzo Life Science reagents withTECAN analyser and the results are presented as pgdl CRPwas assessed by immunoturbidimetry using Human reagentsand HumaStar300 analyser the results are presented as mgdl Statistical Analysis All the results were analysed usingIBM SPSS Statistics We evaluated the normality of datadistribution using the KolmogorovSmirnov test The variationbetween groups was determined using the parametric teststtest when two groups were compared or ANOVA test whenmore groups were compared and nonparametric tests likeMannWhitney or Wilcoxon The correlation between groupswas evaluated using linear regression and Pearson coefficientp was considered with statistical significance ResultsAntiGM1 GM2 GM3 GD1a GD1b GT1b andGQ1b autoantibodies determined in primary metastaticmelanoma had diï¬erent serological profiles compared tothe control group The presence of IgG and IgM antiganglioside antibodies in primary melanoma was as followsantiGM1 and antiGM2 and antiGM3 and antiGD1a and antiGD1b and antiGT1b and antiGQ1b and Inmetastatic melanoma IgG and IgM antiganglioside antibodies had the following profile antiGM1 and antiGM2 and antiGM3 and antiGD1a and antiGD1b and antiGT1b and and antiGQ1b and In the control group antiganglioside autoantibodies were absentThe assessment for IgG antiGM1 antiGM2 antiGM3antiGD1a antiGD1b antiGT1a and antiGT1b showedextremely low signal intensity in all groups Figure Whencomparing the mean of signal intensity for IgG no statisticaldiï¬erences were observed between groups We obtained a statistically significant diï¬erence in IgM antiGM1 antiGM2antiGM3 antiGD1a antiGD1b antiGT1a and antiGT1b when comparing primary melanoma respectively metastatic melanoma to the control group and once more whencomparing primary versus metastatic melanoma Table To evaluate if the presence of IgM antibodies was associatedwith melanoma development we determined their relation to‚ammatory factors IL8 and CRP recommended by AJCCfor melanoma staging Table IL8 levels were statistically significantly increased in primary melanoma ± pgmland in metastatic melanoma ± pgml when compared with the control group ± pgml CRP levelswere found in primary ± ngml and metastaticmelanoma ± ngml significantly higher when compared with the control group ± ngml IL8 andCRP had no statistically significant variation when comparedto primary versus metastatic melanoma groups Positive correlations with statistical significance were determined betweenantiGM1 and CRP respectively IL8 between antiGD1aand CRP respectively IL8 Figure Negative significant correlations were observed between antiGM3 antiGT1b andCRP respectively IL8 Figure High levels of CRP and IL were associated with an increase in antiGM1 antiGD1aand a decrease in antiGM3 antiGM2 antibodies of IgM typeTable DiscussionsMelanoma the most aggressive skin tumour is a multifactorial cancer being the result of the interplay between geneticimmunological and environmental factors [“] Gangliosides due to their expression on tumor cells have beeninvolved in tumor biology and immunogenicity and hence 0cJournal of Immunology ResearchAntiGM1 IgM and IgG signalintensity in all groups AntiGM2 IgM and IgG signalintensity in all groups ControlPrimary melanomaMetastatic melanomaControlPrimary melanomaMetastatic melanomaAntiGM1 IgM Ž AntiGM1 IgGaAntiGM3 IgM and IgGintensity levels in all groups AntiGM2 IgM Ž AntiGM2 IgGbAntiGD1a IgM and IgGintensity signal in all groups ControlPrimary melanomaMetastatic melanomaControlPrimary melanomaMetastatic melanomaAntiGM3 IgM Ž AntiGM3 IgGcControlPrimary melanomaMetastatic melanomaAntiGD1b IgM and IgG signalintensity in all groupsControlPrimary melanomaMetastatic melanomaAntiGD1b IgM Ž AntiGD1b IgGAntiGD1a IgM Ž AntiGD1a IgGdAntiGT1b IgM and IgGintensity levels in all groups AntiGT1b IgM Ž AntiGT1b IgGefAntiGQ1b IgM and IgGintensity levels in all groupsControlPrimary melanomaMetastatic melanomaAntiGQ1b IgM Ž AntiGQ1b IgGgFigure Antiganglioside signal intensity in all groups ˆ—p have been considered as targets for cancer immunotherapy[“] The probability thatsome tumourassociatedganglioside determinants induce a human immune responsegenerated much interest in medical research [ “]If endogenously synthesized antiganglioside antibodies reactonly with human cancer cells these antibodies could play animportant role in the host™s protective immunity to thetumor There is little information about quantitative variations of serum antigangliosides their origin and progressionof melanoma Tumour ganglioside antigens generate a significantly increased immune response in patients with primarymelanoma versus metastatic melanoma In our study thehost™s ability to generate an early antiganglioside responseis supported by a significantly increased titter of IgMantibodies in patients with primary versus metastatic melanoma and the control groupfor antiGM1 antiGM2antiGM3 antiGD1a antiGD1b antiGT1b and antiGQ1b Figure The range of antiganglioside antibodiescould serve as an indicator of diï¬erentiation between patientswith primary melanoma and metastatic melanoma Based on 0cJournal of Immunology ResearchTable IgM antiganglioside signal intensity in all groupsAntibodies Classp significancePM vs MM PM vs control MM vs controlIgMAntiGM1IgMAntiGM2IgMAntiGM3AntiGD1aIgMAntiGD1b IgMAntiGT1bIgMAntiGQ1b IgMMM metastatic melanoma PM primary melanoma psignificancestatisticalTable IL8 and CRP in all studied groupsStudy groupPrimarymelanomaMetastaticmelanomaControlgroupCRPmgdl ± ± ± psignificance”IL8pgml ± ± ± psignificance”our findings we estimate that the levels of the antigangliosideantibodies could provide information regarding the clinicalstaging of melanomaIn addition the capacity of patients to develop an antiganglioside response in the early stage of development of melanoma could be understood as a mean of defence of the bodythrough eliminating a danger signal from the tumour microenvironment represented by the stimulation of glycosphingolipid synthesis [ ] Synthesis of antiganglioside antibodiescould confer a survival advantage in patients with primarymelanoma [] In metastatic melanoma patients we observeda reduction of antiganglioside antibody synthesis a result thatcould suggest the immunosuppressive eï¬ect exerted by theoverproduction of gangliosides associated with tumour metastasis andor due to the overall decreased immune responseIt has been shown in a previous study that in patients withuntreated primary melanoma there is a significant statisticalcorrelation between antiGM1 type IgM level and clinicalstage of the disease Breslow index Clark level tumour localization histologic type presenceabsence of ulceration [ ]In our study patients with cutaneous melanoma had detectable levels of antiGM1 in primary stages The presence of apositive significant relationship between IgM antiGM1 leveland IL8 and CRP in our study justified our statement regarding the involvement of these antibodies in tumour proliferation by stimulating ‚ammation [ “] The presentstudy is the first one that evaluated the relation betweenantiganglioside antibodies and IL8 and CRP based on theirrole in melanoma diagnosis progression and outcome [“] in metabolic disorders [ ]Previous studies in patients with prostate cancer [] orsarcoma [] have shown that antiGM1 antibodies had nodiagnostic or prognostic value in these pathologies In patientswith diï¬erentiated thyroid cancer antiGM1 type IgG andIgM were associated with carcinogenesis but the lack of correlation between antibody level and clinical status indicated thatantiGM1 had no diagnostic value in diï¬erentiated thyroidcancer []Another study performed by our group showed thatpatients with primary melanoma with a high level of IgMantiGM3 had a favourable prognosis compared withpatients displaying a low antibody titer [] In a study onpatients with primary untreated melanoma stages I andII lymph nodes clear of metastasis it was shown that theantiGM2 antibody titer for IgMtype was not diï¬erentiatedin correlation to the tumor thickness For antiGM3 it wasobtained a direct relationship between the serum titer andthe thickness of the tumor [] AntiGM2 and antiGM3antibodies have no diagnostic significance in thyroid cancerdue to the low prevalence of these antibodies [] In ourpresent study antiGM3 negative correlations with IL8and CRP are suitable with the hypothesis that patients withprimary melanoma with a high level of IgM antiGM3 havea favourable prognosisGD1a was thought to generate an immune response inpatients with earlystage melanoma [] In patients withT1T2 stage prostate cancer there were identified increasedIgM antiGD1a values compared to the T3T4 stage whichsustains the development of an early endogenous immuneresponse able to eliminate the danger signal from the tumormicroenvironment These data support the role of antiGD1ain the early diagnosis of localized prostate disease [] TheantiGD1a IgMtype titer was defined as a negative predictivefactor of survival in patients with soft tissue sarcoma [] andin patients with primary melanoma [] In patients™ serumdiagnosed with ovarian cancer an increased titer of IgMantiGD1 was found the authors pointing out that theseantibodies could represent immunological markers associated with ovarian cancer progression [] In cutaneousmelanoma IgM antiGD1a could be considered a markerassociated with melanoma progression based on the negativecorrelation with CRP and IL8 as shown in our studyThe antiGD1b immune response in patients with gastricneoplasm can be used as a prognostic marker [] On thecontrary the lack of correlation between the presence ofantiGD1b and the clinical status of patients with thyroidcancer has indicated that antigangliosides do not have diagnostic significance in this neoplasm []The antiGT1b titer can be an overall positive factor associated to global survival in sarcoma [] AntiGD1b GT1band GQ1b antibodies that are negatively correlated with IL8and CRP suggest that they could indirectly suppress tumorgrowth and angiogenesis AntiGD1b GT1b and GQ1bIgM type ‚uence the progression of melanoma [ ]soft tissue sarcomas [ ] Ehrlich subcutaneous solidtumors [ ] Ehrlich carcinomas accompanied by ascites[ ] and gastric cancer []Our study limitations could be considered the semiquantitative assessment method of antiganglioside antibodies as aquantitative determination technique could oï¬er more sensitive data ing the door for further studies One more 0cJournal of Immunology ResearchMGitnAMGitnAaDGitnA CRP CRP CRP MGitnAMGitnAaDGitnAIL8IL8IL8Figure AntiGM1 antiGM3 and antiGD1a in relation to CRP and IL8 in the primary melanoma groupimportant limitation of the study is that we evaluated thecorrelation between antiganglioside antibodies and ‚ammation markers in melanoma IL8 and CRP only after orbetween the surgical treatment of melanoma newer therapiesbeing also in place This could be the first study which otherresearchers and clinicians can use and analyze in order toevaluate the ‚uence of diï¬erent melanoma treatments onantiganglioside antibodies The pathogenic mechanismsinvolved in melanoma are complex [“] therefore theevaluation during the followup period of melanoma patientsat diï¬erent points is needed for a better antigangliosideprofile characterization 0cJournal of Immunology ResearchTable AntiGM1 GM2 GM3 GD1a GD1b GT1b andGQ1b relation with ‚ammatory markers in primary melanomagroupAntiGM1AntiGM2AntiGM3AntiGD1aAntiGD1bAntiGT1bAntiGQ1bCRPr p ‰¤ r ˆ’p r ˆ’p r p ‰¤ r ˆ’p ‰¤ r ˆ’p ‰¤ r ˆ’p IL8r p ‰¤ r ˆ’p r ˆ’p r p ‰¤ r ˆ’p ‰¤ r ˆ’p ‰¤ r ˆ’p ConclusionsAntiganglioside antibodies antiGM1 and GD1a werepositively correlated while antiGM3 GD1b and GT1bwere negatively associated with the ‚ammatory markersIL8 and CRP The host™s ability to elaborate an early antiganglioside response could be considered as a defence mechanism directed toward eliminating a danger signal from thetumour microenvironment Moreover our results suggest thattumour ganglioside antigens generate a significantly increasedimmune response in patients with primary versus metastaticmelanoma Antiganglioside antibodies associated with ‚ammation markers could be used as diagnostic monitoring andtreatment tools in patients with cutaneous melanomaData AvailabilityThe data used to support the findings of this study areincluded within the articleConflicts of InterestThe authors declare no conflicts of interestAuthors™ ContributionsAll authors have equally contributed to the writing and editing of the manuscriptAcknowledgmentsThis research and article processing charges were funded by agrant of the Romanian Ministry of Research and InnovationCCCDIUEFISCDI project number 61PCCDI2018 PNIIIP112PCCDI2017034References[] YH Xu S Barnes Y Sun and G A Grabowski œMultisystem disorders of glycosphingolipid and ganglioside metabolism Journal of Lipid Research vol no pp “ [] I Horwacik and H Rokita œTargeting of tumorassociatedgangliosides with antibodies aï¬ects signaling pathways andleads to cell death including apoptosis Apoptosis vol no pp “ [] J L Daniotti R D Lardone and A A Vilcaes œDysregulatedexpression of glycolipids in Tumor cells from negative modulator of Antitumor immunity to promising targets for developing therapeutic agents Frontiers in Oncology vol p [] I Nicolae A Caragheorgheopol S Schipor œGnagliosides and sex hormones in human melanoma Acta Endocrinologica vol no pp “ [] Y Ohmi M Kambe Y Ohkawa œDiï¬erential roles ofgangliosides in malignant properties of melanomas PLoSONE vol no article e0206881 [] M H Ravindranath S Muthugounder N Presser A D Santin R S Selvan and D L Morton œGanglioside GD1a present in ovarian cancer cells ascites and sera of patients elicitsendogenous IgM response Proceedings of the American Association for Cancer Research vol p [] V B Doronin T A Parkhomenko M Castellazzi et alœComparison of antibodies hydrolyzing myelin basic proteinfrom the cerebrospinal fluid and serum of patients with multiple sclerosis PLoS One vol no article e107807 [] C D Ene and I Nicolae œGangliosides and Antigangliosidesin Malignant Melanoma Melanoma Current Clinical Management and Future Therapeutics M Murph Ed MandiMurph Intech ISBN [] N AlvarezRueda S Leprieur B Clemenceau œBindingactivities and antitumor properties of a new mousehumanchimeric antibody specific for GD2 ganglioside antigen Clinical Cancer Research vol no pp 5613s“5620s [] K Bennaceur I Popa J A Chapman œDiï¬erent mechanisms are involved in apoptosis induced by melanoma ganglicellsosidesGlycobiology vol no pp “ on human monocytederived dendritic[] C D Nicolae and I Nicolae œAntibodies against GM1 gangliosides associated with metastatic melanoma Acta Dermatovenerologica Croatica vol no pp “ [] S GrouxDegroote Y Guerardel and P Delannoy œGangliosides structures biosynthesis analysis and roles in cancerChembiochem vol no pp “ [] C Tringali I Silvestri F Testa œMolecular subtyping ofmetastatic melanoma based on cell ganglioside metabolismprofiles BMC Cancer vol no p [] M Neagu C Constantin C Caruntu C Dumitru M Surceland S Zurac œInflammation a key process in skin tumorigenesis Oncology Letters vol no pp “ [] R Takeuchi M Kambe M Miyata œTNFαsignal andcAMPmediated signals oppositely regulate melanomaassociated ganglioside GD3 synthase gene in human melanocytes Scientific Reports vol no p [] A H Otake R de Freitas Saito A P M Duarte A F Ramosand R Chammas œGD3 gangliosideenriched extracellular vesiclesetstimulate melanocyte migration Biochimica 0cJournal of Immunology ResearchBiophysica Acta Molecular and Cell Biology of Lipidsvol no pp “ [] K Hamamura K Furukawa T Hayashi œGangliosideGD3 promotes cell growth and invasion through p130Casand paxillin in malignant melanoma cells Proceedings of theNational Academy of Sciences of the United States of Americavol no pp “ [] M Neagu C Constantin C Caruntu M Surcel D Boda andS Zurac œCytokine pattern for improving immunoscore inmelanoma patients European Journal ofImmunologyvol pp [] M Neagu C Constantin and C Longo œChemokines in themelanoma metastasis biomarkers portrait Journal of Immunoassay Immunochemistry vol no pp “ [] K A Timani B Gyorï¬y Y Liu K S Mohammad and J J HeœTip110SART3 regulates IL8 expression and predicts theclinical outcomes in melanoma Molecular Cancer vol no p [] S J Wigmore K C H Fearon J P Maingay P B S Lai andJ A Ross œInterleukin8 can mediate acutephase protein production by isolated human hepatocytes American Journal ofPhysiologyEndocrinology and Metabolism vol no pp E720“E726 [] M Bickel œThe role of interleukin8 in ‚ammation andJournal of Periodontologyregulationmechanisms ofvol no pp “ [] M Neagu C Constantin and S Zurac œImmune Parametersin The Prognosis and Therapy Monitoring of Cutaneous Melanoma Patients Experience Role and Limitations BioMedResearch International vol Article ID pages[] S R Georgescu M R Ioghen M I Sarbu œBiologicaltherapy in the treatment of melanoma Journal of Mind andMedical Sciences vol no pp “ [] A V Dumitru M Tampa S R Georgescu œImmunohistochemical mismatch in a case of rhabdomyoblastic metastaticmelanoma Romanian Journal of Morphology and Embryology vol no pp “ [] S N Pavri J Clune S Ariyan and D Narayan œMalignantMelanoma Plastic and Reconstructive Surgery vol no pp 330e“340e [] M Rastrelli S Tropea C R Rossi and M Alaibac œMelanoma epidemiology risk factors pathogenesis Diagnosis andClassification In Vivo vol no pp “ [] L LugovićMihić D Ćesić P Vuković G Novak BilićM Å itum and S Å poljar œMelanoma development currentknowledge on melanoma pathogenesis Acta Dermatovenerologica Croatica vol no pp “ [] M Costache A V Dumitru O M Pătraşcu œA challenging case of ocular melanoma Romanian Journal of Morphology and Embryology vol Suppl pp “ [] R Ancuceanu and M Neagu œImmune based therapy for melanoma Indian Journal of Medical Research vol no pp “ [] C A Perez M H Ravindranath D Soh A Gonzales W Yeand D L Morton œSerum antiganglioside IgM antibodies insoft tissue sarcoma clinical prognostic implications CancerJournal vol no pp “ [] B Mondal and S Sahal œInhibition of subcutaneous growth ofEhrlich ascites carcinoma EAC tumor by postimmunizationwith EACcell gangliosides and its antiidiotype antibody inrelation to tumor angiogenesis apoptosis cell cycle and ltration of CD4 CD8 lymphocytes NK cells suppressorcells and APCcells in tumor Indian Journal of ExperimentalBiology vol no pp “ [] S Sahal and S Mondal œSupression of Ehrlich subcutaneoussolid tumor growth by immunization with ganglioside GT1bof its origin its IgM antibody or antiidiotype antibody Journal of Experimental Clinical Cancer Research vol no p [] M M Konstandoulakis K N Syrigos M LeandrosA Charalabopoulos A Manouras and B C GolematisœAutoantibodies in the serum of patients with gastric cancertheir prognostic importance Hybridoma vol no pp “ [] M H Ravindranath S Muthugounder X Ye and D L Morton œInnate immune response to gangliosides of primary melanoma favors danger hypothesis Proceedings of the AmericanAssociation for Cancer Research vol p [] M H Ravindranath S Muthugounder and N Presser œGanglioside signatures of primary and nodal metastatic melanomacell lines from the same patient Melanoma Research vol no pp “ [] A Lewartowska T Pacuszka G Adler M Panasiewicz andW Wojciechowska œGanglioside reactive antibodies of IgGand IgM class in sera of patients with diï¬erentiated thyroidcancer Immunology Letters vol no pp “ [] I Nicolae C D Nicolae O A Coman M StefanescuL Coman and C Ardeleanu œSerum total gangliosides levelclinical prognostic implication Romanian Journal of Morphology and Embryology vol no pp “ [] C Nicolae and I Nicolae œHeterogeneity of gangliosides inmelanocytic tumors Acta Endocrinologica vol no pp “ [] S GrouxDegroote M RodríguezWalker J H Dewald J LDaniotti and P Delannoy œGangliosides in cancer cell signaling Progress in Molecular Biology and Translational Sciencevol pp “ [] I Nicolae C D E Nicolae and E Ceauşu œInvestigation onantigangliosides antibodies in asymptomatic HIV patientsBMC Infectious Diseases vol no S4 p [] G N Tzanakakis M Neagu A M Tsatsakis and D NikitovicœProteoglycans and immunobiology of cancer therapeuticimplications Frontiers in Immunology vol p [] Q Li M Sun M Yu œGangliosides profiling in serum ofbreast cancer patient GM3 as a potential diagnostic biomarker Glycoconjugate Journal vol no pp “[] C D Ene A E Anghel M Neagu and I Nicolae œ25OHvitamin D and interleukin8 emerging biomarkers in cutaneous melanoma development and progression Mediators ofInflammation vol Article ID pages [] N R Sproston and J J Ashworth œRole of Creactive proteinat sites of ‚ammation and infection Frontiers In Immunology vol p [] A E Anghel C D Ene M Neagu and I Nicolae œThe relationship between interleukin8 and Ki67 in cutaneous malignant melanoma HVM Bioflux vol no pp “[] C D E Nicolae and I Nicolae œInterleukin 8serumconcentration but not lactate dehydrogenase activity positively correlates to CD34 antigen in melanoma tumors Journal of 0cJournal of Immunology ResearchImmunoassay and Immunochemistry vol no pp “ [] A E Anghel C D Ene I Nicolae V A Budu C Constantinand M Neagu œInterleukin major player in cutaneous melanoma metastasic process Romanian Biotechnological Letters vol no pp “ [] M Neagu œMetabolic traits in cutaneous melanoma Frontiers in oncology vol p [] C D Ene A E Anghel M Neagu and I Nicolae œInterleukin and diabetic nephropathy Human and Veterinary Medicine vol no pp “ [] M Neagu C Constantin I D Popescu œInflammationand metabolism in cancer cell “ mitochondria key playerFrontiers in Oncology vol p [] C Nicolae I Nicolae and O Coman œGD1b GT1b

Thyroid_Cancer

Genetic alterations in the 3q263132 locus conferan aggressive prostate cancer phenotypeBenjamin S SimpsonSusan Heavey Jason Pitt5 Caroline M Moore6 Hayley C Whitakerœ‰ Niedzica Camacho234 Hayley J Luxton Hayley Pye Ron Finn1Largescale genetic aberrations that underpin prostate cancer development and progressionsuch as copynumber alterations CNAs have been described but the consequences ofspecific changes in many identified loci is limited Germline SNPs in the 3q2631 locus areassociated with aggressive prostate cancer and is the location of NAALADL2 a gene overexpressed in aggressive disease The closest gene to NAALADL2 is TBL1XR1 which is implicated in tumour development and progression Using publiclyavailable cancer genomic datawe report that NAALADL2 and TBL1XR1 gainsamplifications are more prevalent in aggressivesubtypes of prostate cancer when compared to primary cohorts In primary disease gainsamplifications occurred in CI“ and CI“ for NAALADL2 and TBL1XR1 respectively increasing in frequency in higherGleason grade and stage tumours Gainsamplifications result in transcriptional changes andthe development of a proproliferative and aggressive phenotype These results support apivotal role for copynumber gains in this genetic region Molecular Diagnostics and Therapeutics Group Research Department of Targeted Intervention Division of Surgery Interventional Science UniversityCollege London London UK Human Oncology and Pathogenesis Program Memorial Sloan Kettering Cancer Center New York NY USA MarieJoseand Henry R Kravis Center for Molecular Oncology Memorial Sloan Kettering Cancer Center New York NY USA Department of Pathology MemorialSloan Kettering Cancer Center New York New York for Genomics Research Discovery Sciences Biopharmaceutical RD AstraZeneca Cambridge UK Cancer Institute of Singapore National University of Singapore Singapore Singapore Department of Urology UCLH NHS Foundation Trust London UKœ‰email HayleyWhitakeruclacukCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xProstate cancer PCa is the most common noncutaneouscancer in developed countries12 and is defined by dynamicgenome alterations and both its pathological and geneticheterogeneity3 An important pathological predictor of prostatecancer aggressiveness is Gleason grade used to assess risk ofprogression and stratify patients for treatment however theunderlying genomic changes which accompany more aggressivetumours remains incompletely definedOverall copynumber alteration CNA burden has been linkedto poorer prognosis in prostate cancer associating with Gleasongrade biochemical recurrence and prostate cancer specific deathhowever the exact mechanism driving these prognostic changes isunknown and thought to be primarily driven by general chromosomal instability4“ Changes in specific loci have also beenlinked to aggressiveness in particular gains in proliferative geneseg MYC 8q24 and loss of tumour suppressors PTEN 10q23and NKX31 8p2178 Many genetic alterations have been linkedwith prostate cancer such as point mutations in SPOP FOXA1and IDH19 Largescale oncogenic structural rearrangementstranslocations and copynumber changes are also common oftenleading to the coordinated dysregulation of multiple elements forexample the loss of 21q which is associated with the TMPRSSERG fusion rearrangement and the subsequent rearrangement ofSMAD410 Improved understanding of the mechanisms governing disease pathogenesis and progression may allow for bettertherapeutic exploitation for example genetic alterations in theDNA repair machinery have been linked to susceptibility toPARP inhibitors in a range of tumour types and alterations in ARconfer sensitivity or resistance to androgen deprivation therapy inmetastatic castrate resistant prostate cancer mCRPC11NAALADL2 is located on 3q2631 and is a member of theglutamate carboxypeptidase II family along with the widely studied PCa marker PSMA NAALAD112 and its expression haspreviously been associated with prostate tumour stage andgrade13 with expression predicting poor survival following radicalprostatectomy13 A large genomewide association study GWASof prostate cancer cases found rs78943174 a SNP withinthe 3q2631 NAALADL2 locus was associated with high Gleasonsum score14 A further rs10936845 SNP was identified within aGATA2 motif that increases NAALADL2 expression in prostatecancer patients where increased expression also predicted biochemical reccurence15 The same study showed even higherbinding preference to HOXB13 and FOXA1 to this site suggestingcooccupancy by these important transcription factors both ofwhich have been shown to be involved in AR cistromereprogramming1516functionsAdjacent to NAALADL2 in the genome is TBL1XR1 a corecomponent of nuclear receptor corepressor NCoR complex thatacts as a coregulator of nuclear receptors ‚uencing severalcellularand‚ammation17 TBL1XR1 is also an androgen receptor AR coactivator18 Expression of TBL1XR1 has been associated withpoor prognosis in several cancers predicting poor overall survivaland lymph node metastasis in gastric19 and ovarian cancers20 andrecurrence in colorectal21 breast22 and liver cancers23antiapoptosisincludinggrowthHere we utilise largescale publicly available genomic data tobetter characterise the broad somatic copynumber changesoccurring within the 3q263132 locus particularly centredaround gainsamplifications in NAALADL2 and TBL1XR1 andlinking them to the clinical characteristics of aggressive prostatecancerResults3q263132 gain frequency is increased in aggressive PCaCopynumber alterations often alter the expression of the gene inwhich they occur with gene dosage known to correlate withmRNA expression Genetic structural variants are also known toalter transcriptional regulation by altering cisregulatory elementssuch as promotors and enhancers resulting in differentialexpression2425 Increased NAALADL2 and TBL1XR1 expressionhave previously been linked to poor prognosis in cancers leadingus to examine the frequency of somatic copynumber gains inthese genes across various prostate cancer subtypes19“Alteration frequency was assessed using data from cBioportalFig 1a and all study data was processed using a standardisedpipeline to ensure comparable results Alteration frequency wasassessed in a total of patients samples in nonoverlapping studies Appendix eleven studies focused onprimary prostate cancer four on metastatic prostate cancer andone on neuroendocrine and castrateresistant cancers Significantcopynumber increases above a derived background thresholdwere categorised as ˜gains™ and copynumber decreases as ˜deletions™ Overall the distribution of NAALADL2 and TBL1XR1alterations were significantly different between disease subtypesto that which is expected Chisquared goodnessoffittestFig Somatic alteration frequency of NAALADL2 and TBL1XR1 across prostate cancer subtypes in publically available genomic studies n a NAALADL2 genetic alteration frequency across different subtypes of prostate cancer b TBL1XR1 genetic alteration frequency across differentsubtypes of prostate cancer P primary prostate cancer M metastatic prostate cancer NE neuroendocrine prostate cancer and castrate resistantprostate cancer CRPC All annotations were assigned using Genome Nexus and CNAs are called using GISTIC or RAE algorithms Pvalues show theresults of a Chisquared goodnessoffit test to determine if the number of observed patients with each alteration type is different from that which isexpected across each cancer subtype Results detailed in Supplementary data COMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xp — ˆ’ and p — ˆ’ Fig 1a b with gains beingmost frequent in castrateresistant prostate cancer and respectively followed by neuroendocrine and metastatic and then primary prostatecancer and Fig 1a b3q263132 gains extend across an oncogenerich region ofChr3 As CNA™s are known to associate with more aggressivesubtypes of prostate cancer we investigated their association withclinical characteristics to establish if changes can be detected earlyin the life history of cancer predicting more aggressive diseaseWe utilised copynumber data from primary an confineddisease from both the UK and Canadian International CancerGenome Consortium ICGC cohorts and The Cancer GenomeAtlas TCGA These studies use intermediatehigh risk prostatecancer patients with no treatment prior to radical prostatectomyTo allow comparisons between the studies data were reanalysedusing the Genomic Identification of Significant Targets in Cancer GISTIC2 methodfavoured by the broad institute andTCGA27 as it distinguishes between lowlevel copy numberincreases gains and highlevel copynumber increases amplifications Within the three cohorts we found that copynumbergains across both genes were frequent with gains in NAALADL2ranging from Canada to UK and between“ for amplifications Table TBL1XR1 had an almostidentical CNA frequency of between UK to Canada Table We fitted a randomeffects model to more accurately estimatethe frequency of NAALADL2 and TBL1XR1 gainamplificationscombining the data from all three cohorts which estimated thetrue frequencies to be CI “ and CI “ for NAALADL2 and TBL1XR1 respectively Supplementary Fig Leaveone out analysis and adiagnostic plots revealed that the ICGC Canada study was asignificant source of heterogeneity thereforethe study wasremoved and the model refit The final estimated frequency ofNAALADL2 and TBL1XR1 gainsamplifications was CI “ and CI “ respectivelyin primary prostate cancerDue to their close proximity in the genome we investigated ifgainsamplifications in NAALADL2 and TBL1XR1 cooccurred inthe same patients using a genomewide Fisher™s exact test with afalse discovery rate correction NAALADL2 and TBL1XR1significantly coamplified in all three cohorts ICGC UK p — ˆ’ ICGC Canada p 158e — ˆ’ and TCGA p — ˆ’testingconfirmed that widespanning gainsamplifications occurred inneighbouring regions in the majority of patients In the ICGC UKSupplementary Fig Additionallycohort n there was a significant cooccurrence of somaticcopynumber gainsamplifications in NAALADL2 with TBL1XR1FDRcorrected Fisher™s exact test — ˆ’ Fig 2a Gainsin this region also significantly correlated with two regionsspanning chromosomes and both gains previously describedas being abundant in prostate cancer Supplementary Data The Canadian cohort n showed a similar pattern of cooccurrence with gainsamplifications spanning the region surrounding NAALADL2 and TBL1XR1 3p253 to 3q29 Fig 2bThere was also a significant cooccurrence with gains in thebeginning of chromosome as well as some sporadic cooccurrence across the genome Fig 2b Supplementary Data These results were supported by the outcome of the same analysisin the TCGA cohort n although several large spikes ofcooccurrence were also observed in regions nottoNAALADL2 and TBL1XR1 as these spikes were not present inthe other two cohorts they most likely represent artefacts Fig 2cSupplementary Data Overall across the three cohorts therewere was a consistent coamplification in region spanning genes between 3p141 and 3q29 While a number of patients hadmultiple CNAs we found no consistent cooccurrence withcommon CNAs such as MYC gain FGFR1 gain PTEN loss RB1loss or NKX31 loss FDRcorrected Fisher™s exact test p The 3q26 region where NAALADL2 and TBL1XR1 are locatedis rich in oncogenes such as PIK3CA SOX2 ECT2 and PRKCIwhich may act to drive tumorigenesis29 We determined thenumber of known oncogenes within this defined region bycomparing the overlapping genes that coamplified withNAALADL2 and TBL1XR1 in alltheNetwork of Cancer Genes database30 This revealed that of genes are known oncogenes including BCL6 ATRand PI3K family members Supplementary Data These resultsconfirm that a high proportion of prostate cancer patientsdevelop large copynumber gains across multiple oncogenes inthis genetic regionthree cohorts againstlocalGains in 3q263132 associate with adverse clinical featuresCommon prostate cancer CNAs such as those in MYC andPTEN are known to associate with higher Gleason grade31Consistent with these findings we also found NAALADL2 andTBL1XR1 amplifications were highly correlated with GradeGroup GG showing that the frequency of NAALADL2 andTBL1XR1 gains tripling between GG1 and GG2 lesions and morethan doubling between GG2 and Table A Chisquaredgoodnessoffit test showed that the distribution of gainsamplifications between Grade groups was significantly different to thedistribution of diploid patientsfor both NAALADL2 andTBL1XR1 p — ˆ’ and p — ˆ’ WhenTable Alteration frequency of NAALADL2 and TBL1XR1 called via the GISTIC2 method in three nonoverlapping primary anconfined radical prostatectomy cohorts from the International Cancer Genome Consortium ICGC and The Cancer GenomeAtlas TCGAICGC UKICGC CANADATCGANAALADL2TBL1XR1NAALADL2TBL1XR1NAALADL2TBL1XR1AlterationDeep DelShallow DelDiploidGainAmplificationTotalnnnnnnThe degree of copy number alteration is discretised into five categories amplification gain representing low and high level copy number increase diploid no significant CNA and shallow and deepdeletion representing low and high level copy number lossCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xand compared to those without and Moreover ofthe patients who had their lymph nodesexamined the percentage of patients with lymph node positivitydefined through positivity on haematoxylin and eosin stainingHE was more than double in patients with NAALADL2 orTBL1XR1 gains and compared to those withouta gain and Chisquared goodnessoffit test p and p Finally while only one man in the cohorthad evidence of positive findings in his bone scan we did observea significant between the number of equivocal bone scans inpatients with gains and compared to and in those patients without gainsChisquaredgoodnessoffit test p and p for NAALADL2and TBL1XR1 respectively however the number of expectedcases in each of these categories was less than adding someuncertainty to this result We found no significant difference inthe mean age between patients with different copynumbers ofNAALADL2 or TBL1XR1 KruskallWallis rank sum test p and As gainsamplifications in NAALADL2 and TBL1XR1 coincidewith a cluster of known oncogenes and coincide with clinicalvariables linked to more aggressive disease we also compareddiseasefree survival Comparing patients with gainsamplifications in NAALADL2 and TBLXR1 to those with diploid copies weobserved no significant association in the ICGC UK cohort n although there was a trend towards reduced diseasefreesurvival Supplementary Fig 4A In the larger TCGA cohortn there was a significant reduction in diseasefree survivalin patients with a gain in either NAALADL2 Logrank MantelCox p or TBL1XR1 Logrank MantelCox p Supplementary Fig 4BUnivariable Cox regression confirmed that carrying a gainamplification in NAALADL2 and TBL1XR1 in the TCGA cohortresulted in reduction in diseasefree survival hazard ratio HR CI “ p Forreference weperformed a similar analysis of patients with PTEN deletion orMYC gains two common copy number alterations with provenin prostate cancer3233association with diseasefree survivalWhen patients were stratified solely by CNA status and survivalcompared using the KaplanMeier method those patients withMYC gain or PTEN deletion homo or hemizygous showed nosignificant difference in diseasefree survival Logrank MantelCox p and p respectively while those stratified byNAALADL2 gain TBL1XR1 gain or both NAALADL2 andTBL1XR1 gain showed significant differences in survival Logrank MantelCox p Supplementary Fig 5A“E Univariable Cox regression estimated the hazard ratios for thesecopynumber alterations as CI “ CI “ and CI “ for MYCPTEN and NAALADL2TBL1XR1 respectively We also comparedthe diseasefree survival of patients with only a copynumberalteration in each of the four genes where each group wasmutually exclusive Supplementary Fig 5F G This showed thaton the whole patients with CNAs in NAALADL2TBL1XR1 hadreduced or equal diseasefree survival as those with either onlyMYC gain or only PTEN loss Patients with copy number gains inboth had a worse prognosis All clinical data is available inSupplementary Data Fig Genomewide cooccurrence with NAALADL2 and TBL1XR1 gainsamplifications The Y axis shows “log10 qvalues from a Fishers exact testbetween gainamplifications in NAALADL2 and cooccuring genes Thedotted line represents the threshold for statistical significance aftercorrection for multiple testing a Significantly cooccurring gains across thegenome in the ICGC UK cohort b Significantly cooccurring gains acrossthe genome in the ICGC Canada cohort c Significantly cooccurring gainsacross the genome in the TCGA cohort NAALADL2 and TBL1XR1 cytobandpositions are labelled All Fisher tests use NAALADL2 gain or amplificationas the altered group Full results are detailed in Supplementary Data compared to common CNAs such as PTEN loss and MYC gainthe alteration frequency of NAALADL2 and TBL1XR1 was morecorrelated with higher Gleason grade groups Spearmans rho was p p for NAALADL2 and TBL1XR1and p for PTEN and MYC respectivelySupplementary Fig 3AMoreover we also noted the same pattern ofincreasingfrequency of gains with T stage Chisquared goodnessoffit testp and p respectively Table Patients with gains exhibited differences in the location of thetumour within the prostate with and of thosewith NAALADL2 and TBL1XR1 gains having tumoursinoverlapping and multiple zones compared to just and for those without gains Chisquared goodnessoffit testp and p There was also an increased relativenumber of positive surgical margins Chisquared goodnessoffittest p and p in patients with gains As CNAs in NAALADL2 and TBL1XR1 were associated withclinical characteristics such as Gleason grade group and T stagewe used multivariable Cox regression models to confirm that anychanges in survival were driven by these associations and foundthat copy number gains in NAALADL2 and TBL1XR1 were nolonger significant once corrected for Gleason grade and T stagep Supplementary Data These results suggest thatthe differences in diseasefree survival seen when stratified byCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xTable The frequency of NAALADL2 and TBL1XR1 gainamplifications by Gleason Grade Group in the TCGA cohortGrade groupGG1ObservedExpected within GGGG2ObservedExpected within GGGG3ObservedExpected within GGGG4ObservedExpected within GGGG5ObservedExpected within GGTotal nNAALADL2DiploidGainTBL1XR1DiploidGainTotalGrade groups defined as Grade Group Gleason score ‰¤ Grade Group Gleason score Grade Group Gleason score Grade Group Gleason score Grade Group Gleason scores and Displayed are the numbers of patients observed with gain or without diploid a gainamplification in this region in each Grade Group Additionally the expected number ofpatients estimated to be within each category is also shown along with the percentage of each Grade Group which is made up by patients with or without a gain Bold values indicate the overallpercentage of the group with a given copynumber state All clinical data detailed in Supplementary Data Table The frequency of NAALADL2 and TBL1XR1 gainamplifications by T stage in the TCGA cohortT stageT2aObservedExpected within T stageT2bObservedExpected within T stageT2cObservedExpected within T stageT3aObservedExpected within T stageT3bObservedExpected within T stageT4ObservedExpected within T stageTotal nNAALADL2DiploidGainTBL1XR1DiploidGainTotalDisplayed are the numbers of patients observed with gain or without diploid a gainamplification in this region in each T stage Additionally the expected number of patients estimated to be withineach category is also shown along with the percentage of each T stage which is made up by patients with or without a gain Bold values indicate the overall percentage of the group with a given copynumber state All clinical data detailed in Supplementary Data COMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xgainamplification status are driven by strong association withthese clinical variablesIn the ICGC cohortsindividuals with somatic singlebasealterations in NAALADL2 also associated with reduced diseasefree survival in a combined ICGC cohort as well as associatingwith reduced diseasefree and overall survival in an early onsetprostate cancer cohort ICGC EOPC Denmark Singlebasesubstitutions in TBL1XR1 were only associated with diseasefreesurvival in the ICGC EOPC cohort Supplementary Fig Singlebase alterations did not occur with a frequency greater than onein any single base in NAALADL2 or TBL1XR13q263132 gains cooccur with proproliferative transcriptionTo determine the potentialfunctional consequences of gainswithin the NAALADL2 and TBL1XR1 amplicon mRNA expression profiles were explored using the TCGA RNAseq dataDESeq2 was used to determine differentially expressed genesbetween patients with copynumber gains for both NAALADL2and TBL1XR1 compared to those without For NAALADL2 therewere differentially expressed genes DEGs and DEGsfor TBL1XR1 when the two groups were compared FDR Supplementary Data Our previous study on NAALADL2identified nine genes which were reciprocally regulated by overexpression or knockdown of NAALADL226 Of these nine wefound that three cancer antigen XAGE1B adhesionmotiliy regulator SPON2 and AR regulator HN1 were significantly differentially expressed p in patients with a NAALADL2 gainand in the same direction as the overexpression model2634“When comparing the DEGs between patients with and withoutin either NAALADL2 or TBL1XR1 wegainsamplificationsobserved that of the DEGs overlapped between NAALADL2 and TBL1XR1 Fig 3a of the geneswere located within the locus we identified as coamplified withNAALADL2 and TBL1XR1 and were differentially expressedconsistent with a mechanism of selfregulating expression2425TBL1XR1 was one ofsignificant overlapping DEGsNAALADL2 was just on the boundary of statistical significanceFDR corrected Wald test p Supplementary Fig theNAALADL2 has been shown to be coexpressed with numberof androgen regulated proteins and contains a number of ARbinding sites and TBL1XR1 is an AR coactivator and may beinvolved in AR cistrome reprogramming18263738 We thereforelooked at overlap between androgen regulated genes with ARbinding sites full or partial and genes demonstrated to beandrogen regulated following R1881 stimulation in at least twoindependent studies3739 shared genes were differentiallyexpressed in patients with NAALADL2 and TBL1XR1 gainsamplifications that contained AR binding sites and demonstratedandrogen regulation by R1881 genes had either aAR binding motif were androgen regulated in two or morestudies or both Fig 3bOf the overlapping DEGs a total of were knownoncogenesSupplementary Data which may drive anaggressive clinical phenotype Of note was PI3K family membersPIK3C2G PIK3CA PIK3CB PIK3R4 Mucin family membersMUC1 MUC4 and MUC6 and other prostate cancer associatedgenes such as SMAD4 SOX9 and SPOP794041 Additionallyseveral genes which form commercial prognostic assays were alsodifferentially expressed such as the Decipher assay NFIB LASP1ZWILCH THBS2 COL1A2 and COL5A142 Oncotype DX assaySFRP4 COL1A1 KLK2 TPX24344 and the Prolaris assayASPM BUB1B CENPF and FOXM145We inspected of the top most significant shared DEGs usingunsupervised hierarchal clustering Fig 3b SupplementaryData DEGs mostly displayed upregulation consistent with agenedosage effect Fig 3b24 Enrichment for biological processes was assessed by Geneset enrichment analysis GSEA forNAALADL2 and TBL1XR1 gainsseparately and by overrepresentation analysis ORA on the shared DEG list usingWebGestalt46GSEA on the individual lists of DEGs showed that despite a largeoverlap the enriched biological processes did differ between the twogenes patients with a gain in NAALADL2 showed enrichment inprocesses related to NADH dehydrogenase complex assemblyFDR mitochondrial respiratory chain complex assemblyFDR translational initiation FDR cytochromecomplex assembly FDR protein localisation toendoplasmic reticulum FDR and cytoplasmic translationFDR Supplementary Data Patients with a gain inTBL1XR1 showed enrichment in mitotic cell cycle phase transitionchromosome segregation actin filamentbased movement microtubule cytoskeleton anisation involved in mitosis regulation ofcell cycle phase transition cell cycle G1S phase transition FDR as well as a number of other processes SupplementaryData To understand the combined effect of gainsamplification inthese genes we investigated overrepresentation of processes in theDEGs which were common to both NAALADL2 and TBL1XR1In the shared DEG list the significantly enriched Gene OntologyGO biological processes were all involved in the cell cycle cyclepathway including mitotic regulation and chromosome segregation Fig 3c Supplementary Data These findings support ahypothesis whereby gains in NAALADL2 and TBL1XR1 concomitantly bring about mRNA expression changes which supportan aggressive proproliferative phenotype in primary prostatecancerDiscussionIn this study we present evidence that somatic copynumber gainsin NAALADL2 and TBL1XR1 are more frequent in high gradeand aggressive forms of prostate cancer These results are bolstered by studies which have identified CNAs in this region inmCRPC however to our knowledge this is the first time thesegains have been reported in neuroendocrine disease47 We alsodemonstrate that NAALADL2 and TBL1XR1 gains occur in anearlier setting cooccurring with gains in neighbouring genes Amajor barrier to the adoption of CNA based tests in the clinic isthe reliance on expensive NGS approaches as well as sufficientsequencing depth and coverage to assess overall copynumberburden The discovery of smaller clinically significant loci couldallow for cheaper quicker targeted approaches particularly if asingle loci can elude to gainsamplifications in a larger regionsurvivalto diseasefreeIn primary prostate cancer Gainsamplifications in this regionassociated with Gleason grade tumour stage number of positivelymph nodes bone scan results and as these variables contributetostratified byNAALADL2TBL1XR1 status also have altered diseasefree survival times Our work is supported by previous studies that haveeluded to the clinical significance of this locus particularly asgermline SNPs within this locus have been associated with higherGleason grade tumours and more aggressive disease14 This alsosupports smaller studies such as those by HeselmeyerHaddadet al who identified two out of seven patients with gains inTBL1XR1 in recurrent prostate cancer48 However these studiesinvestigated these genes in isolation na¯ve to the larger context inwhich these alterations occur Here we have found that gainsamplifications atthis locus not only coamplify with otherdescribed oncogenes but associate with much larger transcriptional changes which are consistent with the observed aggressiveclinical phenotypetimepatientsCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xFig Transcriptomic changes in patients with NAALADL2TBL1XR1 gains a Venn diagram showing the number and percentage of overlapping DEGsbetween patients with NAALADL2 gainamplification and TBL1XR1 gainamplification overlap b Venn diagram showing the number ofNAALADL2 and TBL1XR1 DEGs and genes with identified AR binding sites determined through ChIPSeq and AR knockdown and genes shown to beandrogen regulated following R1881 stimulation c Unsupervised hierarchal clustering of the top most significant DEGs bar beneath upper dendrogramshows copynumber status of patients where red is patients with a gain in both NAALADL2 and TBL1XR1 and grey represents those without gainamplification in these genes Heatmap represents meancentred z scores derived from RKPM values d Chord diagram showing significantly overrepresented GO biological processes and key genes within these processes All clinical data detailed in Supplementary DataCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xOverall changes in copynumber burden have been shown to beindicative of genetically unstable tumours and predict prostatecancer relapse5 Many single CNAs have already been describedthat predict PSA recurrence after radical prostatectomy includingPTEN loss cooccurrence of PTEN FAS 10q2331 and PAPSS210q232“10q2331 loss a loss of 16q with or without a loss ofPTEN a loss within 6q 13q gains in MYC 11q1317 7q and aconcurrent loss of 8p22 with a gain of 8q2487“ Compared towellknown CNAs such as PTEN loss and MYC amplification wehave observed that Gainsamplifications in NAALADL2TBL1XR1equally or better segregate patients who will have reduced diseasefree survivalThe gainsamplifications in NAALADL2TBL1XR1 also corresponded to a significantincrease in both NAALADL2 andTBL1XR1 mRNA supporting previous studies that have described upregulation of these genes and linked them to poor prognosis in various cancers19“ This suggests that gains in thesegenes may cause increased expression of NAALADL2 andTBL1XR1 in cancers We also noted a number of the differentiallyexpressed genes between patients with and without a gainamplification in NAALADL2TBL1XR1 have been shown to beandrogen regulated however further work is required to determine if gainsamplifications in this region cause changes in ARtranscriptional regulation through cis regulatory elements or as adirect consequence of the genes altered in this region1837In those patients with these gains we noted transcriptionalchanges in several genes associated with aggressive prostatecancer including differential expression of genes appertaining toprognostic assays such as Decipher Oncotype DX and Prolaris aswell as families such as mucins50“ This may explain theaggressive clinical phenotype observed in these patients We alsoobserved that when weighted individually there were differencesin enrichment of biological processes between those with NAALADL2 gains and TBL1XR1 gains suggesting that each generesults in some unique cellular changesOur finding that gains in the 3q26 locus result in concurrentexpression of oncogenes located within this region and theirdownstream targets identifies multiple potential therapeutic avenues warranting further investigations This study centred aroundtwo genes NAALADL2 and TBL1XR1 both of which areattractive therapeutic targets with TBL1XR1 previously suggestedas a potential cancer target operating via the TGFβ signallingpathway and potentially regulating AR signalling5354 Additionally the tumour specificity of NAALADL2 and basal membranouslocalisation makes it potentially accessible using antibodydrugconjugates13 This approach may be feasible if like other familymembers such as PSMA antibody binding results in subcellularinternalisation12 Moreover several of the oncogenes in whichgains cooccur as well as the downstream oncogenes activatedfrom gains in the 3q26 region such as ATR PI3K family members PIK3C2G PIK3CA PIK3CB PIK3R4 MUC4 BCL6 SOX9can be therapeutically targeted or have been suggested as therapeutic targets in cancer5155“ In the PI3K pathway PIK3CBspecific inhibitors may have utility in patients with mutationsamplifications andor fusion of this gene59 These findings mayhave clinical relevence as it has been reported by de Bono et althat many individuals who had durable year responses toPIK3CBspecific inhibition harboured activating mutation oramplification in PIK3CB60 and phase II trials of ipatasertib anAkt inhibitor targeting the PI3KAkt axis has shown promise inlate stage mCRPC61 Together our results suggest that largescalegenomic gainsamplifications occur in the 3q26 region in a hi

Thyroid_Cancer

"radiotherapy in combination with anti cytotoxic T lymphocyte associated antigen monoclonal antibody ipilimumab in patients with metastatic melanoma Nathalie Chaput Gras2 Emilie Lanoy3 Alicia Larive3 Celine Boutros Christine Mateus4 Emilie Routier4 Roger Sun5 Yun Gan Tao5 Christophe Massard6 Rastilav Bahleda6 Dominique Schwob3 Nathalie Ibrahim7 Rita Maria Khoury Abboud7 Caroline Caramella8 Andrea Lancia Lydie Cassard2 Severine Roy4 J C Soria10 Caroline Robert11 Eric Deutsch12 To cite Boutros a0C Chaput Gras a0N Lanoy a0E et a0al Dose escalation phase study of radiotherapy in combination with anti cytotoxic T lymphocyte associated antigen monoclonal antibody ipilimumab in patients with metastatic melanoma Journal for ImmunoTherapy of Cancer 20208e000627 101136jitc2020000627 –º Additional material is published online only To view please visit the journal online http dx jitc CR and ED contributed equallyAccepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJFor numbered affiliations see end of Correspondence toDr Eric Deutsch eric deutsch gustaveroussy frBackground A synergy between radiotherapy and anti cytotoxic T lymphocyte associated antigen anti CTLA4 monoclonal antibody has been demonstrated preclinically The Mel Ipi Rx phase study aimed to determine the maximum tolerated dose MTD and safety profile of radiotherapy combined with ipilimumab in patients with metastatic melanomaPatients and methods A dose escalation design was used with and Gy dose of radiotherapy at week combined with mgkg ipilimumab every weeks for four doses Patients with evidence of clinical benefit at week were eligible for maintenance with ipilimumab mgkg every weeks starting at week until severe toxicity or disease progression The database lock occurred on April Tumor growth rate of irradiated lesions and non irradiated lesions were analyzed to assess the systemic immunologic antitumor response Blood immune monitoring was performed before and during treatment to determine if radiotherapy could modify ipilimumab pharmacodynamicsResults patients received ipilimumab between August and July Nine patients received the four doses of ipilimumab All patients received the combined radiotherapy Grade adverse events occurred in nine patients the most common being colitis and hepatitis No drug related death occurred Dose limiting toxicity occurred in two of six patients in the cohort receiving Gy The MTD was Gy Two patients had complete response three had partial response response and seven had stable disease giving an objective response rate of and a clinical benefit rate of at week The median duration of follow up was years Q145 Q368 The median overall survival CI was estimated at years “ The median progression free survival PFS CI was “ Radiotherapy combined with ipilimumab was associated with increased CD4 and CD8ICOS T cells Increased CD8 was significantly associated with PFSConclusion When combined with ipilimumab at mgkg the MTD of radiotherapy was Gy This combination of ipilimumab and radiotherapy appears to be associated with antitumor activity Increased CD8 was significantly associated with PFS Thus immune biomarkers may be useful for early response evaluationTrial registration number NCT01557114INTRODUCTIONImmunotherapy using immune checkpoint inhibition has revolutionized the management of patients with advanced stage melanoma and is an emerging approach for many other cancers1 The first immune checkpoint inhibitor that was developed was ipilimumab2 Ipilimumab targets the cytotoxic T lymphocyte associated antigen CTLA4 and significantly improves the overall survival in patients with metastatic melanoma2 However the objective response rate ORR and the disease control rates proportion of patients with a partial response PR or complete response CR or stable disease SD are relatively low and respectively3 As such increased interest has been drawn to enhance the induction of systemic immune responses of ipilimumab by combining it with radiotherapy4“ One of the rationale put forward to combine these therapies is that ipilimumab is able to deplete T regulatory cells Treg cells through antibody dependent cell cytotoxicity and consequently increases the CD8 T cell to Treg ratio whereas radiotherapy promotes the diversity of the T cell receptor TCR Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access repertoire of intratumoral T cells7“ When combined ipilimumab promotes the expansion of T cells while radiation enhances the TCR repertoire of the expanded peripheral clones4 Ipilimumab at a dose of mgkg is approved in several countries for the treatment of unresectable or metastatic melanoma In a recent prospective phase study patients were treated with ipilimumab mgkg combined with concurrent or sequential stereotactic ablative radiotherapy11 Dose limiting toxicities DLTs and grade toxicities were reported in and of patients respectively PR and clinical benefit were reported in and of patients respectively However few clinical studies were conducted to assess the efficacy of ipilimumab mgkg combined with radiotherapy11“ although overall survival was significantly improved with the ipilimumab mgkg monotherapy compared with the mgkg dose2 Here we present the phase ˜Mel Ipi Rx™ study investigating the safety and efficacy of ipilimumab mgkg combined with radiotherapy in patients with metastatic melanoma Although the treatment landscape of patients with advanced melanoma has changed since this study was initiated the increased survival benefit of ipilimumab mgkg compared with mgkg suggests that the clinical utility of ipilimumab in refractory patients with high unmet medical need could warrant further assessment The primary objective of this study was to determine the maximum tolerated dose MTD DLT and the recommended phase dose RPTD of radiotherapy administered in combination with ipilimumab at a dose of mgkg in patients with metastatic melanoma The secondary objectives were to determine the adverse event AE profiles to describe the preliminary antitumor activity following escalating doses of radiation combined to ipilimumab using the immune related response criteria irRC and to evaluate the overall survival in patients treated with this combination The exploratory objectives were to evaluate the systemic immunologic antitumor response and factors influencing this responseMETHODSPatientsEligible patients had unresectable locally advanced or metastatic melanoma with at least one measurable metastasis accessible to radiotherapy Subcutaneous nodules and lymph nodes were considered as targets for irradiation Tumor lesions located within vital ans or gastrointestinal tract were not considered as target for radiotherapy All patients had measurable and non measurable disease evaluable according to irRC Patients were ‰¥ years of age were able to give written informed consent had Eastern Cooperative Oncology Group ECOG performance status of to and had normal renal and liver functions on blood tests Patients were excluded if they had one of the following criteria suspected or known central nervous system tumors including brain metastasis any other malignancy with a disease free for less than years an autoimmune disease a history of prior treatment with ipilimumab prior radiotherapy within the same body area or radiotherapy in fields containing flat bones volume If chemotherapy or immunotherapy were previously used a wash out period of weeks at least was required before the first administration of ipilimumabStudy designThis phase I dose escalation study evaluated the MTD of radiotherapy in combination with ipilimumab in patients with unresectable locally advanced or metastatic malignant melanoma Eligible patients received ipilimumab at mgkg at weeks and for a total of four doses at week intervals online supplementary figure S1 Patients without progressive disease PD who tolerated the treatment continued ipilimumab dosing in week intervals until progression or withdrawal of consent Radiotherapy was delivered on week on Monday Tuesday and Friday at the time of the second cycle of ipilimumab on measurable superficial lesions including subcutaneous nodules and lymph nodes Radiotherapy was delivered using MV photons or electrons with standard field encompassing Maximal field had to be at least × cm but not be more than × cm maximal dimensions on a lesion Millimetric margins were used to take into account microscopic spreading and patient movements clinical and planning target volumes of mm Dose escalation of ionizing radiation was planned in cohorts of three to six patients depending on the occurrence of DLTs during the first combination treatment cycle A hypofractionated radiation regimen higher doses per fraction was used Dose escalation was used with total doses of and Grays Gy administered in three fractions every Monday Wednesday and Friday and planned in cohorts of three to six patients depending on the occurrence of DLTs from week to week A minimum deadline of hours between two radiotherapy sessions had to be respected If necessary the radiotherapy could be put back to Tuesday Thursday Saturday Given the small size of the radiation field and the fact that irradiated lesions were superficial vital ans and gastrointestinal tract were not involved in the irradiated fieldToxicities were evaluated according to the Common Terminology Criteria for Adverse Events CTCAE version DLT observation period ranged from week to Any toxicity before the start of radiotherapy was not considered as a DLT but as a toxicity from ipilimumab alone The DLT was defined by the appearance of at least one of the following study combination related event within weeks grade vomiting diarrhea or gastrointestinal bleeding grade or non hematologic toxicity excluding grade nausea vomiting diarrhea and transient fever or grade or radiation dermatitis except if return to grade ‰¤ within weeks If none of the first three subjects in a given dosing cohort had experienced DLT dose escalation was realized If Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cone of the first three subjects in a given dosing cohort had experienced DLT an additional three subjects were enrolled to that dose level before further escalation was considered A dose escalation was realized if none of these additional three subjects had experienced DLT If two of the first three subjects in a given dosing cohort had experienced DLT dose escalation was stopped Dose escalation was continued until one third or more of the subjects at a particular dose level experienced DLT or up to the fourth cohort This was considered the maximum administered dose MAD The MTD was defined as the highest dose at which less than one third of the subjects experienced DLT The RPTD was defined as the MTD or the dose that was considered to give the optimal clinical andor immunological results Tumor assessments consisting of CT scans and assessment of skin lesions were performed at weeks and every weeks thereafter until the patient withdrew from the study or entered the follow up phase MRI or CT scan of the brain had to be realized when clinically indicated Evidence of PD was confirmed by a second assessment performed “ weeks later Definitions of lesions were based on irRC Measurable lesions were defined as lesions that could be accurately measured in two perpendicular diameters with at least one diameter ‰¥ mm and the other dimension ‰¥ mm It was possible to consider skin lesions if they were measurable All measurable lesions up to a maximum of lesions per an and lesions in total were identified as index lesions measured and recorded at screening and follow up The index lesions were representative of all involved ans Non index lesions corresponded to measurable lesions that were recorded and evaluated at the same assessment time points as the index lesions and that were irradiated according to the trial irradiation designBlood immune monitoringBlood samples were collected at baseline T0 at week W4 before second injection of ipilimumab and at week W6 after radiotherapy and before third injection of ipilimumab online supplementary figure S2 Phenotyping was performed on fresh whole blood and peripheral blood mononuclear cells PBMCs isolated by Ficoll density gradient were frozen for later analyses Data analysis from standard blood tests was realized to estimate the derived neutrophil to lymphocyte ratio dNLR at baseline W4 and W6 to see whether or not granulocytes could impact the prognosis of patients Whole blood or PBMCs were incubated with fluorochrome conjugated antibodies for min at room temperature or at °C respectively followed by min of lysis Versalyse Beckman Coulter Mervue Galway Ireland The following fluorochrome conjugated antibodies were used fluorescein isothiocyanate anti ICOS CD278 clone DX29 phycoerythrin PE conjugated anti CD25 clone B1499 allophycocyanin cyanine APC Cy7 conjugated CD45RA clone HI100 phycoerythrin cyanine PE Cy7 conjugated anti CD45RA clone access2H4 allophycocyanin Alexa Fluor AA700 conjugated anti CD3 clone UCHT1 Pacific Blue PB conjugated anti CD4 clone 13B82 and Krome orange conjugated anti CD8 clone B911 were obtained from Beckman Coulter Stained cells were acquired using a FACS Canto II cytometer BD Bioscience or a Gallios Cytometer Beckman Coulter and analyzed using Kaluza software Beckman Coulter Conventional T cells CD3CD4 and CD3CD8 were respectively defined by CCR7CD45RA for naïve T cells CCR7CD45RA for central memory T cells TCM CCR7CD45RA for effector T cells TEM and CCR7CD45RA for terminally differentiated T cells TEMRA Treg cells were defined as CD3CD127lowCD25Tumor growth rate analysisTo assess the systemic immunologic antitumor response tumor growth rate TGR of irradiated lesions and non irradiated lesions were analyzed Briefly TGR estimates the variation in tumor volume over time using each patient as his own control and has shown to be interesting to identify the specific therapeutic effect of a treatment regardless of the disease course of each patient16 TGR was expressed as a percentage increase in tumor volume during month in accordance with Hiniker et al11 using the following formula TGR100 [exp[3LogDtDt0t] ˆ’] with D0tumor size defined as the sum of the longest diameters of the lesions at baseline Dttumor size defined as the sum of the longest diameters of the lesions at the time t of evolution evaluation in monthsTGR100 [exp3LogDtDt0tˆ’]The TGR was computed for irradiated lesions TGRirr and non irradiated target lesions defined by the radiologist for the irRC evaluation TGRnon irr for two treatment periods when data were available the Reference TGR REF TGR assessed before the onset of the treatment using the baseline CT and a CT realized before the baseline prebaseline CT the Experimental TGR EXP TGR assessed between the onset of the treatment and the first evaluation The TGR was computed using the same target lesions at each evaluation time A positive value of the EXP TGR reflected a bigger lesion at the first evaluation than at baseline Therefore new lesions related to PD were not included in the TGR computation Difference between EXP TGR and REF TGR was used to assess the effect of the treatment ΔTGREXP TGRˆ’REF TGR a negative value reflecting a slowdown of the natural course of the disease ie a slower tumor growth or a tumor response between the two periodsStatistical analysisDemographic and baseline characteristics were summarized for all registered subjects using descriptive statistics Toxicity grades per subject were tabulated for AEs and on study laboratory measurements by using the NCI CTCAE version Analyses of efficacy endpoints were based on all subjects evaluable for efficacyBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access The Kaplan Meier method was used to estimate overall survival and immune related progression free survival PFS Overall survival was defined as the time from treatment initiation to the date of death or date of last follow up in persons alive at last follow up Immune related progression was defined according to irRC An event was defined as progression or death whichever comes first PFS was defined as the time from treatment initiation to the date of occurrence of the event Follow up of patients who did not experience was censored at the date of last evaluation Duration of follow up was estimated using the Schemper and Smith method18 All statistical analyses were done using SAS software V94Evolution of innate immune cells was analyzed using GraphPAD PRISM software values at W4 and at W6 were described and compared with baseline by using Friedman test followed by Dunn's multiple comparisons testStudy oversightThe study was conducted in accordance with the protocol Good Clinical Practice guidelines and the provisions of the Declaration of Helsinki All patients provided written informed consentRESULTSPatient populationNineteen patients with advanced melanoma were treated at Gustave Roussy in the Mel Ipi Rx phase trial between August and July online supplementary table S1 The database lock occurred on April Nine patients received the four doses of ipilimumab and two patients received maintenance ipilimumab one and two cycles respectively All patients received the combined radiotherapy at week in three fractions Thirteen patients were enrolled at dose level Gy and six patients at dose level Gy Demographic data of patients are summarized in table Overall patients presented visceral metastases M1c had elevated level of lactate dehydrogenase LDH tumors were BRAF mutated and none had a history of brain metastases All patients had received systemic therapy previously Prior therapy included chemotherapy in patients BRAF inhibitors in patients radiotherapy in patient and surgery in patients respectively and all of them were immune checkpoint inhibitor naïve Irradiation was delivered on subcutaneous lesions in patients and patients in the cohorts treated with and Gy respectively and on lymph nodes in patients and patients in the cohorts treated with and Gy respectively online supplementary table S2 The average irradiated tumor volumes were relatively homogeneous with a median of mm IQR “SafetyAll patients presented at least one AE of any grade These AEs were felt to be probably related to ipilimumab The role of concurrent radiotherapy on these AEs was difficult Table Patient baseline characteristicsCharacteristics SexMaleFemaleAge yearsMedianRangeMutation BRAF statusNon mutantMutantUnknownLactate dehydrogenaseNormalElevatedM staging extent of metastasesM0M1aM1bM1cBrain metastasesNoYesSite of irradiated lesionsLymph nodesSubcutaneous nodulesVisceral ansPrevious treatments No prior systemic treatmentIpilimumabChemotherapyAnti BRAFRadiotherapySurgeryOverall populationN19 No “ The normal range for lactate dehydrogenase is UIL All patients were naïve to immune checkpoint inhibitorsto assess However no radiation induced necrosis or local symptoms were observed inside the radiation field AEs of all grades are summarized in table regardless of their attributionAsthenia was the most commonly reported AE of any grade It occurred in patients Among them nine patients were treated in the Gy cohort and five patients in the Gy cohort The median time to onset of asthenia was Q1 Q34“ weeks and the median duration was Q1 Q34“ weeks The other most common AEs of Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cTable Adverse events of all grade safety populationNo of patients with at least one adverse eventsFatigueDiarrheaDisease related painFeverPainNauseavomitingAnorexiaConstipationColitisPruritusWeight lossAnemiaEdema of limbsDyspneaSkin eruptionVitiligoHypereosinophiliaAlanineaspartate aminotransferase increasedCoughLymphedemaLevel dose N13 Level dose N6 TotalN19 any grade included diarrhea disease related pain fever nausea and vomitingThirteen patients discontinued the study owing to treatment related AEs nine patients in the cohort receiving Gy and four patients in the cohort receiving Gy Nine of these patients had AEs of grade Multiple AEs of grade occurred in some patients AEs of grade included colitis n2 hepatitis n2 asthenia n2 thyroid disorders n1 DRESS drug rash with eosinophilia and systemic symptoms syndrome n1 and nauseavomiting n1 Online supplementary table S3 presents the grade and grade events for each dose level for all events There were no treatment related deathsFour of the patients who discontinued the study did not have grade or AEs Indeed three patients had grade colitis associated with either contraindication to corticosteroids because of uncontrolled diabetes mellitus or complete remission after four cycles of ipilimumab leading to regular follow up without maintenance ipilimumab One patient had a grade asthenia after four cycles of ipilimumab leading to regular follow up without maintenance ipilimumab accessDose escalation and DLTsEighteen patients were evaluable for DLTs in this study patients in the cohort treated with Gy and patients in the cohort treated with Gy Among them four patients experienced DLTs All the DLTs occurred outside the radiation fieldIn the cohort treated with Gy of evaluable patients experienced DLTs after two cycles of ipilimumab combined with radiotherapy Indeed one of these patients presented grade hepatitis and the other one presented grade colitis with grade hypokalemia grade anorexia and grade thyroid disorders In both patients the three fractions of radiotherapy on Monday Wednesday and Friday were delivered on week DLTs led to permanent ipilimumab discontinuation in both patientsIn the cohort treated with Gy two of the six evaluable patients experienced a DLT after two and three cycles of ipilimumab respectively combined with radiotherapy One of these patients presented grade hepatitis and the other one presented grade colitis with unusually normal macroscopic colonoscopy but a total villous atrophy mimicking celiac disease on biopsies In both patients the radiotherapy fractions on Monday Wednesday and Friday were delivered on week DLTs led to permanent ipilimumab discontinuation in both patientsThe MTD of radiotherapy was thus Gy when combined with ipilimumab mgkg in the present design Consequently the RPTD of radiotherapy administered in combination with ipilimumab at mgkg in patients with metastatic melanoma was GyClinical outcomesAt the time of analysis the median duration of follow up was years Q145 Q368 The median overall survival CI was estimated at years “ The median PFS CI was “ Figure shows Kaplan Meier median overall survival and PFS curves with CI According to irRC the best response within the trial was CR for two patients both in the cohort receiving Gy PR for three patients two patients in the cohort receiving Gy and one patient in the cohort receiving Gy and SD for seven patients six patients in the cohort receiving Gy and one patient in the cohort receiving Gy giving an ORR of and a clinical benefit rate of at week online supplementary table S2 The initial melanoma staging of the patients who had CR was M1a for one patient multiple subcutaneous nodules and M1c multiple subcutaneous nodules and lymph nodes associated with an elevated LDH for the other patient The initial melanoma staging of the patients who had PR was M1a for one patient multiple subcutaneous nodules and M1c for two patients one of these patients had a subcutaneous nodule a lymph node and elevated LDH The other patient had multiple lymph nodes and bone metastasisBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access Figure A Waterfall plot of patients according to the variation of tumor growth rate ΔTGR between reference before treatment and experimental period on treatment For each patient specific ΔTGR of irradiated and non irradiated lesions are represented ΔTGR on treatment lesions at the first evaluation are bigger than at baseline ΔTGR of non irradiated lesions was superior to the irradiated lesion B Changes of the sum of diameters of the target lesions irradiated and non irradiated respectively at months in compared with the baselineVariation of tumor growth rate across lesionsTGR variation for irradiated and non irradiated lesions before and after the treatment was evaluable for patients figure A total of non irradiated lesions with up to lesions for one patient were evaluated for the REF and EXP TGRnon irr median IQR “ non irradiated lesions by patient and irradiated lesions for the REF and EXP TGRirr one patient had two irradiated lesions Median reference and experimental period were IQR “ and IQR “ months respectively The sum of diameters of lesions at the three evaluation times prebaseline baseline first evaluation and corresponding TGR are presented in table The EXP TGR on treatment was not correlated with the REF TGR before treatment Spearman™s rho011 p073 Decrease of the TGR was ˆ’ of the tumor sizemonths IQR “ to “ for irradiated lesions and ˆ’ month IQR “ to “ for non irradiated lesions although the difference was not significantly different p082 Response Figure Kaplan Meier median overall survival A and progression free survival B curves with CINo pseudoprogression was observed among the patients treated in this study nor radiation induced necrosis or edema Of note three patients were not evaluable for response because they progressed and died before the radiologic assessment scheduled in this studyTreatments administered after the ˜Mel Ipi Rx™ study in patients with disease progression are summarized in online supplementary figure S3Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cTable Characteristics of lesions evaluated for tumor growth rate TGRPatients nLesions n sumLesions n median IQRSum of diameters prebaseline median IQRSum of diameters baseline median IQRSum of diameters at first evaluation median IQRREFperiod median IQREXPperiod median IQRREF TGR median IQREXP TGR median IQRTGRdiff median IQR accessWilcoxon p valueOverallIrradiated lesionsNon irradiated lesions to to to to to to to to to to to ˆ’ ˆ’ to to ˆ’ to ˆ’ ˆ’ to ˆ’ˆ’ ˆ’ to ˆ’ to to to ˆ’ ˆ’ to ˆ’ˆ’ ˆ’ to ˆ’REF TGR corresponds to TGR before the start of the treatment EXP TGR corresponds to TGR between the start of the treatment and the first evaluation at monthsof non irradiated lesions seemed more representative of patient outcomes vs irradiated lesion EXP TGRnon irr was significantly higher in patients with PD Although the number of patients was too low for statistical test p values are shown for information in table figure and online supplementary figure S4Immune analysesWe analyzed immune parameters in the blood that have been previously described to be changed during ipilimumab treatment to determine if radiotherapy could modify ipilimumab pharmacodynamics19 For CD4 T cell counts and as expected ipilimumab alone W4 could favor accumulation of TEM Treg and ICOSCD4 T cells Interestingly at W6 after ipilimumabradiotherapy only TEM and ICOSCD4 T cells remained significantly increased suggesting that combination favored accumulation of activated memory CD4 T cells rather than Treg cells For CD8 T cell counts no accumulation could be observed at W4 while augmentation of TCM and TEMRA could be depicted between W4 and W6 suggesting that adjunction of radiotherapy to ipilimumab was more prone to boost these CD8 T populations online supplementary figure S2 High fold change in CD8 from baseline to week was significantly correlated with PFS p00223 but not significantly correlated with overall survival p02355 online supplementary figure S5Innate immune cells and NLR absolute neutrophils count ANC divided by the number of lymphocytes or dNLR ANC divided by the number of white blood cellsˆ’ANC have been shown to have a prognostic role in patients treated with immunotherapy and even might represent a predictive biomarker of response We took advantage of standard blood tests to determine if Table Univariate analysis of tumor growth rate TGRAll lesions n37Progressive disease WilcoxonNop valueYesIrradiated lesion n13Progressive diseaseNoYesWilcoxon p valueNon irradiated lesions n24Progressive diseaseNoWilcoxon p valueYesSum of diameters prebaseline meanSum of diameters baseline meanSum of diameters at first evaluation meanREF TGR meanExp TGR meanΔTGR meanˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’TGR is evaluated in percentage per months ΔTGR EXP TGR “ REF TGR A negative value corresponds to a slowdown of the tumor growthBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access neutrophils monocytes NLR or dNLR at baseline W4 and W6 could correlate with the prognosis in our study ANC or monocytes did not correlate with the prognosis of patients while absolute count of lymphocytes significantly increased at W6 compared with baseline in only patients with a clinical benefit CRPRSD Both NLR and dNLR were significantly lower at W6 only in patients with clinical benefit online supplementary figure S6 Note that we did not found an association with the dose of radiotherapy data not shownDISCUSSIONIn this dose escalation phase study four patients experienced DLTs All the DLTs occurred outside the radiation field Therefore it was difficult to assess the role of concurrent radiotherapy on the DLTs The MTD of radiotherapy combined with ipilimumab at mgkg was Gy The RPTD of radiotherapy administered in combination with ipilimumab at mgkg in patients with metastatic melanoma was Gy A hypofractionated radiation regimen higher doses per fraction was used in this study It is usually preferred for melanoma which displays low alphabeta ratio Our three fractions hypofractionated regimen is in line with the radiation standard in the metastatic setting Moreover it has been shown recently that radiation doses per session inferior to Gy combine more favorably with immunotherapy through interferon type induction21 Of note our study is the only one that combines radiotherapy and high dose of ipilimumab mgkgThe incidence of treatment related AEs was high with this combination in our study but numerically similar to the incidence reported previously with ipilimumab monotherapy at mgkg Grade or AEs occurred in of patients in our study whereas they occurred in “ of patients treated with ipilimumab monotherapy at mgkg2 The overall AE spectrum of the combination in this study was consistent with previous findings with the drug™s immune based mechanism of action The high rate of AEs might be partially attributed to the high dose of ipilimumab The dose of mgkg was chosen based on data from the randomized phase trial that compared various

Thyroid_Cancer

lack of COVID19 diagnostic tests for the whole Spanish population thecurrent strategy is to identify the disease early to limit contagion in the communityAimTo determine clinical factors of a poor prognosis in patients with COVID19 infectionDesign and settingDescriptive observational retrospective study in three primary healthcare centres with anassigned population of MethodExamination of the medical records of patients with COVID19 infections confirmed by polymerase chain reaction Logistic multivariate regression models adjusted for age and sexwere constructed to analyse independent predictive factors associated with death ICUadmission and hospitalizationResultsWe included patients mean age years female aged � years were health workers doctors nurses auxiliaries Predictors of ICUadmission or death were greater age OR 95CI to male sex OR 95CI to autoimmune disease OR 95CI to bilateral pulmonary infiltrates OR 95CI to elevated lactatedehydrogenase OR 95CI to elevated Ddimer OR 95CI to and elevated Creactive protein OR 95CI to Myalgia or arthralgia OR 95CI to was protective factor against ICU admission andPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsdeath Predictors of hospitalization were chills OR 95CI to feverOR 95CI to dyspnoea OR 95CI to depressionOR 95CI to lymph ia OR 95CI to andelevated Creactive protein OR 95CI to Anosmia OR 95CI to was the only significant protective factor for hospitalization after adjusting forage and sexConclusionDetermining the clinical biological and radiological characteristics of patients with suspected COVID19 infection will be key to early treatment and isolation and the tracing ofcontactsIntroductionOn December the health authorities of Wuhan city Hubei Province China reporteda cluster of cases of pneumonia of unknown aetiology with onset of symptoms on December including severe cases with a common exposure identified in a city market [] whichwas closed on January On January the Chinese authorities identified a newCoronaviridae family virus initially named coronavirus 2019nCoV and later coronavirusSARSCoV2 as the causal agent [] The genetic sequence was shared by the Chinese authorities on January On January the first case was detected in the USA in Washingtonstate [] On January the World Health anization declared the SARSCoV2 outbreak in China a public health emergency of international concern [] Subsequently the outbreak has spread outside China with Europe especially affected []The first positive case diagnosed in Spain was confirmed on January on the islandof La Gomera while the first death occurred on February in Valencia city the date was confirmed twenty days later The first confirmed case in Barcelona was on February and fromthen until June there have been confirmed cases in Spain []The most common signs of infection are respiratory symptoms fever cough and shortnessof breath In more severe cases the infection may cause pneumonia severe acute respiratorysyndrome renal failure and death [] Transmission appears to be mainly persontopersonvia the airway through respiratory droplets measuring microns when the patient has respiratory symptoms cough and sneezing and contact with fomites [] Most estimates of theincubation period of COVID19 range from to days with most around five days Evidenceon the transmission of the virus before symptom onset is unclear There is currently no specifictreatment for COVID19 infections To date the most important scientific efforts have focusedon three areas strategies to contain the spread of the disease the initiation of clinical trialswith antivirals and multiple therapies and the design of a new vaccine which is still unclearThese strategies include some of a community nature where primary healthcare plays a centralrole in disease prevention and control [] Few studies have described the clinical characteristics of the disease fewer the predictive factors and virtually none have described the Mediterranean population compared with the rest of the world Therefore this study aimed todescribe the clinical biological and radiological manifestations the evolution treatments andmortality rate of patients with COVID19 infection in the population of Barcelona city anddetermine the most important predictors of a poor prognosisPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsMaterials and methodsA multicentre observational descriptive study was carried out in three urban primary healthcare centres serving an assigned population of with one reference hospital The studyincluded all consecutive adult patients with COVID19 confirmed by polymerase chain reaction PCR from nasal and pharyngeal samples during the study period of February to April Diagnostic confirmation was made in the hospital laboratories as PCR is not available in primary healthcare centres Signs and symptoms the main available haematologicaland biochemical data and the results of imaging tests were recorded as were comorbiditiesthe evolution the hospitalization rate intensive care unit ICU admission and the treatmentsreceived The study population was divided into four age groups “ years “ years“ years and � years Other variables recorded were the type of followup the need fortemporary work disability and the source of possible contacts The time to first medical visitwas defined as the difference in days between symptom onset and medical visit by a familyphysician The factors that determined a poor prognosis hospitalization ICU admissiondeath were collected The data were obtained from the electronic medical record Missingdata were collected by telephone interviews with patients when possible Patients from nursinghomes were excluded as the rate of infections and mortality has been shown to be muchhigher than in the noninstitutionalized population The study was approved by the EthicsCommittee of the Hospital Clinic of Barcelona registration number HCB20200525 Thestudy was conducted according to the Helsinki Declaration and Spanish legislation on biomedical studies data protection and respect for human rightsStatistical analysisCategorical variables are presented as absolute frequencies and percentages and continuous variables as means and standard deviations SD Predictors of death ICU admission andhospitalization were determined using the student™s t test for continuous variables and the chisquare test for categorical variables Logistic multivariate regression models adjusted for ageand sex were constructed to analyse independent predictive factors associated with death ICUadmission and hospitalization Odds ratios OR and their confidence intervals 95CIobtained in the adjusted regression analysis were calculated Forest plots were used to represent OR and 95CI Values of p005 were considered statistically significant The statisticalanalysis was performed using the R version for WindowsResultsClinical characteristics and comorbiditiesWe included patients mean age years female aged � yearsThe mean time from symptom onset to the medical visit was SD days Clinical characteristics are shown in Table Notably were health workers doctors nurses auxiliaries The most frequentclinical symptoms were cough fever general malaise fatigue myalgia or arthralgia dyspnoea diarrhoea headache anosmia and dysgeusia Physical examination in patients showed had auscultatory alterations tachypnoea and an oxygen saturation of � ICU admission and death were associated with a greater mean age years vs yearsp male sex vs p dyspnoea vs p fever vs p auscultatory alterations vs p and low oxygen saturation vs p Table Myalgia or arthralgia vs PLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Clinical and exploratory factors predicting hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admissionHospitalizationAge”yearsDistribution”no “ years“ years“ years� yearsMale”no Occupation”no n No n YesPAdjusted ORNoYesPAdjusted OR ± ± n ± [“][ CI]n ± n ± [“][ CI] [“] [“]Other type of exposure Health professionalOther health workersSmoking exsmokersmoker”nototal no Temperature at admission” ˚C Patients with fever �˚C”nototal no Time from symptom onset tomedical visit”days¡Symptoms”no ¶ ± ± ± ± ± ± NANANA [“] [“] [“] [“] ± ± ± [“] [“] ± [“] NANANA [“]CoughGeneral malaiseFatigue [“] [“] [“] [“] [“] [“]Myalgia or arthralgia [“] [“]DyspnoeaDiarrhoeaHeadacheAnosmiaDysgeusiaSore throatBlocked noseNausea or vomitingSputum productionChillsAstheniaChest painAlterations in physical examination”nototal no §Auscultatory alterationsTachypnoeaTachycardiaPharyngitis [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] NA [“]Continued PLOS ONE 101371journalpone0237960 August PLOS ONE 0cTable ContinuedVariablesTotalDeath or ICU admissionHospitalizationPrognostic factors in Spanish COVID19 patientsOxygen saturation �”nototalno n n No n YesPAdjusted ORNoYes[ CI]n n [“] PAdjusted OR[ CI] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Temperature distribution was ˚C “ËšC “ËšC and ˚C ¡ In patients™ data on period between symptom onset and medical visit were lacking¶ Symptoms with a frequency of patients were disorientation n conjunctivitis n haemoptysis n and cutaneous lesions n § patients had a physical examination The alterations with a frequency of patients were cutaneous lesions n and tonsillopharyngitis n Ref reference NA not applicable101371journalpone0237960t001p headache vs p dysgeusia vs p andanosmia vs p were less frequent in patients admitted to the ICU or whodied than the remaining patients Age OR 95CI to and male sexOR 95CI to were independent predictors of ICU admission and deathMyalgia or arthralgia OR 95CI to was the only significant protective factor against ICU admission and death after adjusting for age and sex Fig The best clinicalpredictors of hospitalization were chills OR 95CI to fever OR 95CI to and dyspnoea OR 95CI to Anosmia OR 95CI to was the only significant protective factor for hospitalization after adjusting for age and sex Table and Fig Comorbidities were presented by patients the most common were hypertension in diabetes mellitus in and obesity in Table Heartdisease vs p autoimmune disease vs p diabetes vs p hypertension vs p and chronic kidney disease vs p were the comorbidities significantly associated with ICUadmission and death Table Autoimmune disease was the only significant predictivecomorbidity for ICU admission and death after adjusting for age and sex OR CI to Fig Depression was the best predictor of hospitalization among allcomorbidities OR 95CI to Fig Having � comorbidity was associated with ICU admission and death OR 95CI to and hospitalizationOR 95CI to independently of age and sexImaging and laboratory testsChest Xray was necessary in patients and showed lobar pulmonary infiltrates in bilateral pulmonary infiltrates in and an interstitial pattern in Table Chest CT was required in patients and pulmonary ultrasound in Biologically of patients had lymph ia mm3 Likewise had a lactate dehydrogenase LDH Uml and liver test alterations were commonelevated ASTGOT in and ALTGPT in In of cases Ddimer waselevated 500mgL The most important factors for ICU admission and death were bilateralpulmonary infiltrates OR 95CI to elevated lactatedehydrogenaseOR 95CI to elevated Ddimer OR 95CI to andelevated Creactive protein OR 95CI to Fig Significant predictivePLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsFig Prognostic factors for death and ICU admission �The upper limits of the confidence intervals were restricted to in order not to mask the significant effects of other variables with smaller ranges101371journalpone0237960g001factors associated with hospitalization after adjusting for age and sex were lymph iaOR 95CI to and elevated Creactive protein OR 95CI to Fig Treatment complications and evolutionTreatment included hydroxychloroquine in patients azithromycin in lopinavirritonavir in glucocorticoids in and tocilizumab in among others Table and of patients required hospitalization Phone follow up was registered in patients patients were monitored at homePLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsFig Prognostic factors for hospitalization �The upper limits of the confidence intervals were restricted to inorder not to mask the significant effects of other variables with smaller ranges101371journalpone0237960g002 of the patients of working age sought work disability due to COVID19 TheICU admission rate was The evolution included pneumonia in patientsadult respiratory distress syndrome in severe renal failure in pulmonarythromboembolism in and sepsis in patients Occupational contact with persons with confirmed or suspected COVID19 infection was reported by patientswhile reported that contact occurred in the family setting Occupational contactwas a protective factor against hospitalization OR 95CI to ICU admission and death OR 95CI to after adjusting for age and sex The mortalityrate to date was PLOS ONE 101371journalpone0237960 August PLOS ONE 0cTable Comorbidities associated with hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admissionHospitalizationn NoYesPAdjusted OR [NoYesPAdjusted OR [n n CI]n n CI]Prognostic factors in Spanish COVID19 patientsComorbidities”no  Any comorbidityHypertensionDiabetesObesityDyslipidaemiaCancer [“] [“] [“] [“] [“] [“] [“] [“] Chronic kidney disease Heart disease Autoimmune disease Chronic obstructive pulmonary diseaseDepressionCardiac arrhythmiaThyroid alterationsAsthmaLiver diseaseCerebrovascular diseaseAlzheimer disease [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Comorbidities with a frequency of patients were bronchiectasis n fibromyalgia n anaemia n arthritis n HIV n syphilis n andtuberculosis n 101371journalpone0237960t002DiscussionThis study summarizes the clinical biological and radiological characteristics evolution andprognostic factors of patients with COVID19 disease in primary and community healthcareTo date we are aware of three published Spanish studies [“] The first reported data from patients on ICU admissions in a region where the pandemic was reported early [] Thestudy by Borobia [] describes the first adult patients with COVID19 consecutivelyadmitted to a University Hospital in Madrid The third focuses on the differences by agedependent categories in the clinical profile presentation management and shortterm outcomes [] Although there have been two systematic reviews and metaanalysis that analysethe clinical characteristics of COVID19 they are limited to Chinese cohorts or case series [] and a large USA cohort [] that did not analyse clinical predictors of a poor prognosisClinically the same main symptoms of cough and fever are reported in all series Howeverin Barcelona city we have observed diarrhoea anosmia and dysgeusia which is hardlyreported in the Chinese series [] which unlike ours comes principally from hospitals diarrhoea occurred in of cases very similar to the in New York [] and clearly higherthan the reported in China Nearly of patients had anosmia and dysgeusia similar tothe results obtained in French patients [] In contrast expectoration was found in only compared with in the Chinese seriesPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Analytical and radiological predictors of hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admission n Hospitalization n n NoYesPAdjusted ORNoYesn n [ CI]n n PAdjusted OR[ CI]Alterations in chest Xray”nototalno  Bilateral pulmonary infiltratesInterstitialground glass patternLobar pulmonary infiltrateAlterations in chest CAT scan”nototal no ¡ [“] [“] [“] [“] [“] [“]Bilateral pulmonary infiltrates Interstitialground glass pattern Laboratory parameters”nototalno [“] [“] [“] [“]Leukocytes mm3 [“] [“]Lymphocytes mm3Platelets mm3 [“] [“] [“] [“]Haemoglobin gdl [“] [“]Creactive protein mglitreProcalcitonin ngmlLactate dehydrogenase UlitreAminotransferase aspartate UlitreAlanine aminotransferase UlitreTotal bilirubin mgdL [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]Creatine kinase Ulitre [“] [“]Creatinine 15mgdL Ddimer mglitreSodium mEqlitrePotassium mEqlitre [“] [“] [“] [“] [“] [“] [“] [“]In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  patients had a chest Xray The alterations with a frequency patients were pneumothorax n and pleural effusion n Chest Xray resultswere not available in patients¡ patients had a chest CAT scan Alterations with a frequency of patients were pulmonary thromboembolism n emphysema n lobarpulmonary infiltrates n pneumonia n atelectasis n and pleural effusion n CAT scan results were not available in five patients101371journalpone0237960t003PLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Predictors of the evolution complications and treatment in patients hospitalized or with ICU admissiondeathVariablesTotalDeath or ICU admission n Hospitalization n n NoYesPAdjusted OR [NoYesPAdjusted OR [n n CI]n n CI]Complications”no  Any complicationPneumonia NA [“] [“] [“]Adult respiratory distress [“ [“]syndromeRenal failurePulmonary thromboembolismTreatments”no ¡HydroxychloroquineAzithromycinLopinavirRitonavir [“] [“] [“]NA] [“] [“] [“] [“] [“] [“]Oxygen therapy [“] [“]Intravenous antibiotics [“] [“]GlucocorticoidsTocilizumabCephalosporinsLow molecular weight heparin Remdesivir Covid19 infection”no [“] [“] [“] [“] [“] [“] [“] [“] [“] [“]Any cohabitant [“] [“]Any work colleague [“] [“]Any contact person in other [“] [“]settingsIn bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex  Complications in patients were sepsis n multian failure n electrolyte alterations n hematologic alterations n and lung cancer n ¡ Treatments with a frequency of patients except remdesivir were amoxicillin n interferon n rituximab n darunavir n and entecavirn NA not applicable101371journalpone0237960t004Chinese patients had a mean age of years ten years lower than our series and ofour patients were aged � years compared with and in China Germany andthe USA respectively but in Italy [ “] Older age and male sex predisposed to ahigher mortality rate in our and all large series [ ] In our patients comorbidities werethree times higher than in the Chinese cohort [] and were similar to the findings of the NewYork study [] Any comorbidity was a risk factor for hospitalization ICU admission anddeath Depression was an independent risk factor for hospitalization which has not beenobserved in other cohorts studied Depression was often accompanied by a vulnerable socialsituation which may have justified hospitalization Likewise autoimmune diseases were independent risk factor for ICU admission and death Various hypotheses have been postulated onpossible autoimmune alterations in the pathogenic evolution of the disease With respect toPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientstreatment no drug has proved effective against Covid19 until now Moreover many treatments were unavailable in the outpatient setting Currently we are only certain that treatmentwith tocilizumab showed better survival rates in retrospective cohorts [] although its efficacy has not been tested in randomized clinical trials Therefore the results on the outcomesassociated with treatment should be interpreted with cautionThe same comorbidities were identified with hypertension and diabetes being the twomost common while in the USA and Italy obesity seems to be higher Our results show thatobesity was close to being an independent risk factor for hospitalization OR CI to Strikingly of our patients were healthcare workers compared with in Wuhanand in Germany [ ] Although these studies recognized an important degree ofunderreporting of cases in health workers the difference remains important There are at leasttwo possible explanations first the lack of personal protective equipment in the initial phaseof the epidemic a constant revindication of health professionals who felt undersupplied Secondly many cases were health professionals from primary healthcare or the reference hospitalwho reside in the same area where they workIn all reported series bilateral pneumonia was the most common radiological finding waspresent in more than half the cases [] and was a factor of a poor prognosis and mortality Incontrast an interstitial radiological pattern did not confer an increased risk of mortality TheWuhan study reported a CAT scan use of compared with in Barcelona In contrast chest Xrays were carried out in and respectively the availability of diagnostic means was higher in China A recent international consensus states that radiologicalassessment is not necessary in asymptomatic patients or those with mild disease but is requiredin patients with moderate or severe disease regardless of whether a definite diagnosis ofCOVID19 has been made [] In addition simple chest Xrays are preferable in a resourceconstrained environment with difficulties in accessing CAT scans [] The possible use of pulmonary ultrasound for the pointofcare diagnosis of COVID19 pneumonia has not been sufficiently analysed but might be an efficient alternative due to its portability and reliability []In fact the regional Catalan government has recently acquired ultrasound machines toenable family physicians to make doctors can make pointofcare home or nursing homediagnoses of pneumonia [] Biologically lymph ia and increased CRP LDH and Ddimer were usually constant and similar in all series and were associated with an increased riskof mortality A differential variable in our series is a greater number of alterations in liver testswhich was present in “ of patients data similar to the USA and Italian cohorts but different from the Chinese cohort where it was [] We also found hypokalaemia in of patients a factor not reported in other studiesWe found a hospitalization rate of compared with “ in the USA and inChina and an ICU admission rate of which was similar to the Chinese USA “ and German results While the protocols of action and admission are similarand depend on the level of clinical involvement the therapeutic protocols differ between hospitals cities and countries There remain many unknowns in the treatment of COVID19The only truth is that we do not have a vaccine an etiological treatment or a treatment withsufficient scientific evidence to generalize its use Currently the systematic review of antiretroviral treatments has not offered conclusive results [] and despite in vitro results for hydroxychloroquine COVID19 infections are currently intractable [ ]The mortality rate in our study was compared with in New York in hospitalized patients in China in Germany and in Italy Different informationand recording systems the availability of diagnostic tests and above all the anization ofnational health systems may have contributed to the differences observedPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsThe study had some limitations due to the observational retrospective design However itis sufficiently representative of the population with confirmed COVID19 to permit betteridentification of the factors of a poor prognosis of the disease from a clinical perspective Wecannot rule out some heterogeneity in data codification due to observers™ interpretations of themedical records However this bias is minimal as most clinical factors included are clearlydefined in the electronic medical record Another limitation of this study is the percentage ofpatients without laboratory parameters more than Even though in real clinical practicethese percentages may be expected the results corresponding to laboratory parameters shouldbe interpreted with cautionFour months after the declaration of the pandemic there is not a sufficiently reliable available and generalizable diagnostic test that can analyse the seroprevalence of COVID19 evenin the most industrialized countries Given this lack determining the clinical biological andradiological characteristics of probable cases of COVID19 infection will be key to the initiation of early treatment and isolation and for contact tracing especially in primary healthcareSupporting informationS1 DatasetXLSXAcknowledgmentsThe authors thank David Buss for editorial assistanceAuthor ContributionsConceptualization Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuData curation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuFormal analysis Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuInvestigation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuMethodology Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuProject administration Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuResources Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuSupervision Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuValidation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsVisualization Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuWriting “ original draft Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuWriting “ review editing Antoni Siso´Almirall Belchin Kostov Minerva MasHerediaSergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana AugustAnguitaGuimet Jaume BenaventÃreuReferences World Health anization Pneumonia of unknown cause”China wwwwhointcsrdon05january2020pneumoniaofunkowncausechinaen accessed April Centers for Disease Control and Prevention Novel Coronavirus 2019nCoV Situation Summarystackscdcgovviewcdc84806cdc_84806_DS1pdf accessed April Holshue ML DeBolt C Lindquist S Lofy KH Wiesman J Bruce H First Case of Novel Coronavirus in the United States N Engl J Med “ 101056NEJMoa2001191 PMID World Health anization Novel Coronavirus2019nCoV wwwwhointdocsdefaultsourcecoronavirusesituationreports20200130sitrep10ncovpdfsfvrsnd0b2e480_2 accessed AprilJohns Hopkins University CSSE COVID19 Dashboard by the Center for Systems Science and Engineering CSSE at Johns Hopkins University JHU gisanddatamapsarcgiscomappsopsdashboardindexhtmlbda7594740fd40299423467b48e9ecf6 accessed April Ministerio de Sanidad Gobierno de España Situacio´n del COVID19 en España covid19isciiies accessed April Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Clinical Characteristics of Coronavirus Disease in China N Engl J Med 101056NEJMoa2002032 PMID Yeo C Kaushal S Yeo D Enteric involvement of coronaviruses is faecaloral transmission of SARSCoV2 possible Lancet Gastroenterol Hepatol “ 101016S2468 PMID Chang BB Chiu TY Ready for a long fight against the COVID19 outbreak an innovative model oftiered primary health care in Taiwan BJGP 103399bjgp 20X101068PMID Barrasa H Rello J Tejada S Martı´n A Balziskueta G Vinuesa C SARSCov2 in Spanish Intensive Care Early Experience with 15day Survival In Vitoria Anaesth Crit Care Pain Med 101016jaccpm202004001 PMID Borobia AM Carcas AJ Arnalich F A´ lvarezSala R MonserratVillatoro J Quintana M A Cohortof Patients with COVID19 in a Major Teaching Hospital in Europe J Clin Med Martı´nSa´nchez FJ Del Toro E Cardassay E Valls Carbo´ A Cuesta F Vigara M Clinical presentation and outcome across age categories among patients with COVID19 admitted to a Spanish Emergency Department Eur Geriatr Med 101007s41999020003592 PMIDFu L Wang B Yuan T Chen X Ao Y Fitzpatrick T Clinical characteristics of coronavirus disease COVID19 in China A systematic review and metaanalysis J Infect 101016jjinf202003041 PMID RodriguezMorales AJ CardonaOspina JA Gutie´rrezOcampo E VillamizarPeña R HolguinRiveraY EscaleraAntezana JP Clinical laboratory and imaging features of COVID19 A systematicreview and metaanalysis Travel Med Infect Dis 101016jtmaid2020101623PMID Richardson S Hirsch JS Narasimhan M Crawford JM McGinn T Davidson KW PresentingCharacteristics Comorbidities and Outcomes Among Patients Hospitalized With COVID19 inthe New York City Area JAMA 101001jama20206775 PMID Goyal P Choi JJ Pinh

Thyroid_Cancer

" COVID19 pathways for brain andheart injury in comorbidity patients A role of medical imaging and artificial intelligencebased COVIDseverity classification A review Computers in Biology and Medicine 101016jcompbiomed2020103960 0cThis is a PDF file of an that has undergone enhancements after acceptance such as the additionof a cover page and metadata and formatting for readability but it is not yet the definitive version ofrecord This version will undergo additional copyediting typesetting and review before it is publishedin its final form but we are providing this version to give early visibility of the Please note thatduring the production process errors may be discovered which could affect the content and all legaldisclaimers that apply to the journal pertain Published by Elsevier Ltd 0cCOVID19 Pathways for Brain and Heart Injury in Comorbidity Patients A role of Medical Imaging and Artificial Intelligencebased COVID severity Classification A Review Jasjit S Suri PhD MBA FAIMBE FAIUM FAPVS1 Anudeep Puvvula MBBS FCD12 Mainak Biswas PhD3 Misha Majhail14 Luca Saba MD5 Gavino Faa MD6 Inder M Singh MD7 Ronald Oberleitner BS MBA8Monika Turk MD PhD9 Paramjit S Chadha BE1 Amer M Johri MD FASE10 J Miguel Sanches PhD11 Narendra N Khanna MD DM FACC12 Klaudija Viskovic MD PhD13 Sophie Mavrogeni MD PhD FESC14 John R Laird MD FACC FACP15 Gyan Pareek MD16 Martin Miner MD17 David W Sobel MD16 Antonella Balestrieri MD5 Petros P Sfikakis MD18 Gee Tsoulfas MD19 Athanasios Protogerou MD PhD20 Durga Prasanna Misra MD FRCP21 Vikas Agarwal MD FRCP21 Gee D Kitas MD PhD FRCP22 Puneet Ahluwalia MS MCh24 Raghu Kolluri MD25 Jagjit Teji MD26 Mustafa AlMaini MD27 Ann Agbakoba MD28 Surinder K Dhanjil MSc FSVU29 Meyypan Sockalingam MBBS FRCR30 Ajit Saxena MD12 Andrew Nicolaides MS PhD FRCS31 Aditya Sharma MD32 Vijay Rathore MD33 Janet N A Ajuluchukwu MD34 Mostafa Fatemi PhD FAIMBE FIEEE FASA FAIUM35 Azra Alizad MD FAIMBE FAIUM36 Vijay Viswanathan MD PhD37 P K Krishnan MD38 Subbaram Naidu PhD Life FIEEE39 1Stroke Monitoring and Diagnostic Division AtheroPoint„¢ Roseville CA USA 2Annu™s Hospitals for Skin and Diabetes Nellore AP INDIA 3JIS University Kolkata West Bengal INDIA 4Oakmont High School and AtheroPoint„¢ Roseville CA USA 5Department of Radiology Azienda Ospedaliero Universitaria Cagliari ITALY 6Department of Pathology AOU of Cagliari Italy 7Stroke Monitoring and Diagnostic Division AtheroPoint„¢ Roseville CA USA 8Behavior Imaging USA 9The HanseWissenschaftskolleg Institute for Advanced Study Delmenhorst GERMANY 10Department of Medicine Division of CardiologyQueen™s University Kingston Ontario CANADA 11Institute of Systems and Robotics Instituto Superior Tecnico Lisboa PORTUGAL 12Department of Cardiology Indraprastha APOLLO Hospitals New Delhi INDIA 13University Hospital for Infectious Diseases Zagreb CROTIA 14Cardiology Clinic Onassis Cardiac Surgery Center Athens GREECE 15Heart and Vascular Institute Adventist Health St Helena St Helena CA USA 16Minimally Invasive Urology Institute Brown University Providence Rhode Island USA 17Men™s Health Center Miriam Hospital Providence Rhode Island USA 18Rheumatology Unit National Kapodistrian University of Athens GREECE 19Aristoteleion University of Thessaloniki Thessaloniki GREECE 20National Kapodistrian University of Athens GREECE 21Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow UP INDIA 22Academic Affairs Dudley Group NHS Foundation Trust Dudley UK 23Arthritis Research UK Epidemiology Unit Manchester University Manchester UK 24Max Institute of Cancer Care Max Superspeciality Hospital New Delhi INDIA 25OhioHealth Heart and Vascular Ohio USA 26Ann and Robert H Lurie Children™s Hospital of Chicago Chicago USA 27Allergy Clinical Immunology and Rheumatology Institute Toronto CANADA 28University of Lagos NIGERIA 29AtheroPoint LLC CA USA 30MV Center of Diabetes Chennai INDIA 31Vascular Screening and Diagnostic Centre and University of Nicosia Medical School CYPRUS Journal Preproof 0c32Division of Cardiovascular Medicine University of Virginia Charlottesville VA USA 33Nephrology Department Kaiser Permanente Sacramento CA USA 34Department of Medicine Lagos University Teaching Hospital Lagos NIGERIA 35Dept of Physiology Biomedical Engg Mayo Clinic College of Medicine and Science MN USA 36Dept of Radiology Mayo Clinic College of Medicine and Science MN USA 37MV Hospital for Diabetes and Professor M Viswanathan Diabetes Research Centre Chennai INDIA 38Neurology Department Fortis Hospital Bangalore INDIA 39Electrical Engineering Department University of Minnesota Duluth MN USA Corresponding Author Dr Jasjit S Suri PhD MBA FAIMBE FAIUM FAPVS Fellow American Institute of Medical and Biological Engineering Fellow American Institute of Ultrasound in Medicine Fellow Asia Pacific Vascular Society Stroke Monitoring and Diagnosis Division AtheroPoint„¢ Roseville Roseville CA USA Phone Email jasjitsuriatheropointcom Journal Preproof 0cCOVID19 Pathways for Brain and Heart Injury in Comorbidity Patients A role of Medical Imaging and Artificial Intelligencebased COVID severity Classification A Review Abstract Artificial intelligence AI has penetrated the field of medicine particularly the field of radiology Since its emergence the highly virulent coronavirus disease COVID19 has infected over million people leading to over deaths as of July 1st Since the outbreak began almost s about COVID19 have been published pubmedncbinlmnihgov however few have explored the role of imaging and artificial intelligence in COVID19 patients”specifically those with comorbidities This paper begins by presenting the four pathways that can lead to heart and brain injuries following a COVID19 infection Our survey also offers insights into the role that imaging can play in the treatment of comorbid patients based on probabilities derived from COVID19 symptom statistics Such symptoms include myocardial injury hypoxia plaque rupture arrhythmias venous thromboembolism coronary thrombosis encephalitis ischemia inflammation and lung injury At its core this study considers the role of imagebased AI which can be used to characterize the tissues of a COVID19 patient and classify the severity of their infection Imagebased AI is more important than ever as the pandemic surges and countries worldwide grapple with limited medical resources for detection and diagnosis We conclude that imaging and AIbased tissue characterization when considered alongside COVID19 symptoms and their pretest probabilities offer a compelling solution for assessing the risk of comorbid patients These methods show the potential to become an integral part of tracking and improving the healthcare system both during the pandemic and beyond Keywords COVID19 comorbidity pathophysiology heart brain lung imaging artificial intelligence risk assessment Journal Preproof 0c Introduction In December a novel coronavirus referred to as œsevere acute respiratory distress syndrome coronavirus  SARSCoV2 [] appeared in Wuhan the capital of Hubei Province in PR China The disease caused by the virus was initially named œnovel coronavirus pneumonia NCP by the Chinese government but was subsequently renamed œcoronavirus disease  COVID19 by the World Health anization WHO On January 30th it was declared a public health emergency of international concern PHEIC [] It is believed that SARSCoV2 is primarily transmitted through saliva droplets or nasal discharge [] The first evidence of humantohuman transmission was found by Jasper FukWoo Chan et al in their study at The University of Hong KongShenzhen Hospital [] Due to its contagiousness Ro27 the virus has reached epidemic levels affecting countries and causing over million infections and more than deaths as of July 1st [] shown in Figure Recent literature suggests that patients with preexisting diseases are likely to experience severe complications from COVID19 [] In one study on admitted diabetic and nondiabetic COVID19 patients the mortality rate vs and the rate of admission to the intensive care unit ICU vs were significantly higher for diabetic patients Diabetic patients also experienced severe inflammatory responses and cardiac hepatic and renal coagulopathy [] The prevalence of heart and brain injuries was also higher in COVID19 patients with concomitant chronic conditions like diabetes kidney disease dyslipidemia hypertension [] chronic obstructive pulmonary disease COPD and cardiovascular diseases [] Recent studies have shown that SARSCoV2 invades the thin lining of the epithelial cells of the arteries leading to atherosclerosis [] and arterial inflammatory disease”one of the major causes of cardiovascular diseases CVDs which also causes heart and brain injuries [ ] This could be due to a reduced expression of angiotensinconverting enzyme ACE2 causing endothelial dysfunction which in turn aggravates existing Journal Preproof 0catherosclerosis [ ] It has also been observed that comorbid patients when subjected to imagescreening show mild to severe pretest probability PTP for COVID19 [] The conventional cardiovascular risk factors CCVRF in these comorbid patients appear strongly correlated either to their heart imaging or to surrogate biomarkers of coronary artery disease such as carotid artery disease Both imaging and biomarkers could be helpful in severity predictions for COVID19 [] Figure illustrates the associations between SARSCoV2 and other comorbidities such as diabetes as well as the comparative survival rates for COVID19 patients with and without diabetes Figure World map showing COVID19 spread over countries courtesy John Hopkins University ACE2 expression causes scars in the vessels and can even rupture the walls of the arteries [] For this reason CCVRF should be considered alongside imaging in patients who present with COVID19 and many comorbidities [] The second stage is the one at which a patient is most severely affected by Journal Preproof 0cCOVID19 and has the highest probability of cardiac injury or release of troponin T TnT Imaging has been shown to offer benefits in monitoring the tissue scars caused by COVID19 [] Figure a Association of SARSCoV2 with other comorbidities and b comparison of the mortality rate of diabetic and nondiabetic COVID19 patients reproduced with permission [] Multiple modalities can be utilized to determine whether a patient has the sequelae of COVID19 including magnetic resonance imaging [] computed tomography [] and ultrasound [] The advantage of these imaging modalities is the visual access they provide to the scar tissue caused by the disease A disadvantage however is their inability to provide a œrisk assessment The application of artificial intelligence AI can enhance the information provided by these imaging modalities resulting in a more accurate characterization of the tissue and the disease process [] In fact the combination of AI and medical imaging has been shown to improve diagnosis and risk stratification speed up patient evaluation enhance disease monitoring and accelerate early intervention [ ] Thus this review will focus on the use of AIbased tissue characterization of images of comorbid patients affected by COVID19 The layout of this paper is as follows Section presents the pathophysiology of the four pathways leading to brain and heart injury Section summarizes the evidence related to the use of Journal Preproof 0cimaging during the COVID19 pandemic Section elaborates on the use of AIbased tissue characterization for risk assessment Finally the paper concludes with a critical discussion The Pathophysiology of SARCoV2 Leading to Brain and Heart Injury Several studies suggest that SARSCoV2 uses the ACE2 receptor to gain access to cells by binding to the SPIKE protein ˜S™ protein on their surface [] see Figure ACE2 and angiotensinconverting enzyme ACE1 are homolog carboxypeptidase enzymes that have different vital functions in the reninangiotensinaldosterone system RAAS pathway [] ACE2 is widely expressed in myocardial cells [] type pneumocytes enterocytes and astrocytes in the brain [ ] Thus it is recognized as a cause of extrapulmonary complications Figure shows the overall picture of how SARSCoV2 causes brain and heart injuries via four different pathways These include i the neuronal pathway ii the hypoxia pathway iii the RAAS pathway and iv the immune pathway We will discuss these pathways and the injuries they lead to which may manifest as viral encephalitis infectious toxic encephalopathy or acute cerebrovascular disease i The Neuronal Pathway Figure the pathway I Recent epidemiological studies have demonstrated similarities at the genomic level between SARSCoV1 MERS and SARSCoV2 [ ] Meanwhile previous experimental studies have shown that beta coronaviruses in general”such as SARSCoV1 and MERS”can spread into and directly infect the brain when inhaled as droplets via the nasal epithelium [ ] Figure depicts the olfactory nerve and the olfactory bulb []”labeled as œa and œb respectively”on the image of the sagittal brain in the neuronal pathway Based on recent reports we are aware that patients infected by SARSCoV2 show symptoms of dysgeusia loss of taste and anosmia loss of smell [ ] Bohmwald et al further validate that coronaviruses that infect through the olfactory nerve and bulb can reach the brain and cerebrovascular fluid CSF within seven days Additionally these viruses have been observed to cause inflammation and demyelination [] The Journal Preproof 0cauthors demonstrated in an experimental study of mice that removing the olfactory bulb from the pathway can lead to the restriction of CoV in the central nervous system CNS [] Based on this evidence we believe that the neuronal pathway is one possible track for SARSCoV2 ii The Hypoxia Pathway Figure pathway II In this pathway decreased levels of ACE2 proliferate in the lung parenchyma cells after the coronavirus has passed through causing exaggerated neutrophils accumulation enhanced vascular permeability and the formation of diffuse alveolar and interstitial exudates This ultimately leads to pulmonary edema and acute respiratory distress syndrome ARDS [] ARDS is characterized by severe abnormalities in blood gas composition resulting from an oxygen and carbon dioxide mismatch which leads to low blood oxygen levels [ ] This ongoing hypoxia can lead to myocardial ischemia and heart injury [ ] see Figure pathway IIA Hypoxia in the brain increases anaerobic metabolism in the mitochondria of the brain cells [] leading to cerebral vasodilatation edema and impaired flow This can result in cerebral ischemia and acute cerebrovascular diseases such as acute ischemic stroke [ ] see Figure pathway IIB iii The RAAS Pathway after SARSCoV2 Figure pathway III The RAAS pathway is crucial in regulating blood pressure as well as the balance of fluid and electrolytes Any disturbance in this pathway can trigger the pathogenesis of cardiovascular diseases [] Before a SARSCoV2 infection triggers the RAAS Angiotensin I Ang I cleaves to Angiotensin II Ang II via ACE1 Ang II causes vasospasm It is also a proinflammatory agent with prothrombotic and proliferative effects that are detrimental to vascular tone and hemostasis [ ] Thus as a counterregulatory mechanism ACE2 degrades Ang II and generates Ang which counteracts the negative impacts of Ang II [ ] Both ACE2 and Ang have cardiocerebral vascular protective effects [] After the triggering of SARSCoV2 infection it results are in the deregulation of RAAS causing heart and brain injury in two different pathways The main culprit is an increase in Ang II which is caused by the decrease in ACE2 levels Figure pathway IIIA First an increase in Ang II levels stimulates the adrenal cortex of the kidney resulting in an increased production of aldosterone Aldosterone is a steroid hormone that causes sodium and water Journal Preproof 0creabsorption to increase at the distal tubule and collecting duct of the nephron [] This reabsorption increases blood volume and causes an elevation in blood pressure which results in the endothelial dysfunction that causes brain and heart injury [] The second effect of an increase in Ang II levels ie as a consequence of decreased ACE2 levels is endothelial dysfunction leading to intimal damage in the arterial walls [] which can be seen during the imaging of the arterial wall see Figure pathway IIIB This pathway can also trigger a cytokine storm as high levels of Ang II can cause an increase in proinflammatory cytokines see the bridge line between the RAAS and immune pathways iv The Immune Pathway Figure pathway IV Several recent studies have reported SARSCov2 viral pneumonia [ ] having an exaggerated inflammatory response known as a œcytokine storm This response appears to present at advanced stages of severe COVID19 with increased levels of inflammatory cytokines leading to multiplean failure [] The rise in inflammatory markers”including IL6 IL7 IL12 IL15 IL22 TNFα and CXCL10”results in the destabilization of plaque This in turn can cause plaque rupture resulting in heart and brain injury [ ] The Role of Imaging in Comorbid Patients with COVID19 As the previous section discussed COVID19 uses four pathways ie neuronal hypoxia RAAS and immune to cause critical heart and brain injuries in patients with comorbidities The prevalence of myocardial injury and brain injury caused by COVID19 [ ] points to a need for increased use of medical imaging to expedite assessments differential diagnoses and patient management [ ] with proper safety measures [] The seriousness of a patient™s COVID19 symptoms helps to determine which imaging modality is appropriate portable or nonportable and invasive or noninvasive BMode ultrasound imaging is portable and can be used for lowrisk patients Meanwhile Xray magnetic resonance imaging [] and computed tomography [] are nonportable and can be used for mediumrisk patients Intravascular ultrasound IVUS [] and ventriculography are invasive imaging modalities used in highly critical cases [ ] Amongst all the imaging modalities ultrasound is noteworthy because it is radiationfree portable quick repeatable inexpensive Journal Preproof 0cand can be performed in isolation thus lowering the chance of spreading the COVID19 infection [ ] There are several examples of medical imaging that have led to proper treatment and healthcare management during the pandemic ultimately reducing the mortality rate Xray imaging of the chest has demonstrated irregular patchy hazy reticular and widespread groundglass opacities indicating the progression of COVID19 at various stages this information can support the healthcare team in developing the most appropriate treatment plan [] Chest CT scans of COVID19 patients revealed in almost of the patients that the disease was affecting at least one of the five lobes of their lungs [] Chest MRI scans of COVID19 patients showed pulmonary tissue consolidation in six diffusionrestricted regions in six and lung damage in seven [] Meanwhile heart MRI studies of recovered patients showed that of the patients had myocardial edema At the same time late gadolinium enhancement was found in implying that COVID19“related cardiac injury is longstanding and requires frequent monitoring even after recovery [] In a different study MR scans of a COVID19 patient revealed myocardial inflammation signifying myocardial injury due to a cytokine storm related to the SARSCoV2 infection as discussed in Section Pathway IV [] Several studies have also evaluated the effects of COVID19 on the brain In one MRI scans revealed hemorrhagic rim enhancing lesions within the bilateral thalami medial temporal lobes and subinsular regions [] shown in Figure In another brain MRI scans were completed for patients of which produced abnormal findings [] Additionally evidence of liver injury and gall bladder abnormality was found in a joint CT and ultrasound study of the abdomen [] Recent MRI scans of COVID19 patients™ olfactory bulbs have revealed the cause of olfactory function loss to be the interaction between SARSCoV2 and the ACE2 protein expressed by the olfactory epithelium which leads to inflammatory obstruction [] Journal Preproof 0cFigure We have shown in four pathways how COVID19 can cause Brain and heart injury Brain image in pathway I httpdebugliescom20200123olfactorydisturbanceshaveimplicationsinmentalandemotionalwellbeinghealth Courtesy of Debug Lies Invasive imaging is another option for diagnosing COVID19 patients who have critical comorbidities In one such study IVUS along with stenting was performed with precautions on a COVID19 patient with myocardial infarction [] shown in Figure A detailed discussion of precautions is included in section In another study takotsubo syndrome a form of myocardial injury triggered by COVID19 was detected using ventriculography [] In various studies the medical imaging of COVID19 patients had been crucial to ascertaining the extent of tissue damage and critical infection although there were no visible symptoms [ ] Therefore medical imaging is the preferred way to ascertain the extent of cardiac and brain tissue damage throughout the lifetime of COVID19 patients COVID19 Journal Preproof 0cpatients with comorbidities are especially vulnerable and so they need to be screened through medical imaging from the first day of their diagnosis Medical imaging can help patients with deep vein thrombosis DVT as they are highly susceptible to severe tissue damage from COVID19 A study showed that COVID19 patients suffering from DVT had a worse prognosis than patients without DVT Patients with DVT were admitted to the ICU more frequently discharged less frequently and suffered more deaths than those without DVT [] Figure MRI scan of COVID19 patient showing hemorrhage MRI images demonstrate T2 FLAIR hyperintensity within the bilateral medial temporal lobes and thalami A B E F with evidence of hemorrhage indicated by hypointense signal intensity on susceptibilityweighted images C G and rim enhancement on postcontrast images D H reproduced with permission [] Journal Preproof 0c Figure Application of chest CT and IVUS for a COVID19 patient suffering from myocardial infarction a Chest CT scan with viral pneumonia showing fibrinous focal exudative changes b when the patient complained of chest pain the ECG report showed the STsegments elevations in V1V5 lead c d CAG radiology that the proximal segment of LAD was occluded e f The blood flow of LAD restoration after DESs was implanted g the dissection distal shown by IVUS to the stent in LAD from “ o'clock h the low echogenic shadow with scattered higher echogenic flicker indicating a thrombus i after a DES was implanted and the stent was well expanded the dissection could not be seen j The thrombus disappearance after the intervention reproduced with permission [] Journal Preproof 0c Although medical imaging can be very useful to patients and doctors alike the exponential pandemic curve inadequate medical facilities [] and a limited number of radiologists make the assessment diagnosis and management processes challenging tedious and errorprone Therefore although medical imaging can make diagnoses faster as stated above it will be of limited use Given this fact new age techniques such as artificial intelligence AI [] applications in medical imaging for tissue characterization can make computeraided assessments and diagnoses faster The main reason for this is that the AI can be scaled up to match the pandemic curve thereby meeting the immediate demands of medical image diagnoses during the COVID19 pandemic AIbased tests can categorize the nature of a patient™s risk in one of the categories namely norisk low lowmedium LM highmedium HM lowhigh LH or highhigh HH risk depending on the patient™s symptoms and their severity [ ] as shown in Figure The imaging modality also varies with the degree of risk as follows no imaging for norisk portable imaging for low and LM risk nonportable imaging for HM and LH and invasive imaging for HH A probability PTP is performed to accurately interpret diagnostic results to categorize the patient into one of the four groups [] After that for norisk patients nonimaging biomarkers can be collected for risk assessment using AIbased data protocols For lowrisk patients portable 2D3D imaging such as ultrasound is used whereas nonportable and invasive 2D3D imaging such as MRICTXRayechocardiography can be used for LM patients For HH patients invasive imaging techniques such as IVUS and ventriculography can be used Based on the data provided by various 2D3D scans AIbased medical imaging is applied for risk assessment Further treatment is then planned based on this imaging process In the next section deep learning DLbased medical imaging is proposed for medical imaging scans particularly ultrasounds for COVID19 patients Machine Learning and Deep Learning for Tissue Characterization Using AI and associated technologies in healthcare can significantly slow down diagnosis times Journal Preproof 0cespecially during the COVID19 pandemic as patient numbers are continually growing and there are few specialists available [] Figure Role of AIbased risk assessment on COVID19 patients having comorbidity Journal Preproof 0cAlthough some caution must be exercised regarding its fullscale deployment [] its overall usefulness in healthcare management during times of crisis cannot be ignored [ ] In general AI in healthcare refers to all artificial intelligencebased technologies that make educated decisions regarding a patient™s diagnosis monitoring treatment and management The importance of AI has specifically increased many folds when imaging comes into play mainly because of large volumetric data sizes and the extensive need to characterize and quantify the disease via lesion images [] Tissue imaging and its characterization is of prime importance since it has a direct influence on decisions related to COVID19 severity for a patient [] The main benefit of AI methods is that the machines can be used to train by mimicking the physician™s cognitive actions and such trained models can be used to predict the disease™s severity in asymptomatic patients Within a short period several machine learning MLbased techniques used the power of AI to manage COVID19 [ ] ML and DL Architectures ML Architecture ML is a twostage process In stage I different features are extracted from the lesion COVID images the extractions are then operated on by an ML statistical model called a training system to generate offline coefficients These coefficients are then transformed by the test lesion images which yield an intelligent classification or inference A typical ML system for predicting risk class is shown in Figure CUSIP is an imagebased phenotype that uses the event equivalent gold standard EEGS [ ] model DL Architecture DL refers to a visual cortex that imitates multiple layers of a neural network applied directly to tissue images to extract features and for characterization purposes [] The convolution neural network CNN [] shown in Figure is one such DL network architecture that is widely used to characterize medical images It performs a series of convolution maxpooling operations to extract features and perform characterizations ML and DL both follow the supervised learning Journal Preproof 0capproach by which models are trained using offline data Figure Typical lowcost machine learning architecture utilizing EEGS model As discussed in previous sections there are four pathways through which a COVID19 infection leads to heart and brain injuries AI can be used via medical imaging to detect the extent of tissue damage in these pathways and help medical professionals to develop an effective treatment plan for patients There are several instances in which the AI paradigm has been used for tissue characterization based on Journal Preproof 0cmedical images during the pandemic as well as during standard times Some uses of AI are described anwise following a proposed model for characterizing DLbased tissues Figure A convolution neural network courtesy of AtheroPoint„¢ CA USA ML Architecture used for Tissue Characterization for Stroke Risk Stratification There are two types of tissue characterization that can be carried out using AI i MLbased [ ] and ii DLbased [] Various MLbased technologies have been developed to classify symptomatic and asymptomatic plaque from ultrasound images For example an MLbased technique based on support vector machines SVM was developed to characterize the symptomatic and asymptomatic plaques of carotid scans that indicated the presence of plaque [ ] SVM classifiers work by determining the maximum margin between two data clusters First a texture analysis [] is used to extract the features ie standard deviation entropy symmetry and run percentage in

Thyroid_Cancer

role of miR1179 in the development of cancer has been proved by different studies However the expression profile and role of miR1179 is yet to be explored in human oral cancer Consistently this study was undertaken to explore the molecular role of miR1179 in regulation of the human oral cancer development and progression The results showed miR1179 to be significantly p overexpressed in all the oral cancer cell lines relative to normal cells The repression of miR1179 transcript levels not only suppressed the proliferation of oral cancer cells but also increased their sensitivity to vincristine The decline in proliferative rates was attributed to induction of autophagy in oral cancer cells as confirmed by transmission electron microscopic analysis Western blot analysis showed that the expression of LC3BII increased and that of beclin decreased while LC3BI expression remained constant upon miR1179 inhibition Inhibition of miR1179 caused significant decrease in the migration and invasion of the oral cancer cells The migration and invasion found to be and for SCC9 and and for SCC25 cells upon miR1179 inhibition At molecular level the miR1179 was shown to exert its anticancer effects via deactivation of MEKERK and PI3KAKT signalling cascades In the findings point towards the potential of miR1179 in the treatment of oral cancerKeywords Oral cancer Micro RNA Proliferation Autophagy MetastasisIntroductionDespite advancement in science and technology the current strategies employed for the treatment of human oral cancer are still far from descent As such presently the oral cancer is still ranked 6th most prevalent type of cancer globally Peers et a0al The survival rates of oral cancer are comparatively lower than breast and prostate cancers The overall 5year survival rate of oral Correspondence MonicaHendrixaxoyahoocom Yanmei Gao and Hanmei Xu contributed equally to this workDepartment of Stomatology Affiliated Hospital of Jilin Medical University NO of Road Huashan Fengman Area Jilin Chinacancer is only as against and for breast and oral cancers respectively Gupta et a0al Additionally the recurrence of human oral cancer further adds to the problem Borsetto et a0al Hence it is need of the hour to look for the alternative approaches for the management of oral cancer As revealed by the recent research reports the molecular regulators of human cancers have emerged as vital targets to be studied for their usage against these deadly ailments Cao et a0 al Among these molecular agents the small RNA entities called microRNAs miRs which do not code for proteins but have molecular regulatory roles have been shown to be involved in the development and tumorigenesis The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 0cGao a0et a0al AMB Expr Page of of number of human cancers including the human oral cancer Gaezon et a0 al Wu et a0 al The miRs have not only been shown to regulate the proliferation of human cancers but have also been shown to have influence the metastasis to neighbouring tissues Peng et a0al MicroRNA1179 miR1179 has been implicated in the regulation of proliferation and metastasis of different human cancers Jiang et a0al Krutovskikh et a0al Song et a0 al Lin et a0 al The miR1179 has been shown to inhibit the growth of the glioblastoma cells by inducing cell cycle arrest Xu et a0al The inhibition of metastasis of hepatocellular carcinoma has been reported to be due to interaction of miR365 with ZEB2 Gao et a0al In yet another study miR targets HMGB1 to suppress the proliferation of gastric cancer cells Li and Qin Nonetheless the role and therapeutic implications of miR1179 has not be reported This study was therefore designed to investigate the role of miR1179 in human oral cancer cells via modulation of the MEKERK and PI3KAKT signalling pathwaysMaterials and a0methodsCell lines and a0culture conditionsThe oral cancer cell lines SCC4 SCC9 SCC15 and SCC25 along with normal EBTr cell line were procured from ATCC USA The culturing of cell lines was performed using Dulbeccoʼs modified Eagleʼs medium DMEM Thermo Scientific The cell lines were maintained in a CO2 incubator at a0°C with CO2 concentration and relative humidity of TransfectionTo stably transfect the oral cancer cells with miRNC and miRinhibitor Lipofectamine Thermo Scientific was used and the procedure was carried as per the manufacturer guidelinesExpression analysisThe RNeasy Mini Kit Qiagen and miScript Reverse Transcription Kit Qiagen were respectively used to isolate the RNA from cancer and normal cell lines and for cDNA synthesis The SYBR Green mix Thermo Scientific was used for performing the expression analysis of miR1179 through quantitative realtime polymerase chain reaction qRTPCR on QuantStudio real time PCR Thermo Scientific The cycling conditions were as follows °C for a0 s followed by cycles of °C for a0s and °C for a0min The expression values were quantified using ˆ’ ddCt method The real primer sequences were miRF ² GCG GAA GCA TTC TTT CAT ² and miRR ² CAA GGG CTC GAC TCC TGT ²Cell viability assayTo assess the proliferation rates of the oral cancer cells transfected with miRNC and miRinhibitor for a0 h and administered withwithout a0 µM vincristine the cells were cultured in 96well plate for a0h a0h or a0h at °C Following this the 345dimethylthiazol2yl25diphenyl tetrazolium bromide MTT reagent Thermo Scientific was added at final concentration of After a0 h a0 µl dimethyl sulfoxide DMSO was added for dissolving the crystals of formazan Absorbance at a0 nm was taken using spectrophotometer to determine the proliferation rates of oral cancer cellsElectron microscopyFor the assessment of induction of cell autophagy the cancer cells transfected with miRNC or miRinhibitor for a0 h were examined using Transmission electron microscope TEM Trypsin treatment was used to make the collection of cancer cells which were then washed and then fixed with glutaraldehyde in phosphate buffer a0m The Osmium tetroxide was then used for postfixing of cells The cells were then treated with ethanol and embedded in resin The ultramicrotome was used for section cutting which was followed by electron microscopyTranswell chamber assayThe migration and invasion of oral cancer cells transfected with miRNC or miRinhibitor were determined by using Transwell chamber without or with matrigel coating Here the cell suspension containing approximately cells was poured into the upper portion of transwell chamber and lower portion was supplemented with a0µl DMEM medium with fetal bovine serum FBS Following a0 h incubation at a0 °C and CO2 concentration the cancer cells from the surface of membrane™s upper side were swabbed away with cotton swabs while the cells sticking to lower surface of membrane were fixed with ethanol and the stained with crystal violet The cells were then examined and photographs were taken using — light microscopeWestern blottingFor the estimation of protein concentrations the western blotting technique was used Precisely after transfection with miRNC or miRinhibitor the oral cancer cells were cultured for a0h at a0°C Centrifugation was used to collect the cells which were then washed using PBS buffer This was followed by treatment with RIPA buffer containing a0 mM TrisHCl pH a0 mM NaCl Triton X100 SDS a0mM EDTA a0mM Na2HPO4 a0mM NaF a0mM NaVO4 a0mM phenyl 0cGao a0et a0al AMB Expr Page of methylsulfonyl fluoride and protease cocktail inhibitor The total protein concentrations were estimated using Bradford assay Equal protein concentrations were loaded on the Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDSPAGE from each sample The gel was blotted to PVDF membrane followed by treatment with primary antibodies Santa Cruz Biotechnology Inc Dallas TX USA overnight at °C The blots were washed in tris buffered saline TBS incubated with horseradish peroxidaseconjugated secondary antibody Santa Cruz Biotechnology Inc for a0h at °C washed again three times with TBS and chemiluminescence was captured on hyperfilm following incubating the blots in enhanced chemiluminescence reagentStatistical analysisThe experiments were performed in triplicate and expressed as mean ± standard deviation SD The Student™s ttest was performed through GraphPad Prism software The pvalues were taken as statistically significant differenceResultsmiR‘ is a0upregulated in a0oral cancer cellsThe qRTPCR analysis showed miR1179 to be significantly upregulated in all the oral cancer cell lines studied SCC4 SCC9 SCC15 and SCC25 relative to normal oral cell line EBTr The maximum transcript upregulation of miR1179 was observed in SCC9 oral cancer cells 65fold and SCC25 cells 45fold relative to normal EBTr cells Fig a01a As such SCC9 and SCC25 cell lines were taken for further experimentationmiR‘ inhibition suppresses the a0proliferation and a0enhances chemosensitivity of a0oral cancer cellsTo reveal the molecular functionality of miR1179 in oral cancer the suppression of miR1179 was achieved by transfecting the SCC9 and SCC25 cancer cells with miR1179 inhibitor Using miRNC transfected as control the repression of miR1179 transcript levels was confirmed by qRTPCR method which showed significant p decrease of miR1179 expression in SCC9 and SCC25 cells Fig a01b Additionally the results showed that suppression of miR1179 resulted in remarkable decline of proliferation rates of SCC9 and SCC25 cancer cells Fig a01c Interestingly the antiproliferative effects of vincristine were shown to be greatly enhanced when SCC9 and SCC25 cancer cells were transfected with miRinhibitor construct Fig a0 Together the results clearly indicate that miR1179 inhibition suppresses proliferation and enhances the chemosensitivity of the human oral cancer cellsFig miR1179 regulates the proliferation of human oral cancer cells a Expression of miR1179 in human oral cancer and normal cells b Expression of miR1179 in miRNC and miR1179 inhibitor transfected SCC9 and ACC25 cells c Cell viability of miRNC and miR1179 inhibitor transfected SCC9 and ACC25 cells Individual experiments were performed in triplicate and expressed as mean ± SD P 0cGao a0et a0al AMB Expr Page of Fig Inhibition of miR1179 enhances the Vincristine sensitivity of the human SCC9 and SCC25 oral cancer cells Individual experiments were performed in triplicate and expressed as mean ± SD P by the western blotting of LC3BI LC3BII and beclin autophagy related proteins The LC3BI protein levels remined constant beclin protein levels declined and the LC3BII protein levels increased in miR1179 inhibitor transfected SCC9 and SCC25 oral cancer cells Fig a03b Hence it is evident that the transcript repression of miR1179 in human oral cancer cells declines the cell proliferation rates via induction of cell autophagymiR‘ suppression reduces oral cancer cell metastasisThe assessment of migration and invasion capabilities of SCC9 and SCC25 oral cancer cells transfected with miR inhibitor or miRNC constructs by transwell chamber assay showed that the miR1179 inhibitor mediated suppression of miR1179 expression resulted in significant decline in the migratory and invasive potential of SCC9 and SCC25 cancer cells Fig a0 The migration and invasion found to be and for SCC9 and and for SCC25 cells upon miR1179 inhibition Taken together the results indicate that miR1179 has a regulatory role on cancer cell metastasismiR‘ modulates MEKERK and a0PI3KAKT pathways in a0oral cancerIn order to analyse the molecular mechanics of miR1179 in oral cancer the repression of miR1179 was made in SCC9 and SCC25 oral cancer cells The assessment of MEKERK signalling pathway revealed that the decline in miR1179 expression reduced the phosphorylatedMEK and ERK pMEK and pERK protein levels Fig a0 5a However the protein levels of nonphosphorylated versions of MEK and ERK remained almost unchanged Similarly the reduction in phosphorylated protein versions of PI3K and Akt proteins pPI3K and pAkt was Fig Inhibition of miR1179 induces autophagy in oral cancer cells a Ultrastructural analysis of the miRNC and miR1179 transfected SCC9 and SC25 cells b Western blot analysis of miRNC and miR1179 transfected SCC9 and SCC25 cells showing the expression of LC3B I II and Beclin Individual experiments were performed in triplicateInhibition of a0miR‘ induces autophagy in a0the a0oral cancer cellsThe electron microscopic examination of SCC9 and SCC25 human oral cancer cells transfected with miR inhibitor or miRNC clearly revealed the presence of autophagy vesicular structures in miRinhibitor transfected cancer cells Fig a0 3a However such structures were totally lacking from the cancer cells transfected with miRNC The results were further supported 0cGao a0et a0al AMB Expr Page of Fig Inhibition of miR1179 inhibits the migration and invasion of miRNC and miR1179 transfected SCC9 and SC25 cells Individual experiments were performed in triplicate and expressed as mean ± SD P observed under miR1179 repression Fig a05b The protein level of PI3K and Akt remained unchanged Taken together the results reveal that the suppression of miR expression in oral cancer results in the blocking of phosphorylation of MEK ERK PI3K and Akt proteinsDiscussionOral cancer is one of the lethal human disorders and this malignancy is responsible for a considerable level of human mortality and morbidity Warnakulasuriya Peterson Overall the oral cancer ranks 6th in terms of its incidence rates globally One more worrying fact about human oral cancer is its recurrence and development of drug resistance Silva et a0 al So an urgency is felt in the scientific community to devise more robust measures for the oral cancer management Recently miRs have emerged as potent regulators of various human cancers as they have been seen to have profound role in human physiology and disease development Gong et a0al The deregulation of miR1179 has been implicated to be associated with a number of human cancers Peng et a0al In the present study miR1179 was found to be upregulated in oral cancer cells which is in accordance with several previous studies Peng et a0 al Krutovskikh et a0 al Further miR1179 downregulation was previously been shown to decline the viability of ovarian cancer cells Zhihong et a0al Similar observations were made from the results of the current study The miR1179 repression was found to inhibit proliferation and enhance the drug sensitivity of oral cancer cells to vincristine Such implications have been drawn also previously Zhihong et a0 al The characterization of miR1179 in oral cancer in this study revealed that autophagy is induced in oral cancer cells when miR1179 transcript levels are repressed The autophagy was confirmed by the presence of autophagy vesicles which was further implicated by the enhancement of LC3II conjugated protein expression level and declining of LC3I and Beclin expression The miR repression in oral cancer cells further declined the migration and invasion rates of cancer cells which is in conformity with the previous studies on miR1179 Jiang et a0al Lastly among the most important molecular events of cancer progression the activation of MEKERK 0cGao a0et a0al AMB Expr Page of upregulated in human oral cancer cells Inhibition of miR1179 overexpression resulted in decline of proliferation and metastasis and enhancement of chemosensitivity of human oral cancer cells Additionally miR1179 also resulted in the blockage of the MEKERK and PI3KAKT signalling pathway pointing towards the therapeutic implications of miR1179 in oral cancer treatmentAcknowledgementsWe acknowledge the Affiliated Hospital of Jilin Medical University Jilin China Shandong China for providing necessary laboratory faciality and supportAuthors™ contributionsYG and TP designed the protocol of the study YG and HX performed the experimental work and collect the data for presented study YG and HX involve in the statistical analysis TP supervised the work and drafted the manuscript although all author contributes for the preparation of manuscript All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsNot applicableEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived June Accepted August ReferencesBorsetto D Higginson JA Aslam A AlQamachi L Dhanda J Marioni G Franchella S Frigo A Praveen P Martin T Parmar S Factors affecting prognosis in locoregional recurrence of oral squamous cell carcinoma J Oral Pathol Med “Cao M Tang Y Tang Y Liang XH Noncoding RNAs as regulators of lymphangiogenesis in lymphatic development inflammation and cancer metastasis Front Oncol Gao HB Gao FZ Chen XF MiRNA1179 suppresses the metastasis of hepatocellular carcinoma by interacting with ZEB2 Eur Rev Med Pharmacol Sci “Garzon R Calin GA Croce CM MicroRNAs in cancer Ann Rev Med “Gong H Liu CM Liu DP Liang CC The role of small RNAs in human diseases potential troublemaker and therapeutic tools Med Res Rev “Gupta N Gupta R Acharya AK Patthi B Goud V Reddy S Garg A Singla A Changing Trends in oral cancera global scenario Nepal J Epidemiol Huang M Huang B Li G Zeng S Apatinib affect VEGFmediated cell proliferation migration invasion via blocking VEGFR2RAFMEKERK and PI3KAKT pathways in cholangiocarcinoma cell BMC Gastroenterol Jiang L Wang Y Rong Y Xu L Chu Y Zhang Y Yao Y miR1179 promotes cell invasion through SLIT2ROBO1 axis in esophageal squamous cell carcinoma Int J Clin Exp Pathol Krutovskikh VA Herceg Z Oncogenic microRNAs OncomiRs as a new class of cancer biomarkers Bioessays “Fig Inhibition of miR1179 blocks MEKERK and PI3K and AKT signalling pathways a Western blots showing the effects of miR1179 inhibition on expression of MEK pMEK ERK and pERK proteins in SCC9 and SCC25 cells b Western blots showing the effects of miR1179 inhibition on expression of PI3K pPI3K AKT and pAKT proteins in SCC9 and SCC25 The experiments were performed in triplicateand PI3KAKT pathways hold a prime essence as these pathways enable the transcription of various downstream regulators of cancer growth and proliferation Huang et a0al These pathways have been shown to be aberrantly activated in different cancer types For example PI3KAKT pathway has been shown to be activated in pancreatic cancer Lan et a0 al In thyroid cancer cells PI3KAKT and MAPKERK pathway has been shown to significantly overexpressed Su et a0 al Similarly MEKERK pathway has shown to be activated in ovarian cancer Zhang et a0al As such the blockage of these signalling pathways is a vital asset to keep the cancer progression at check Zhihong et a0 al The miR1179 suppression renders the MEKERK and PI3KAkt pathways to proceed at fairly diminished rates by preventing the phosphorylation of crucial signalling components like MEK ERK PI3K and AKT Summing up the current study deduced the molecular functionality of miR1179 and implicated its molecular targeting in the management of human oral cancer To conclude the present study showed miR1179 to be significantly 0cGao a0et a0al AMB Expr Page of Lan CY Chen SY Kuo CW Lu CC Yen GC Quercetin facilitates cell death and chemosensitivity through RAGEPI3KAKTmTOR axis in human pancreatic cancer cells J Food Drug Anal “Lin C Hu Z Yuan G Su H Zeng Y Guo Z Zhong F Jiang K He S MicroRNA1179 inhibits the proliferation migration and invasion of human pancreatic cancer cells by targeting E2F5 Chem Biol Interact “Li Y Qin C MiR1179 inhibits the proliferation of gastric cancer cells by targeting HMGB1 Hum Cell “Peng X Guo W Liu T Wang X Tu XA Xiong D Chen S Lai Y Du H Chen G Liu G Identification of miRs143 and145 that is associated with bone metastasis of prostate cancer and involved in the regulation of EMT PLoS ONE 275e20341Peres MA Macpherson LM Weyant RJ Daly B Venturelli R Mathur MR Listl S Celeste RK GuarnizoHerre±o CC Kearns C Benzian H Oral diseases a global public health challenge Lancet “Petersen PE Oral cancer prevention and control“the approach of the World Health anization Oral Oncol ““Silva SD Hier M Mlynarek A Kowalski LP AlaouiJamali MA Recurrent oral cancer current and emerging therapeutic approaches Front Pharmacol Song L Dai Z Zhang S Zhang H Liu C Ma X Liu D Zan Y Yin X MicroRNA1179 suppresses cell growth and invasion by targeting spermassociated antigen 5mediated Akt signaling in human nonsmall cell lung cancer Biochem Biophy Res Co “Su X Shen Z Yang Q Sui F Pu J Ma J Ma S Yao D Ji M Hou P Vitamin C kills thyroid cancer cells through ROSdependent inhibition of MAPKERK and PI3KAKT pathways via distinct mechanisms Theranostics Warnakulasuriya S Global epidemiology of oral and oropharyngeal cancer Oral Oncol ““Wu BH Xiong XP Jia J Zhang WF MicroRNAs new actors in the oral cancer scene Oral Oncol “Xu X Cai N Zhi T Bao Z Wang D Liu Y Jiang K Fan L Ji J Liu N MicroRNA1179 inhibits glioblastoma cell proliferation and cell cycle progression via directly targeting E2F transcription factor Am J Cancer Res Zhang R Shi H Ren F Feng W Cao Y Li G Liu Z Ji P Zhang M MicroRNA3383p suppresses ovarian cancer cells growth and metastasis implication of Wntcatenin beta and MEKERK signaling pathways J Exp Clin Cancer Res “Zhihong Z Rubin C Liping L Anpeng M Hui G Yanting W Zhenxiu S MicroRNA1179 regulates proliferation and chemosensitivity of human ovarian cancer cells by targeting the PTENmediated PI3KAKT signaling pathway Arch Med Sci Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c'

Thyroid_Cancer

distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedInflammatory bowel disease IBD most commonly known as Crohn™s disease CD and ulcerative disease UC is a chronic andrelapsing intestinal disease which cannot be cured completely e prevalence of IBD in Europe and in North America hasincreased over the past years As most IBD patients are young at onset their quality of life QOL can be ‚uenced to varyingdegrees us current treatment goals are typically focused on preventing complications including maintaining clinical remissionand improving the QOL Adjuvant therapies have been widely concerned as an eï¬ective treatment in alleviating IBD symptomsincluding dietary intervention traditional Chinese medicine smoking alcohol and physical activities is review focuses ondiï¬erent ancillary therapies for IBD treatments in particular the mechanism of reducing ‚ammation based on the actual datafrom research studies Moreover comparing the latest data this review also presented potentialfuture prospect foradjuvant therapies IntroductionInflammatory bowel disease IBD is a chronic immunemediated disease mainly consisting of two diseases Crohn™sdisease CD and ulcerative colitis UC [] CD is active in anypart of the gastrointestinal tract from the mouth to the anuswhile UC is restricted to the colonic mucosa [] e etiologyand pathology of IBD remain largely unknown but a complicated interplay of genetic environmental immunologicaland lifestyle factors has been associated with this condition[ ] Clinical symptoms of IBD involved but not limitedmultiple ans and systems throughout the body such asnodular erythema osteoporosis and IBDrelated arthropathye current medical treatment in IBD is available forIBD patients drugbased therapies such as nonsteroidalanti‚ammatory drugs sulfasalazine and masalazinebiological therapies such as antiTNFα antibodies andimmunomodulatorssuch as methotrexate Meanwhilesurgical management is used to control the progression ofsevere disease Unfortunately these drugs also cause adverseeï¬ects in IBD patients and the high disease cost with a longtreatment process has brought economic burden for patients For example biological therapies such as antiTNFαmay increase the risk of infection and the costs of thesedrugs vary from to US dollars [] erefore it isnot surprising that patients seek complementary and adjuvant therapies in IBDIn this review we focus on rational dietary structuresChinese traditional medicine suitable smoking alcohol andphysical activities Rational dietary structure has beenspeculated to be a pivotal factor in the pathogenesis of IBDand may be important in managing disease symptoms IBDpatients choose various dietary strategies to minimize gastrointestinal distress and improve overall health Dietaryproducts are the most common antigens in the intestinealtering the composition of the intestinal flora and changingthe permeability of gastrointestinal tract [] Dietary interventions may not be appropriate alternatives to conventional medical therapy but are eï¬ective complementaryapproaches for IBD treatment In this review we focus ondiï¬erent dietary components which are reported to benefitpatients™ symptoms 0cCanadian Journal of Gastroenterology and Hepatologye popularity of Chinese traditional medicine CAMhas been systematically increasing among various complementary and adjuvant medicine approaches for the treatment of gastrointestinal disorders mainly because it isnatural and eï¬ective CAM products range from homeopathy herbal medicine acupuncture and moxibustion Although the CAM is often of yet unknown efficacy andmechanism the induction and maintenance of disease remission in UC and CD have been investigatedOver the last few years it is controversial whether IBDpatients should quit smoking and alcohol Cigarette smokinghas been shown to worsen disease activity in CD while inUC smoking decreases the extent of disease [] Anotherimportant factor is alcohol alcohol has been previouslyshown to be associated with worsening GI symptoms but acasecontrol study illustrated that moderate red wine consumption by patients is linked with a lower risk in IBD []Physical activity is one candidate complementary intervention that is of potential benefit in various chronicdiseases Previous studies showed that IBD patients perceivephysical activities as a helpful management in reducingsymptoms and complications of IBD [] ere are physiologic benefits to physical activity such as improved bonedensity decreased incident of colitis associated colorectalcancer and the prevention of obesity [] Swimmingwalking and Chinese martial arts such as Qigong and Tai chihave been shown to improve the QOL balance internally forhealing and improve bone density [] We will summarizethe role of diï¬erent physical activities in the developmentand course of IBDs DietaryDiet is closely linked with IBD especially in Western dietCurrently dietary interventions have been studied in IBD toalleviate active disease and maintain remission e Mediterranean diet pattern has been shown to be protective inIBD as the incidence of IBD in the south of Europe is lowerthan in the northern Europe Some of the components in theMediterranean diet pattern such as olive oil fish oil fruitsand vegetables have been shown to be efficacious and patientfriendlyEpidemiological studies illustrated that the intake of ahigher ratio of n ˆ’ polyunsaturated fatty acids n ˆ’ PUFAs and a lower ratio of n ˆ’ polyunsaturated fatty acidsn ˆ’ PUFAs was associated with an increased risk ofdeveloping IBD [ ] Researches to date have demonstrated that n ˆ’ PUFAs may oï¬er a promising approach toimproving dysbacteriosis reducing the likelihood of relapseand lowering the mortality of colitis [ ] Moreover theirprotective eï¬ect in IBD is hypothesized to be derived fromthe balance in the ratio of n ˆ’ 6n ˆ’ PUFAs or higher[] e hypothesized machanism underlying the anti‚ammatory eï¬ect is to release pro‚ammatory mediatorsreduce freeradical generation and platelet activating factorformation all of which are increased in IBD [ ]Currently fish peptide seems to have tissue reparativeproperties based on several studies in rodents and humanSalmon fillets contain n ˆ’ PUFAs and marine collagenpeptide has an antioxidative eï¬ect A previous studyshowed that a regular intake of salmon in patients with UC isbeneficial based on the improved simple clinical colitisactivity index SCCAI and anti‚ammatory fattyacidindex AIFAI [] e combination of fish peptides andfish oil diet was more efficient than pure fish oil in an animalmodels study [] Compared with the pure fish oil addingthe fish peptides diet eï¬ectively reduces the production ofpro‚ammatory cytokines and increases the level of PGE3in plasma [] Additionally other dietary peptides also havedemonstrated an anti‚ammatory eï¬ect in IBD animalmodels Machbank that dish hydrolysate was suggested to have an intestinal protective eï¬ect in mice []Based on these observations the fish peptide diet may be aneï¬ective way to maintain remission IBDDietary fiber is more commonly used as a supplement forthe management of IBD Rational intake of fiber may reduceCD risk especially that which originated from fruits []Fibers from fruit have anti‚ammatory properties andpositive modulation of the intestinal microbiota [] Fibersfermented by bacteria in the colon produce the shortchainfatty acids inhibiting the activation of transcription ofpro‚ammatory mediators [] According to a casecontrol study high fiber intake respondents were percentless likely to develop CD than those with low fiber intakemedian gday [] However fiber intake is controversial Scientists found that the risk of fiber was15 gdaywhile ornton showed there was no diï¬erence betweenpatients and controls [] e future study should pay moreattention to the amount of fiber and IBDVitamin D is a group of fatsoluble vitamins which playsa key role in IBD treatments [] Recent studies have reported that Vitamin D was linked to the protection againstinfection and the control of the gut commensal microbialcomposition [ ] Clinical trials have suggested a positivecorrelation between Vitamin D deficiency and IBD thatnearly half of the patients had hypovitaminosis D [“]Vitamin D deficiency may reduce the expression of a tightjunction in the intestinal epithelium decrease the clearanceof colonic bacteria directly and aï¬ect the gut barrier andimmune system functions that impact the onset and progression of IBD [“] To correct vitamin D deficiencyresearchers found that after ingestion of IU vitaminD3 daily for months and IU vitamin D2 weekly for weeks the vitamin D status was significantly improved and of the patients™ CD activity index CDAI had a drop ofless than points compared with placebo among childrenand adolescents with IBD [] erefore in CD the effective dose of vitamin D3 was determined at IUd [] Traditional Chinese MedicineIn recent years traditional Chinese medicine TCM including herbal medicine acupuncture and hemopathy hasbeen commonly used among IBD patients from all agegroups It is estimated that the percentage of IBD patientsusing TCM in North America at might increase up toeven [] Herbal medicine is the most common TCMmodality with lower cost and higher efficiency TCM herbal 0cCanadian Journal of Gastroenterology and Hepatologyenema has been proved to be the most efficient method fortherapies because of the regulation of immune responses inthe colon mucosa [] e mechanism of TCM in IBDincludes improving anti‚ammatory activities and reducing the level of pro‚ammatory cytokines []Yun Nan Bai Yao YNBY is a Chinese herbal remedyused for treating wounds for its hemostatic properties []In the recent years researchers showed that YNBY caneï¬ectively reduce the severity of experimental colitis by theimmunosuppression and wound healing mechanisms It wasshown that YNBY significantly suppresses the growth ofT lymphocytes and B lymphocytes thus decreasing severalpro‚ammatory cytokines such as TNFα which wereclosely correlated with IBD [] A dosedependent hemostatic eï¬ect was also revealed by researchers through rabbitsmodels [] erefore for patients suï¬ering from gastrointestinal bleeding giving YNBY enterally may serve as aneï¬ective adjunctive therapy YNBY is also capable of reducing intraoperative blood loss e dosage of YNBY onCD is still unclear and future studies should focus on thedosage and indications for IBD patientsTripterygium wilfordii Hook F TWHF aloe vera andtormentil are TCM herbal remedies with anti‚ammatory activities It has been shown that TWHF achieves thesame level in preventing the postoperative recurrence ofCD [] In the previous research the relapsed patients inthe TW and mesalazinetreated groups were versus in months and versus in yearMoreover the CDAI was decreased during the first weeksand reached the minimum in week [] It was alsoreported that respondents™ symptoms are alleviatedthrough using aloe vera [] Tormetil extracts weresuitable for chronic IBD patients which was proved to besafe up to mgday for weeks with minor side eï¬ects[]Another herbal therapy such as Food Allergy HerbalFormula2 FAHF2 which originated from Wu Mei Wanthat has long been used in China to treat colitis may have thepossibility to be served as a novel treatment of CD []Composed of Aconitum Coptis ginger ginseng Cinnamomum Angelica and Ganoderma lucidum it was found to beable to inhibit both adaptive and innate immune pro‚ammatory cytokine responses in ‚amed CD mucosa andvalid in halting progression of colitis in a murine modelwhich indicated that FAHF2 may be safe and eï¬ective forCD to maintain remission and avoid the need for pharmacologic escalation in therapy with medications that havepotentially severe side eï¬ects [ ]C longa L turmeric is a plant whose root segment iscommonly used as a seasoning and in traditional Chinesemedicine for thousands of years Curcumin is the chiefbiologically active derivative of turmeric In a series of invitro and in vivo studies curcumin has recently caught muchattention for its anti‚ammatory characteristic Curcuminis capable of correcting abnormal immune response in IBDthrough decreasing pro‚ammatory cytokines synthesisdownregulating the transcription of the pro‚ammatorygene by inhibiting NFκβ and upregulating antioxidantenzymes []A placebocontrolled doubleblind study proved that aproper combination of curcumin and mesalazine can effectively induce remission in mesalazinetolerant UC patients whose disease course fail to improve under maximumdose of mesalazine for weeks After standard addoncurcumin therapy gday in capsules for month of patients in the curcumintreated group achievedclinical remission and presented ameliorationin endoscopical performance evaluated by the endoscopicMayo index subscore while none achieved remission in the placebo group []Curcumin is nontoxic even at relatively high doses withno known toxic side eï¬ects in humans up to doses of gday and even pediatric patients with IBD are able to tolerate a curcumin dose up to g twiceday and some of themdemonstrate improvement in the disease course [ ]Currently popular anti‚ammatory therapy can leadto tremendous cost while curcumin remedy is much moreaï¬ordable with a price less than per week For its effectiveness safety and aï¬ordability curcumin could be anideal agent for curing IBD However it is hard to maintaintherapeutic curcumin concentration pattern in human bodyfor its rapid metabolism and dissatisfied biodistribution []More eï¬ort should be made to determine a feasible methodof administration as well as improve its absorption andbiodistribution before curcumin can fully benefit IBDpatients SmokingSmoking aï¬ects these CD and UC diï¬erently many casestudies suggest that smoking is a risk factor for CD while ittends to confer a protective eï¬ect against UC [ ]Compared to neversmokers the incidence and severityof UC are lower in smokers in many studies and smokingexerts protective eï¬ects on both the development and theprogression of UC [ ] e relapse rate hospitalizationrates and the need for oral steroids and the colectomy rateswere found to be lower in current smokers rather thannonsmokers [] It was observed that high cigarette dosesuch as cigarettes per day was correlated with less extensive colitis and lower treatment needs [] However theprotective role of smoking in UC sustains until “ yearsafter smoking cessation and then the risk goes up interestingly the risk paralleled past cumulative exposure with anincrease in the disease activity and the need for hospitaladmission and major medical therapy [ ]As for the ‚uence of smoking on CD most studieshave suggested that current smokers have a higher risk ofdeveloping CD than those who have never smoked [ ]Besides previous data have demonstrated that smoking wasassociated more frequently with complicated disease such aspenetrating intestinal complications and a higher relapserate [] It was shown that patients with a high life timetobacco exposure cigarette years and heavy smokers cigarettesday had small bowel disease more oftenthan the patients with both lower life time exposure cigarette years and smoking‰¤ cigarettesday [] Actually the increased risk of disease relapse is significantly 0cCanadian Journal of Gastroenterology and Hepatologyover a threshold cigarettes per day [] In additioncompared with nonsmokers the needs of steroids andimmune suppressants rise in smokers [“] e impact ofcigarette smoking on CD is temporary smoking cessationimproves the course of the disease and it has been estimatedthat after years of smoking cessation former smokersunderwent a process similar to that of patients who havenever smoked and the flareup rate was decreased [ ]Notwithstanding it has been noted that the use of snuswas not associated to the development of either UC or CDwhich underlines diï¬erences in combusted and noncombusted tobacco in the genesis of IBD [ ] In addition the fact that snus users have higher levels of thenicotine metabolite cotinine also implies that nicotine byitself may not be involved in the pathogenesis [] AlcoholAlcohol is another potential trigger for flaring IBD sincealcohol in diï¬erent amount of consumptions aï¬ects theimmune system and results in various imbalanced answith ‚ammation [“] Furthermore alcohol consumption has an eï¬ect on gut permeability and plasma levelsof gutderived bacterial products such as lipopolysaccharides and peptidoglycans [] However compared withnever drinking light drinking has a protective eï¬ect on thedevelopment of UC [] It stands to reason that hazardousalcohol intake it is defined as more than g of alcohol perday for men and g for women is detrimental for IBDpatients while moderate red wine consumption lasted for aweek is linked with a lower risk in IBD e mechanism isthe decrease in stool calprotectin which is known as anantioxidant with anti‚ammatory eï¬ects [ ] A research showed that fecal stool calprotectin was decreasedcompared with baseline after week of drinking in IBDpatients [] Antioxidants with additional anti‚ammatory actions may benefit the treatment in IBD on account ofthe mechanism in ‚ammation caused by oxidative stress[ ] Oxidative stress shows a definition of the imbalancebetween oxidants reactive oxygen species and reactivehydrogen species and antioxidants which is linked tochronic intestinal ‚ammation in the early stage of IBD[] Meanwhile alcohol inhibits the immune system byreducing interleukin IL12 and increasing interleukin IL production which can aï¬ect the induction on or immune responses [ ] Moreover autocrine IL10production can prevent maturation of dendritic cells andinduce anergy in the Tcells responder which is an anti‚ammatory cytokine closely related to the immune system[] However alcohol consumption was controversialnowadays A report showed that although beer is beneficialfor some people though only a few more than percentof the participants reported worsening symptoms fromdrinking Moreover only about percent patients wastolerant to wine while more than percent of the subjectsshowed an increase in red wine symptoms [] ereforethe future study should focus more on the dosage and sideeï¬ect of alcohol consumption Physical ActivityPhysical activity may potentially play a role in alleviatingsymptoms related to extraintestinal manifestations of IBD[] Previous study showed patients with at least metabolic equivalent task MET hours per week of physicalactivity have a reduction in risk of developing Crohn™sdisease compared with those with3 MET·hwk [] Exercise can be beneficial for intestinal and extraintestinalmanifestations of IBD that regular physical exercise couldimprove physiological health maintain their weight improve bone mineral density and alleviate the anorexiacaused by IBD [ ]Lowtomoderate intensity physical activities have beenproved to be safe and suitable that it was well tolerated byIBD patients especially those who were in remission anddid note provoke subjective symptoms [] Moderateintensity physical activity has beneficial eï¬ects on thegastrointestinal system Patients with regular exerciseduring the previous years may have lower chance ofdeveloping CD especially if the exercise is performed dailyAdditionally it was also demonstrated that patients whoperformed exercise were less likely to develop the diseaseactivity in UC [] Several possible mechanisms mayexplain the anti‚ammatory ‚uence of physical activitiesfromskeletal muscleinhibiting the TNF production andstimulating the release of IL10 [ ] However extremeexercise may contribute to intestinal ‚ammation by themeansandCD8 lymphocytes natural killer cells and the level ofreactive oxygen species [] It seems that the eï¬ect ofphysical activity depends on the intensity and duration ofthe physical activityincluding releasing interleukin6 IL6increasingof CD4Some of the aspects were also observed at experimentalconditions because moderate voluntary treadmill exercisecould significantly accelerate the healing of colitis in IBD Arecent study by Cook showed that sessions of forcedtreadmill exercise training exacerbated ‚ammation indextran sodium sulfateinduced colitis proved by excessivediarrhea episodes and increased animal mortality [] In aprevious study a longterm physical activity of 6weekrunning attenuated the colonic TNFα protein contentindicating the anti‚ammatory eï¬ect of physical exercise[ ] According to the previous rodent studies exercisecould downregulate the expression of interleukin 1β IL1βand TNFα in both colonic mucosa and plasma Moreover itwas demonstrated that leptin levels were significantly decreased which diminished the severity of colonic damagemediated and exerted an anti‚ammatory eï¬ect on an‚amed colon []Swimming and cycling are two eï¬ective aerobic exercisesthat can be beneficial with fewer gastrointestinal symptomsby the means of ‚ammatory modulation and apoptosis[ ] In addition walking “ min at of maximalheart rate days per week along with resistance training times per week is advocated in many studies and it may havethe potential to decrease the risk of active disease at sixmonths [ ]ofthenumber 0cCanadian Journal of Gastroenterology and HepatologyQigong and Tai Chi are traditional Chinese physicalactivities which coordinate the body and mind ese approaches have been shown to be eï¬ective in reducingsymptoms such as fatigue depression and pain and improving QOL in a way of moderate exercise [] Qigongcould improve immune functions and reduce ‚ammationprofiles such as pro‚ammatory cytokines TNFα whichwere correlated to IBD Lymphocytes thyroidstimulatinghormone and IgG were found to be modulated in responseto practicing Tai Chi Researchers demonstrated that a minute weekly session of Qigong with a duration of weeksis recommended Moderate Tai Chi and Qigong may enhance physiological and psychological function and thefuture study may concentrate more on the suitable amountof exercise in Tai chi and Qigong [] DiscussionConsidering the past few years the adjuvant treatments forIBD have become increasingly important to better the immunity and ‚ammation in the intestine More and moreinvestigations illuminated the significance of diet traditionalChinese medicines and proper exercise which are capableof taking the edge oï¬ withdrawal symptoms in IBD Furthermore smoking and alcohol mainly act as two environmental factors in adjuvant treatments []ough unable to replace the conventional IBD therapiestreatment still plays an instrumentaltotally adjuvantsupplement role in treating the disease Some patientfriendly components of the Mediterranean diet have beenproven to aï¬ect the intestinal barrier and immune systemfunction thus aï¬ecting the progress of IBD TraditionalChinese medicine can eï¬ectively reduce ‚ammatory cytokines and alleviate IBD Moderate exercise such as QigongTai chi swimming and walking are eï¬ective treatments inreducing the risk of pain and complications with less expenditure [] Moreover achieving smoking cessation is alsoan important goal of IBD treatment for the beneficial eï¬ectsof smoking on disease are oï¬set by the harmful eï¬ects oftobacco on the respiratory and cardiovascular system[ ] whereas reasonable alcohol drinking benefits CDpatients by not aï¬ecting the immune system [ ]However as a novel treatment for CD several weaknesses have been limited by the application of adjuvanttreatments It is of ambiguity whether adjuvant treatment issuitable or curable for all types of IBD patients For examplesome changes associated with diet on the maintenance ofIBD remission are ambiguous and the aï¬ecting dosage forIBD of each beneficial dietary component is unknown Forherbal medicines there has been no specific research toconfirm a particular dose or prescription has significanteï¬ects on patients In addition although previous studieshave vitrified the benefits of exercise in IBD treatment it hasnot been defined as the most appropriate adjuvant for alltypes of IBD especially in Qigong and Tai chi For UCpatients smoking was found to be more conducive while ithas strong linkage with pernicious diseases all the time Soconducting highquality clinicaltrials with appropriateblinding and large number of patients is necessary to obtainmore conclusive results on the curative eï¬ect of adjuvanttreatment in IBDis paper systematically expounds the significance anddiï¬erent factors of IBD adjuvant treatments within the fieldof comprehensive treatments in IBD Adjuvant treatment ismost approbated among the public nowadays for its relatively low cost and minor side eï¬ects compared with traditional remedies Moreover this review also aims to drawthe attention of the public to engage patients in a discussionof adjuvant treatment and underline their role as a complement to conventional IBD therapies Physicians are urgedto explore the use of adjuvant treatment and provide appropriate information and guidance to patients in order todevelop highquality care for patients with IBDData Availabilitye data used to support the findings of this study areavailable from the corresponding author upon requestConflicts of Intereste authors declare that they have no conflicts of interestAuthors™ ContributionsQiyue Wang and Shuyi Mi contributed equally to this workAcknowledgmentsis study was funded by grants from the Zhejiang University student science and technology innovation projectNo 2018R401194Supplementary MaterialsSupplementary materials the supplementary material file isa figure describing the mechanism and eï¬ect of physicalactivities that could alleviate gastrointestinal symptoms andimprove patients™ quality of life Physical activities such asswimming walking Tai Chi and Qigong may induce anti‚ammatory modulation in releasing the interleukin6 IL interleukin10 IL10 lymphocyte and Immunoglobulin G IgG Moreover the expression of interleukin 1βIL1β and the TNFα protein content would be downregulated due to moderate physical activities In additionthe physiological health would be better and the hospitalization time could be shortened through physical activitiesSupplementary MaterialsReferences[] V Andersen A Olsen F Carbonnel A Tjønneland andU Vogel œDiet and risk of ‚ammatory bowel diseaseDigestive and Liver Disease vol no pp “ [] J Bilski B Brzozowski A MazurBialy Z Sliwowski andT Brzozowski œe role of physical exercise in ‚ammatorybowel disease BioMed Research International vol Article ID pages 0cCanadian Journal of Gastroenterology and Hepatology[] D C Baumgart and S R Carding œInflammatory boweldisease cause and immunobiology e Lancet vol no pp “ [] L J Dixon A Kabi K P Nickerson and C McDonaldœCombinatorial eï¬ects of diet and genetics on ‚ammatorybowel disease pathogenesis Inflammatory Bowel Diseasesvol no pp “ [] R Li P Alex M Ye T Zhang L Liu and X Li œAn oldherbal medicine with a potentially new therapeutic application in ‚ammatory bowel disease International Journal ofClinical and Experimental Medicine vol no pp “[] E Cabr´e and E Domenech œImpact of environmental anddietary factors on the course of ‚ammatory bowel diseaseWorld Journal of Gastroenterology vol no pp “ [] K Matsuoka T Kobayashi F Ueno œEvidencebasedclinical practice guidelines for ‚ammatory bowel diseaseJournal of Gastroenterology vol no pp “ [] S L Jowett C J Seal E Phillips W Gregory J R Barton andM R Welfare œDietary beliefs of people with ulcerative colitisand their eï¬ect on relapse and nutrient intake ClinicalNutrition vol no pp “ [] J G Hashash and D G Binion œExercise and ‚ammatorybowel disease Gastroenterology Clinics of North Americavol no pp “ [] V Ng W Millard C Lebrun and J Howard œExercise andcrohn™s disease speculations on potential benefits CanadianJournal of Gastroenterology vol no pp “ [] R Jahnke L Larkey C Rogers J Etnier and F Lin œAcomprehensive review of health benefits of qigong and taichi American Journal of Health Promotion vol no pp e1“e25 [] C A ChapmanKiddell P S W Davies L Gillen andG L RadfordSmith œRole of diet in the development of‚ammatory bowel disease Inflammatory Bowel Diseasesvol no pp “ [] E Scaioli E Liverani and A Belluzzi œe imbalance between n ˆ’ 6n ˆ’ polyunsaturated fatty acids and ‚ammatory bowel disease a comprehensive review and futuretherapeutic perspectives International Journal of MolecularSciences vol no [] P C Calder œPolyunsaturated fatty acids‚ammatoryprocesses and ‚ammatory bowel diseases Molecular Nutrition Food Research vol no pp “ [] R Reifen A Karlinsky A H Stark Z Berkovich andA Nyska œÎ±linolenic acid ALA is an anti‚ammatoryagent in ‚ammatory bowel disease e Journal of Nutritional Biochemistry vol no pp “ [] A Belluzzi C Brignola M Campieri A Pera S Boschi andM Miglioli œEï¬ect of an entericcoated fishoil preparationon relapses in crohn™s disease New England Journal ofMedicine vol no pp “ [] K Hillier R Jewell L Dorrell and C L Smith œIncorporation of fatty acids from fish oil and olive oil into colonicmucosal lipids and eï¬ects upon eicosanoid synthesis in ‚ammatory bowel disease Gut vol no pp “[] D Camuesco M Comalada A Concha œIntestinalanti‚ammatory activity of combined quercitrin and dietary olive oil supplemented with fish oil rich in EPA andDHA n ˆ’ polyunsaturated fatty acids in rats with DSSinduced colitis Clinical Nutrition vol no pp “ [] K Azuma T Osaki and T Tsuka œEï¬ects of fish scalecollagen peptide on an experimental ulcerative colitis mousemodel vol no pp “ [] T Grimstad B Bjørndal D Cacabelos œA salmonpeptide diet alleviates experimental colitis as compared withfish oil Journal of Nutritional Science vol p e2 [] T Marchbank G Elia and R J Playford œIntestinal protectiveeï¬ect of a commercial fish protein hydrolysate preparationRegulatory Peptides vol no “ pp “ [] A N Ananthakrishnan H Khalili G G Konijeti œAprospective study of longterm intake of dietary fiber and riskof crohn™s disease and ulcerative colitis Gastroenterologyvol no pp “ [] A Wedrychowicz A Zajac and P Tomasik œAdvances innutritional therapy in ‚ammatory bowel diseases reviewWorld Journal of Gastroenterology vol no pp “ [] K M Maslowski and C R Mackay œDiet gut microbiota andimmune responses Nature Immunology vol no pp “ [] V Andersen S Chan R Luben œFibre intake and thedevelopment of ‚ammatory bowel disease a Europeanprospective multicentre cohort study epicibd Journal ofCrohn™s and Colitis vol no pp “ [] M T Palmer and C T Weaver œLinking vitamin d deficiencyto ‚ammatory bowel disease Inflammatory Bowel Diseasesvol no pp “ [] A Barb´achano A Fern´andezBarral G FerrerMayaA CostalesCarrera M J Larriba and A Muñoz œe endocrine vitamin D system in the gut Molecular and CellularEndocrinology vol pp “ [] D Statovci M Aguilera J MacSharry and S Melgar œeimpact of western diet and nutrients on the microbiota andimmune response at mucosal interfaces Frontiers in Immunology vol p [] M Ardesia G Ferlazzo and W Fries œVitamin D and ‚ammatory bowel disease BioMed Research Internationalvol Article ID pages [] R Del Pinto D Pietropaoli A K Chandar C Ferri andF Cominelli œAssociation between ‚ammatory boweldisease and vitamin D deficiency Inflammatory BowelDiseases vol no pp “ [] M Sadeghian P Saneei F Siassi and A EsmaillzadehœVitamin D status in relation to crohn™s disease metaanalysisof observational studies Nutrition vol no pp “ [] H M Pappa P D Mitchell H Jiang œTreatment ofvitamin D insufficiency in children and adolescents with‚ammatory bowel disease a randomized clinicaltrialcomparing three regimens e Journal of Clinical Endocrinology Metabolism vol no pp “ [] M Nakano K Tominaga A Hoshino T Sugaya K Kankeand H Hiraishi œerapeutic efficacy of an elemental diet forpatients with crohn™s disease and its association with aminoacid metabolism Saudi Journal of Gastroenterology vol no pp “ [] R Teschke A Wolï¬ C Frenzel A Eickhoï¬ and J SchulzeœHerbal traditional Chinese medicine and its evidence

Thyroid_Cancer

Adjunctive Therapy to Achieve Preoperative Euthyroidism in Graves™ Disease A Case Report Authors™ Contribution Study Design A Data Collection B Statistical Analysis C Data Interpretation D Manuscript Preparation E Literature Search F Funds Collection G ABDEF Noor Abdulghani AlghanimABDEF Shymaa M AlkahtaniAEF Fatimah S AssariAEF Sarah W AlnosaierAEF Reham M BaderAEF ABEF Mariam M HendazAEF Amal AlhefdhiIsra E Elmahi Corresponding Author Conflict of interest Noor Abdulghani Alghanim email nalghanimalfaisaleduNone declared College of Medicine Alfaisal University Riyadh Saudi Arabia Breast and Endocrine Surgery King Faisal Specialist Hospital and Research Center Riyadh Saudi ArabiaPatient Final Diagnosis Symptoms Medication ” Clinical Procedure Specialty Male 37yearoldGraves™ diseaseDifficulty breathing ¢ voice change ¢ weight gainTotal thyroidectomySurgery Objective Background Case Report Conclusions Unusual clinical courseGraves™ disease is an autoimmune disease of the thyroid gland and it is considered the most common cause of hyperthyroidism It is characterized by particular eye manifestations skin changes and pretibial myxedema in addition to the signs and symptoms of hyperthyroidism Graves™ disease can be diagnosed based on clinical presentation and low thyroid stimulating hormone TSH and elevated free T4 FT4 levels Presence of TSH receptor antibody TRAb in the serum confirms the diagnosis of Graves™ disease Imaging studies like radioactive iodine scan will show a high and diffuse uptake Graves™ disease can be managed with three different treatment modalities antithyroid medications radioactive iodine or surgical removal of the thyroid gland Whenever surgery is indicated careful preoperative management to achieve euthyroidism is needed to optimize the surgical outcomeThis is a case of a 37yearold Saudi male known to have Graves™ disease for years who presented to the endocrine surgery clinic with neck swelling difficulty breathing and change in voice After multiple attempts to control his fluctuating thyroid levels the team eventually managed to achieve a euthyroid state in the patient with the addition of saturated solution of potassium iodide SSKI and thus rendering him eligible for urgent surgeryWe report this case to show that SSKI can be used as adjunctive therapy to achieve a preoperative euthyroid state in refractory Graves™ disease MeSH Keywords Graves™ Disease ¢ Hyperthyroidism ¢ Hypothyroidism Fulltext PDF wwwamjcaserepcomindexidArt923342 ” e9233421Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves™ disease¦ Am J Case Rep e923342Figure Typical eye manifestations of Graves™ disease proptosis and periorbital edemaFigure Ultrasound showing an enlarged and hypervascular left thyroid lobe with no suspicious nodulesof the examination was unremarkable except for right scrotal swelling and delayed deep tendon reflexesThe patient then underwent ultrasound US of the thyroid which showed an enlarged and hypervascular gland compatible with Graves™ disease with no suspicious nodules Figure A computed tomography CT scan was done which revealed homogeneous diffuse swelling of bilateral thyroid lobes with no retrosternal extension along with bilateral proptosis Figures Therefore the patient was admitted to achieve a euthyroid state before proceeding for a total thyroidectomy The patient was managed by a multidisciplinary team with the goal of clearing him for surgery During the patient™s 3week hospital stay the dosage of methimazole was continuously altered because serial thyroid function tests showed a change from hyperthyroid to hypothyroid status His TSH and FT4 levels ranged from mUL to mUL and pmolL to pmolL respectively A few days after administration of saturated solution of potassium iodide SSKI was initiated drops three times daily the patient achieved a euthyroid state with TSH mUL and FT4 pmolL so urgent surgery was performed Intraoperatively the patient™s thyroid gland was found to be enlarged and vascular with each lobe measuring approximately to cm The gland was excised bilaterally along with the pyramidal lobe because it was also enlarged The postoperative BackgroundGraves™ disease is an autoimmune disease affecting the thyroid gland [] It is characterized by presence of autoantibodies that target thyroid stimulating hormone TSH receptors causing stimulation of the thyroid gland [] Patients with Graves™ disease usually present with signs and symptoms of hyperthyroidism that include fatigue heat intolerance sweating weight loss palpitations and tremor along with particular eye manifestations and sometimes skin changes [] It is considered the most common cause of hyperthyroidism accounting for approximately to of cases [] The diagnosis of Graves™ disease can be straightforward in the presence of typical signs and symptoms along with low thyroid stimulating hormone TSH and elevated free T4 FT4 levels [] Measuring TSH receptor antibody TRAb is helpful for confirming the diagnosis as it is present in of patients If the cause of hyperthyroidism remains uncertain a radioactive iodine uptake scan should be considered The scan helps to distinguish Graves™ disease from thyroiditis and other causes of hyperthyroidism In Graves™ disease iodine uptake is increased and diffuse [] Treatments of choice for hyperthyroidism include antithyroid medications radioactive iodine and surgical approaches [] The success rate for antithyroid medications is almost compared to and with radioactive iodine and surgery respectively [“] Whenever surgery is indicated careful preoperative management to achieve euthyroidism is needed to optimize the surgical outcome In most cases a euthyroid state is reached within a few weeks of conventional antithyroid medications however in certain conditions as in drug malabsorption and in cases of predominantly high T3 levels it cannot be easily achieved and adjunctive therapy should be considered []Case ReportA 37yearold Saudi male presented to the endocrine clinic with palpitation sweating and weight loss He was diagnosed with Graves™ disease and treated with methimazole mg orally twice daily When symptoms of hypothyroidism developed the dose was decreased to mg orally twice daily The patient was referred to the endocrine surgery clinic complaining of obstructive symptoms in the form of difficulty breathing and voice changes due to neck swelling weight gain of kg during the last month and easy fatigability along with the typical eye manifestations of proptosis and periorbital edema Figure He was otherwise healthy and the rest of his history was unremarkable On physical examination the patient had a hoarse voice fine tremor in both hands and his skin was warm with diaphoresis There was proptosis lid retraction and diplopia involving both eyes Neck examination showed a diffuse tender swelling with bilateral lumps measuring cm on the right and cm on the left along with positive Pemberton sign The rest e9233422Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves™ disease¦ Am J Case Rep e923342Figure CT scan showing diffuse enlargement of the thyroid with no retrosternal extension or invasion of surrounding structurespathology report showed diffuse hyperplasia consistent with Graves™ disease with no evidence of malignancy After the surgery the patient was moved to the Intensive Care Unit ICU where he was assessed and found to be stable with no signs and symptoms of thyrotoxicosis or hypocalcemia Three days later the patient was discharged with orders to take calcium carbonate mg orally three times daily for days acetaminophen mg orally as needed for days levothyroxine mcg orally daily for days and calcitriol mcg orally daily for daysWhen the patient presented to the clinic weeks later for followup he was found to be in good health with no active complaints He had lost weight and there were no voice changes His eye manifestations had decreased but not disappeared completely Laboratory results showed a euthyroid state with a normal calcium levelDiscussionFigure CT scan demonstrating that the distance from the anterior margin of the globe to the interzygomatic line exceeds mm indicating significant bilateral proptosisfor antithyroid medications is almost compared to with radioactive iodine therapy [] Surgical approaches are considered the most successful and definitive treatment with total thyroidectomy being the preferred choice [] A review of the literature done in showed that total thyroidectomy is times more successful than radioactive iodine therapy [] Another study concluded that the highest rates of longterm remission reaching up to are achieved with surgery [] Nonetheless there is no clear consensus on the best treatment modality for Graves™ disease and the choice should be individualized Choice of modality depends on several factors including age comorbidities size of the goiter and severity of thyrotoxicosis [] Surgery is recommended in certain conditions for example in patients with compression symptoms due to presence of a large goiter those with low radioactive iodine uptake suspected thyroid cancer moderate to severe Graves™ ophthalmopathy and patients who cannot tolerate antithyroid medications [] Whenever surgery is selected careful preoperative management is needed to optimize the surgical outcome Preparing a patient with antithyroid medications is recommended by the American Thyroid Association ATA to achieve a euthyroid state and thus lower risk of intraoperative complications []Graves™ disease can be managed with three different treatment modalities antithyroid medications radioactive iodine or surgical removal of the thyroid gland [] The success rate In most cases a euthyroid state is achieved within weeks of antithyroid treatment In certain conditions however that is difficult to achieve with conventional therapy and patients e9233423Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves™ disease¦ Am J Case Rep e923342Moreover the largest case series of patients with severe thyrotoxic Graves™ disease was published in The study involved patients who reached euthyroidism after days of an intensive treatment regimen The authors concluded that patients with severe hyperthyroid Graves™ disease can rapidly achieve preoperative euthyroidism with simultaneous administration of iopanoic acid dexamethasone betablocker and methimazole or propylthiouracil [] Another case of Graves™ disease resistant to antithyroid medications was reported in The patient was promptly managed preoperatively with both iopanoic acid and dexamethasone []Three scientific papers on resistant thyrotoxicosis due to Graves™ disease were published between to In all cases a euthyroid state could not be reached with the usual antithyroid medications and the patients received prednisolone andor lithium which resulted in complete normalization of thyroid function before surgery [“] Furthermore several refractory cases of Graves™ disease unresponsive to usual preoperative management were reported in and The patients were successfully prepared for surgery with use of plasmapheresis []ConclusionsPreoperative management of Graves™ disease can sometimes be challenging There have been many attempts to achieve a euthyroid state with different approaches In the patient described here Graves™ disease was resistant to conventional antithyroid medication for establishment of preoperative euthyroidism Our experience demonstrates that SSKI can be used in a case like ours to not only decrease vascularity of the thyroid gland but also as adjunctive therapy to achieve preoperative euthyroidismshould be prepared for surgery using adjunctive therapy [] Resistance to conventional antithyroid medications is not commonly encountered in clinical practice however there are few reported cases addressing the use of adjunctive therapy to rapidly restore normal thyroid function [] SSKI has been used for many years in management of Graves™ disease [] ATA hyperthyroidism management guidelines recommend preoperative administration of potassium iodide solutions KI for thyroidectomy [] The main rationale of using KI preoperatively is to decrease vascularity and blood loss during the surgery however a few studies suggest that when combined with antithyroid medications it can decrease thyroid hormone levels [“] In a retrospective study showed the effectiveness of adding KI as a rescue preoperative management in uncontrolled Graves™ disease In patients in the study use of KI was safe and effective as preoperative preparation for total thyroidectomy []Cholestyramine was first used to treat Graves™ disease in in Korea [] The patient in that study was a 22yearold female with severe refractory Graves™ disease who was initially managed with a maximal dose of methimazole and propranolol with no improvement She was admitted and treated with methimazole propranolol hydrocortisone and KI The next day cholestyramine was added which resulted in a rapid decline of FT4 Ten days after admission the patient underwent total thyroidectomy [] Several cases of refractory Graves™ disease were reported in the literature between to In all these cases the patients failed to achieve a preoperative euthyroid state with conventional antithyroid medications Within to weeks of administration of cholestyramine as adjunctive therapy they became euthyroid Two studies were published in and to evaluate the effectiveness of adding cholestyramine to the conventional treatment regimen in cases of resistant Graves™ disease The conclusion from these reports is that cholestyramine can be used to safely and rapidly achieve preoperative euthyroidism [“]References Pokhrel B Bhusal K Graves disease In StatPearls Treasure Island FL StatPearls Publishing Barbesino G Tomer Y Clinical review Clinical utility of TSH receptor antibodies J Clin Endocrinol Metab “ DeGroot LJ Graves™ disease and the manifestations of thyrotoxicosis In Feingold KR Anawalt B Boyce A eds Endotext South Dartmouth MA MDTextcom Inc Subekti I Pramono LA Current diagnosis and management of Graves™ disease Acta Med Indones Girgis CM Champion BL Wall JR Current concepts in Graves™ disease Ther Adv Endocrinol Metab “ Wiersinga WM Graves™ disease Can it be cured Endocrinol Metab Seoul “ Wong KK Shulkin BL Gross MD Avram AM Efficacy of radioactive iodine treatment of Graves™ hyperthyroidism using a single calculated I dose Clin Diabetes Endocrinol Piantanida E Preoperative management in patients with Graves™ disease Gland Surg “ Yang Y Hwang S Kim M Refractory Graves™ disease successfully cured by adjunctive cholestyramine and subsequent total thyroidectomy Endocrinol Metab Seoul “ Genovese BM Noureldine SI Gleeson EM What is the best definitive treatment for Graves™ disease A systematic review of the existing literature Ann Surg Oncol “ Bartalena L Diagnosis and management of Graves disease A global overview Nat Rev Endocrinol “ SebastianOchoa A QuesadaCharneco M FernandezGarcia D Dramatic response to cholestyramine in a patient with Graves™ disease resistant to conventional therapy Thyroid “ Calissendorff J Falhammar H Lugol™s solution and other iodide preparations Perspectives and research directions in Graves™ disease Endocrine e9233424Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves™ disease¦ Am J Case Rep e923342 Naafs MA Lugol™s solution in thyroid surgery A minireview Global Journal of Otolaryngology Muldoon BT Mai VQ Burch HB Management of Graves™ disease An overview and comparison of clinical practice guidelines with actual practice trends Endocrinol Metab Clin North Am “ Burch HB Cooper DS Management of Graves disease A review [published erratum appears in JAMA “] JAMA Calissendorff J Falhammar H Rescue preoperative treatment with Lugol™s solution in uncontrolled Graves™ disease Endocr Connect “ Chae SB Kim ES Lee YI Min BR A case of methimazoleresistant severe Graves™ disease Dramatic response to cholestyramine Int J Thyroidol “ Kadem SG Resistant hyperthyroidism responses dramatically to adjunctive oral cholestyramine Jourbnal of Diabetes and Endorinology “ Mercado M MendozaZubieta V BautistaOsorio R EspinozaDe Los Monteros AL Treatment of hyperthyroidism with a combination of methimazole and cholestyramine J Clin Endocrinol Metab “ Tsai WC Pei D Wang TF The effect of combination therapy with propylthiouracil and cholestyramine in the treatment of Graves™ hyperthyroidism Clin Endocrinol Oxf “ Panzer C Beazley R Braverman L Rapid preoperative preparation for severe hyperthyroid Graves™ disease J Clin Endocrinol Metab “ Pandey CK Raza M Dhiraaj S Rapid preparation of severe uncontrolled thyrotoxicosis due to Graves™ disease with Iopanoic acid “ a case report Can J Anaesth “ Saleem T Sheikh A Masood Q Resistant thyrotoxicosis in a patient with Graves disease A case report J Thyroid Res Nair GC C Babu MJ Menon R Jacob P Preoperative preparation of hyperthyroidism for thyroidectomy “ role of supersaturated iodine and lithium carbonate Indian J Endocrinol Metab “ Jude EB Dale J Kumar S Dodson PM Treatment of thyrotoxicosis resistant to carbimazole with corticosteroids Postgrad Med J “ Candoni A De Marchi F Vescini F Graves™ disease thyrotoxicosis and propylthiouracil related agranulocytosis successfully treated with therapeutic plasma exchange and GCSF followed by total thyroidectomy Mediterr J Hematol Infect Dis Ezer A Caliskan K Parlakgumus A Preoperative therapeutic plasma exchange in patients with thyrotoxicosis J Clin Apher “e9233425Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0c'

Thyroid_Cancer

Epidemiologic and clinical features of patients with COVID19 in BrazilCaracter­sticas epidemiolgicas e cl­nicas dos pacientes com COVID19 no BrasilVanessa Damazio Teich1 Sidney Klajner1 Felipe Augusto Santiago de Almeida1 Anna Carolina Batista Dantas1 Claudia Regina Laselva1 Mariana Galvani Torritesi1 Tatiane Ramos Canero1 Ot¡vio Berwanger1 Luiz Vicente Rizzo1 Eduardo Pontes Reis1 Miguel Cendoroglo Neto1 Hospital Israelita Albert Einstein S£o Paulo SP Brazil 1031744einstein_journal2020AO6022 š Objective This study describes epidemiological and clinical features of patients with confirmed infection by SARSCoV2 diagnosed and treated at Hospital Israelita Albert Einstein which admitted the first patients with this condition in Brazil Methods In this retrospective singlecenter study we included all laboratory confirmed COVID19 cases at Hospital Israelita Albert Einstein S£o Paulo Brazil from February until March Demographic clinical laboratory and radiological data were analyzed Results A total of patients with a confirmed diagnosis of COVID19 were included in this study Most patients were male with a mean age of years A history of a close contact with a positivesuspected case was reported by of patients and had a history of recent international travel The most common symptoms upon presentation were fever nasal congestion cough and myalgiaarthralgia Chest computed tomography was performed in patients and of those showed abnormal results Hospitalization was required for patients and were admitted to the Intensive Care Unit Regarding clinical treatment the most often used medicines were intravenous antibiotics chloroquine and oseltamivir Invasive mechanical ventilation was required by of Intensive Care Unit patients The mean length of stay was days for all patients and days for patients requiring or not intensive care respectively Only one patient died during followup Conclusion These results may be relevant for Brazil and other countries with similar characteristics which are starting to deal with this pandemicKeywords Communicable diseases Lung diseasesepidemiology SARSCoV2 COVID19 Coronavirus infections Epidemiology š RESUMOObjetivo Descrever as caracter­sticas epidemiolgicas e cl­nicas de pacientes com infec§£o confirmada pelo SARSCoV2 diagnosticados e tratados no Hospital Israelita Albert Einstein que admitiu os primeiros pacientes com essa condi§£o no Brasil Mtodos Neste estudo retrospectivo de centro ºnico inclu­mos todos os casos com confirma§£o laboratorial de COVID19 no Hospital Israelita Albert Einstein em S£o Paulo SP de fevereiro a mar§o de Foram analisados dados demogr¡ficos cl­nicos laboratoriais e radiolgicos Resultados Foram inclu­dos pacientes com diagnstico confirmado de COVID19 A maioria dos pacientes era do sexo masculino com mdia de idade de anos Foi relatada histria de contato prximo com um caso positivosuspeito por dos pacientes e tinham histria de viagens internacionais recentes Os sintomas mais comuns foram febre congest£o nasal tosse e mialgiaartralgia A tomografia computadorizada de trax foi realizada em pacientes e deles apresentaram How to cite this Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Epidemiologic and clinical features of patients with COVID19 in Brazil einstein S£o Paulo 202018eAO6022 httpdx1031744einstein_journal2020AO6022Corresponding authorVanessa Damazio Teich Avenida Albert Einstein “ MorumbiZip code “ S£o Paulo SP Brazil Phone Email vanessateicheinsteinbrReceived onJuly Accepted onJuly Conflict of interest noneCopyright This content is licensed under a Creative Commons Attribution International LicenseORIGINAL ISSN eISSN 23176385Official Publication of the Instituto Israelita de Ensino e Pesquisa Albert Einsteineinstein S£o Paulo 0cresultados anormais A hospitaliza§£o foi necess¡ria para pacientes e foram admitidos na Unidade de Terapia Intensiva Quanto ao tratamento cl­nico os medicamentos mais utilizados foram antibiticos intravenosos cloroquina e oseltamivir A ventila§£o mec¢nica invasiva foi necess¡ria em dos pacientes na Unidade de Terapia Intensiva O tempo mdio de interna§£o foi dias para todos os pacientes e dias para pacientes que necessitaram ou n£o de cuidados intensivos respectivamente Apenas um paciente morreu durante o acompanhamento Conclus£o Estes resultados podem ser relevantes para o Brasil e outros pa­ses com caracter­sticas semelhantes que come§aram a lidar com essa pandemiaDescritores Doen§as transmiss­veis Pneumopatiasepidemiologia SARSCoV2 COVID19 Infec§µes por coronav­rus Epidemiologia š INTRODUCTIONSince December several cases of pneumonia of unknown origin have been reported in Wuhan China1 The pathogen was further identified as a novel RNA coronavirus currently named as severe acute respiratory syndrome coronavirus SARSCoV22 Huang et al reported the first cases in China with a common clinical presentation of fever cough myalgia fatigue and dyspnea with an dysfunction eg acute respiratory distress syndrome “ ARDS shock acute cardiac and kidney injuries and death in severe cases3Afterwards in January the World Health anization WHO declared the outbreak a Public Health Emergency of International Concern PHEIC and next in March it was characterized as a pandemic4 As of April a total of cases had been reported in countries and regions across all five continents with deaths worldwide5 More recently the Chinese Center for Disease Control and Prevention published data on patients with classified as confirmed cases of coronavirus disease COVID19 Most patients were aged to years with mild clinical presentation ie nonpneumonia and mild pneumonia and overall casefatality rate of increased in elderly population with casefatality rate of in those aged years and older6On February the first Brazilian patient had a confirmed diagnosis of COVID19 at Hospital Israelita Albert Einstein HIAE Hospital Israelita Albert Einstein is a philanthropic hospital in the city of S£o Paulo SP Brazil with twelve health care units including a quaternary hospital with beds and four outpatient emergency care units By the end of this study on March of patients with confirmed COVID19 in Brazil had been diagnosed at HIAE Given the rapid spread of the COVID19 clinical and epidemiological data of several countries are being published on a daily basis79 However no studies have been reported to date presenting the characteristics of COVID19 patients diagnosed in Brazil š OBJECTIVETo describe epidemiological and clinical features of patients with confirmed infection by SARSCoV2 diagnosed and treated at Hospital Israelita Albert Einstein which admitted the first patients with this condition in Brazil š METHODSStudy design and oversightThis was a retrospective observational singlecenter study which included all consecutive patients with a confirmed diagnosis of COVID19 at HIAE between February and March The study was supported by an internal grant from HIAE and designed by the investigators The study was approved by the Research Ethics Committee of the anization protocol number CAAE and the National Commission for Research Ethics PatientsThe diagnosis of the COVID19 disease was performed according to the WHO interim guidance10 A confirmed case of COVID19 was defined as a positive result of realtime reverse transcriptase polymerase chain reaction RTPCR assay of nasal and pharyngeal swab specimens11 All cases included in the current analysis had laboratory confirmationData sourcesThe data were obtained from patients™ electronic medical records EMR including inpatients and outpatients with laboratoryconfirmed COVID19 Data collected included demographic clinical laboratorial and radiological information and was anonymized so that patients could not be identified Demographic characteristics included age sex tobacco smoking weight and body mass index BMI Clinical information included medical travel Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Canero TR Berwanger O Rizzo LV Reis EP Cendoroglo Neto Meinstein S£o Paulo 0cand exposure history signs symptoms underlying comorbidities continuous medication use and treatment measures ie antiviral therapy steroid therapy respiratory support and kidney replacement therapy Laboratory assessment consisted of complete blood count assessment of renal and liver function and measurements of electrolytes Ddimer procalcitonin lactate dehydrogenase Creactive protein and creatine kinase Radiologic abnormality was defined based on the medical report documented in the EMR Disease duration from onset of symptoms hospital and Intensive Care Unit ICU length of stay LOS were also documented Statistical analysis Continuous variables were expressed as means with standard deviations medians minimum and maximum values Categorical variables were summarized as counts and percentages No imputation was made for missing data All statistics are deemed to be descriptive only considering that the cohort of patients in our study was not derived from random selection All analyses were performed using Microsoft Excel š RESULTSDemographic and clinical characteristicsBetween February and March a total of patients were diagnosed with COVID19 at HIAE This study included patients for whom data regarding demographics clinical symptoms laboratory and imaging findings were available in the EMR The remaining patients had only used the hospital laboratory facilities and were followedup by physicians not working in our service network Patients™ demographic and clinical characteristics are shown in table A total of had a recent international travel history and had been at the same marriage celebration in Bahia a state in the Northeast region of Brazil patients had a history of close contact either with a positive or suspected case of COVID19 Most patients were male and the mean age was years Only of patients were younger than years old and were older than yearsFever was present in only of patients upon admission but had a reported history of fever followed by nasal congestion cough Table Clinical and epidemiological characteristicsCharacteristicAge years Mean±SDMedianMinimumMaximumNumber of patientsAge distribution years ‰¥Sex MaleFemaleTravel historyTotal patients n510Total patientsNonhospitalized patients n438Hospitalized patients n72±±± European Union and United Kingdom United States of America and Canada Middle East and IranChina and Japan Latin America Other countries Bahia Brazilian stateNo travel history Exposure source of transmission “ contact with confirmed or suspected casesExposureNo exposureHealthcare professionalYesNoSmoking historyCurrent smokerFormer smokerNever smokedFever on admission YesNoMedianTemperature distribution on admission°C °C°C°CSymptomsNasal congestionHeadacheCoughSore throatSputum production continueEpidemiologic and clinical features of patients with COVID19 in Brazileinstein S£o Paulo 0cContinuationTable Clinical and epidemiological characteristicsContinuationTable Clinical and epidemiological characteristicsCharacteristicFatigueDyspneaNausea or vomitingDiarrheaMyalgia or arthralgiaChillsFeverConjunctival congestionOther symptomsNo symptomsSymptoms duration daysMean±SDMedianMinimumMaximumSigns of infectionThroat congestionTonsil swellingSkin rashOther alterationsNo alterationsCoexisting disordersAny coexisting disorderAsthma or chronic pulmonary obstructive disorderDiabetesHypertensionCoronary heart disease or other heart conditionsCerebrovascular diseaseHepatitis B C HIV or other immunodeficiencyCancerChronic renal diseasean transplantPregnancy Other coexisting disordersNo coexisting disordersMean BMI±SDChronicuse medicationsAny medicationStatinMultivitaminAntidepressantAntihypertensiveAntiplatelet or anticoagulantThyroid hormonesTotal patients n510 Total patientsNonhospitalized patients n438 Hospitalized patients n72 ± ± ± ±± ± continueCharacteristicAntidiabeticPain killersAntibioticsCorticosteroidInhaled medicationsOther medicationsNo use of medicationsTotal patients n510 Total patientsNonhospitalized patients n438 Hospitalized patients n72 Chronicuse medications number of medications distributionOnly type of medication types of medications types of medications or more types of medications “ polypharmacy ESI on arrival Destiny after first evaluation Discharge to homeAdmission on general wardAdmission on ICU Return to the emergency room after first evaluation SimN£oResults expressed by total nn if not otherwise indicatedSD standard deviation BMI body mass index ESI Emergency Severity Index ICU intensive care unitand myalgia or arthralgia The mean duration of symptoms was days which was the same for patients hospitalized or not Upon admission the majority of patients had no significant changes on physical examination Considering all included patients had at least one comorbidity This rate however was far higher in the hospitalized group when compared with the nonhospitalized group the most common comorbidities were hypertension and diabetes The distribution of patients in the Emergency Severity Index ESI differs between the two groups analyzed with the hospitalized group showing a higher rate of ESI indicating that the initial severity was greater in this group since the onset of symptoms Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Canero TR Berwanger O Rizzo LV Reis EP Cendoroglo Neto Meinstein S£o Paulo 0cRadiologic and laboratory findingsTable demonstrates the radiologic and laboratory findings upon admission Only of patients were initially evaluated with chest radiographs whereas were submitted to computed tomography CT Of the radiographs performed had some abnormality while of CT scans showed abnormal results The most common patterns on chest CT were groundglass opacity and bilateral patchy shadowing Table Radiologic and laboratory findingsCharacteristicsRadiologic findings in chest radiographChest radiograph performedAbnormalities on chest radiograph Groundglass opacity Local patchy shadowing Bilateral patchy shadowing Interstitial abnormalities Radiologic findings in chest CT Chest CT performed Abnormalities on chest CT Ground glass opacity Local patchy shadowing Bilateral patchy shadowing Interstitial abnormalitiesLaboratory findingsMedian PaO2FiO2 ratio IQRWhite blood cell countMedian per mm3 Distribution per mm3 Lymphocyte countMedian per mm3 Distribution per mm3 Platelet countTotal number of patients n510 Total number of patientsNonhospitalized patients n438Hospitalized patients n72 Median per mm3Distribution per mm3 Median hemoglobin gdLDistribution of other findings Creactive protein 5mgL Procalcitonin 05ngmLLactate dehydrogenase 214UL Aspartate aminotransferase 40UL Alanine aminotransferase 40UL Total bilirubin 12mgdL Creatine kinase UL Creatinine 1mgdLDdimer 500ngmL Mean sodium mmolLMean potassium mmolL Results expressed by total nn if not otherwise indicatedCT computer tomography PaO2FiO2 oxygen partial pressurefractional inspired oxygen IQR interquartile rangeleukopenia Upon admission lymphocytopenia was identified in of patients thrombocytopenia in and in Most patients had elevated levels of both Creactive protein and lactate dehydrogenase Less common findings were elevated levels of Ddimer aspartate aminotransferase and alanine aminotransferase The hospitalized group had more patients with higher levels of Creactive protein procalcitonin and lactate dehydrogenase The other results do not show any major difference between groups A viral panel was collected in patients and it was positive for rhinovirus in nine cases influenza B in two cases and influenza A in one caseTreatment and complicationsAs shown in table patients had been hospitalized at HIAE by the time of the analysis Among Table Treatments complications and clinical outcomesTotal number of patients n510 CharacteristicDisease severitySevereNot severeIntensive care use during hospital stayYesNoHospital treatments “ medicationsIntravenous antibioticsOseltamivirLopinavir and ritonavirChloroquineCorticosteroidsHospital treatments “ support treatmentsOxygen therapyMechanical ventilationInvasiveNoninvasiveExtracorporeal membrane oxygenationContinuous renal replacement therapyComplicationsSeptic shockAcute respiratory distress syndromeAcute kidney injuryPneumoniaMean length of stay daysLOS all patientsPatients requiring ICU daysICUInpatients unitsPatients not requiring ICU inpatients units daysResults expressed by total nn if not otherwise indicated LOS length of stay ICU intensive care unitEpidemiologic and clinical features of patients with COVID19 in Brazileinstein S£o Paulo 0cthose patients required intensive care during their hospital stay in that were referred from the emergency room to the ICU and eight presented worsening of the clinical condition at inpatients units and were transferred to the ICU The majority of patients received intravenous antibiotic therapy received chloroquine and oseltamivir Oxygen therapy was necessary in of hospitalized patients required mechanical ventilation invasive and noninvasive and extracorporeal membrane oxygenation ECMO was used in only one case Considering patients admitted to the ICU invasive mechanical ventilation was required by of them During hospital admission most patients were diagnosed with pneumonia followed by acute kidney injury and ARDS The mean LOS was days considering only patients requiring intensive care the mean ICU LOS was days and the mean total LOS was days whereas for patients not admitted to the ICU the mean LOS was days Only one patient died in this series that is mortality rate š DISCUSSION It took months from the first diagnosed case of COVID19 in China until diagnosis of patient zero in Brazil on February at HIAE During days after the first diagnosis all cases had a history of recent international travels On March the first case of local transmission was confirmed also at HIAE A relevant proportion of all patients with confirmed COVID19 infection had been diagnosed at HIAE by the time of the analysisThe patients in our series had a mean age of years and were mostly male The studies describing demographic characteristics in the infected general population showed a median age of years712 and the proportion of males was in the Chinese report7 and in the Singapore report The respiratory symptoms were similar to those of patients described in reports from China United States and Europe7913 However the mean days of symptoms was far lower in our series days versus days in Singapore12 days in the United States13 and days in China3 Although fever was reported by the majority of patients it was only present in of patients at the initial assessment at hospital suggesting not only it might not be considered to determine severity of illness but also that diagnostic algorithms using fever for testing may mask the total number of cases and delay diagnosis The prevalence of chronic diseases was far higher in the hospitalized group as compared to nonhospitalized group This prevalence was even higher in the subgroup admitted to the ICU The mean age of hospitalized patients was higher than nonhospitalized patients versus years and the required hospitalization increased with age for patients aged to years for to years and for patients older than years In this Brazilian case series hospitalization was required for patients and of them demanded critical care accounting for of total admissions a number far greater than the Chinese series in which only required ICU7The majority of patients were admitted to the ICU because of acute hypoxemic respiratory failure that required ventilatory support Invasive mechanical ventilation was needed in of ICU patients of total hospitalizations whereas were managed with noninvasive mechanical ventilation The necessity of invasive mechanical ventilation was similar to an ICU series reported from the United States of Washington13 lower than that reported in an Italian publication of Lombardy9 but higher than the Chinese reports and of Wuhan half of these treated with extracorporeal membrane oxygenation31415 Considering the use of noninvasive ventilation the rate was again similar to that reported in Washington and lower than the rates in China and of Wuhan including patients receiving highflow nasal cannula31415 A total of three patients of patients admitted to the ICU developed acute kidney injury and required continuous renal replacement therapy Among those only one patient had chronic kidney disease The prevalence of chronic kidney disease was among hospitalized patients in the Chinese report14 and among patients admitted to the ICU in the series from the United States This study has important limitations First part of the cases had incomplete information documented in the medical records and patient clinical history documentation was not homogeneous among all patients This is a common limitation in retrospective observational studies taking into account that data Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Canero TR Berwanger O Rizzo LV Reis EP Cendoroglo Neto Meinstein S£o Paulo 0cgeneration was clinically driven and not in systematic fashion Second since many patients remained at the hospital and the outcomes were unknown at the time of data collection we censored the data regarding their clinical outcomes as of the time of the analysis Third only patients hospitalized at HIAE were included in the hospitalization group and there is no documentation of hospital admissions outside of our service network Finally this study only included patients attended as outpatients or inpatients at HIAE therefore asymptomatic and mild cases who did not seek medical care were not considered Hence our study cohort may represent more severe COVID19 cases š CONCLUSIONTo date there is no study in Brazil reporting the characteristics of patients diagnosed with COVID19 Brazil is the country in the south hemisphere with the highest number of confirmed cases this disease and Hospital Israelita Albert Einstein is the center where the first patient was diagnosed with a representative sample of all confirmed COVID19 cases in the country The results presented in this study may be relevant for Brazil and other countries with similar characteristics which are starting to deal with this pandemic š CONTRIBUTION OF AUTHORSData were analyzed and interpreted by the authors All authors reviewed the manuscript and checked the exactness and completeness of data š AUTHORS™ INFORMATIONTeich VD httporcid0000000285396037Klajner S httporcid0000000341201047 Almeida FA httporcid0000000171310039 Dantas AC httporcid0000000195056784 Laselva CR httporcid0000000182859633 Torritesi MG httporcid0000000236236475 Canero TR httporcid0000000273994718Berwanger O httporcid0000000249722958Rizzo LV httporcid0000000199499849 Reis EP httporcid000000015110457X Cendoroglo Neto M httporcid0000000281634392 š REFERENCES Lu H Stratton CW Tang YW Outbreak of pneumonia of unknown etiology in Wuhan China The mystery and the miracle J Med Virol Zhu N Zhang D Wang W Li X Yang B Song J Zhao X Huang B Shi W Lu R Niu P Zhan F Ma X Wang D Xu W Wu G Gao GF Tan W China Novel Coronavirus Investigating and Research Team A novel coronavirus from patients with pneumonia in China N Engl J Med Huang C Wang Y Li X Ren L Zhao J Hu Y et al Clinical features of patients infected with novel coronavirus in Wuhan China Lancet Erratum in Lancet Jan World Health anization WHO Coronavirus disease COVID19 outbreak [Internet] Geneva WHO [cited July ] Available from httpswwwwhointwesternpacificemergenciescovid19 The Johns Hopkins Coronavirus Resource Center CRC COVID19 Dashboard by the Center for Systems Science and Engineering CSSE at Johns Hopkins University [Internet] CRC USA [cited July ] Available from httpscoronavirusjhuedumaphtml Wu Z McGoogan JM Characteristics of and important lessons from the coronavirus disease COVID19 outbreak in China Summary of a report of cases from the Chinese Center for Disease Control and Prevention JAMA Feb 101001jama20202648 Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Clinical characteristics of coronavirus disease in China N Engl J Med Holshue ML DeBolt C Lindquist S Lofy KH Wiesman J Bruce H Spitters C Ericson K Wilkerson S Tural A Diaz G Cohn A Fox L Patel A Gerber SI Kim L Tong S Lu X Lindstrom S Pallansch MA Weldon WC Biggs HM Uyeki TM Pillai SK Washington State 2019nCoV Case Investigation Team First case of novel coronavirus in the United States N Engl J Med Grasselli G Zangrillo A Zanella A Antonelli M Cabrini L Castelli A Baseline characteristics and outcomes of patients infected with SARSCoV2 admitted to ICUs of the Lombardy region Italy JAMA World Health anization WHO Clinical management of severe acute respiratory infection when novel coronavirus 2019nCoV infection is suspected interim guidance [Internet] Geneva WHO [cited July ] Available from httpswwwwhointdocsdefaultsourcecoronaviruseclinicalmanagementofnovelcovpdf Brasil Ministrio da Saºde Centro de Opera§µes de Emergªncias em Saºde Pºblica Coronavirus Covid19 Boletim Di¡rio [Internet] Bras­lia DF Ministrio da Saºde [citado Jul ] Dispon­vel em httpswwwsaudegovbrimagespdf2020marco2929COVIDpdf Young BE Ong SW Kalimuddin S Low JG Tan SY Loh J Ng OT Marimuthu K Ang LW Mak TM Lau SK Anderson DE Chan KS Tan TY Ng TY Cui L Said Z Kurupatham L Chen MI Chan M Vasoo S Wang LF Tan BH Lin RT Lee VJ Leo YS Lye DC Singapore Novel Coronavirus Outbreak Research Team Epidemiologic features and clinical course of patients infected with SARSCoV2 in Singapore JAMA Mar 101001jama20203204 Bhatraju PK Ghassemieh BJ Nichols M Kim R Jerome KR Nalla AK Covid19 in critically Ill patients in the Seattle region Case series N Engl J Med Wang D Hu B Hu C Zhu F Liu X Zhang J Clinical characteristics of hospitalized patients with novel coronavirusinfected pneumonia in Wuhan China JAMA Feb 101001jama20201585 Yang X Yu Y Xu J Shu H Xia J Liu H Clinical course and outcomes of critically ill patients with SARSCoV2 pneumonia in Wuhan China a singlecentered retrospective observational study Lancet Respir Med Erratum in Lancet Respir Med 202084e26Epidemiologic and clinical features of patients with COVID19 in Brazileinstein S£o Paulo 0c'

Thyroid_Cancer

Aprospective blinded multicenter studyDear editorThyroid nodules are common and the major point oftheir clinical evaluation rests with the differentiation ofthyroid cancer1“ Herein we present a twostep study todevelop and validate an effective model based on circulating tumor cells CTCs signatures for papillary thyroid cancer PTC diagnosisA total of subjects were clinically evaluated and of which were included in the final analysis A In the first step patients with PTC from a single hospital and healthy subjects formed the test groupMultimarkers reported in the literatures that were relatedto malignancy and thyroid epithelium CK19 SurvivinGalectin3 Tg and TSHR were evaluated and selectedto form a most effective combination for the diagnosticmodel Next the model was evaluated in the validationgroup that was consisted of participants from four different hospitals in China who had one or more thyroidnodules that yielded an ultrasound diagnosis of ThyroidImaging Reporting and Data System45 TIRADS or and measured cm long in diameter Postoperativepathology was considered as the reference standard andonly patients who received surgery and had definite pathological reports were enrolled in the final analysis Thyroidnodule was excluded by ultrasound in healthy subjectsThe process of CTCs separation and enrichmentincluded CD45 negative Dynabeads„¢ M450 CD45 panLeukocyte 11153D Invitrogen by Thermo Fisher Scientific USA and EpCAM positive Invitrogen byThermo Fisher Scientific USA immunomagnetic selections The reliability of CTCs separation and enrichmentwas verified with fluorescence staining in a cell line model PTC cells added into white blood cells separationfrom mL peripheral blood from a healthy subject B mRNA was isolated from the cell fractions Invitrogen by Thermo Fisher Scientific USA and cDNAwas then reverse transcribed with μL of mRNA total μL using the Sensiscript RT Kit QIAGEN Germany mRNA levels of CK19 Survivin Galectin3 Tgand TSHR were analyzed by the QuantiNova SYBR GreenPCR Kit QIAGEN Germany Thermo FisherScientific USA Three independently isolated RNAsamples were measured to determine the threshold cyclevalues CT mRNA level of these markers were measuredwith relative expression level RQ and presented afternormalization against the reference actin and wascalculated with “ΔΔCT method ΔCt Ct target “ Ctactin ΔΔCT ΔCt13 RQ “ΔΔCT The values werepresented as the mean ± standard deviation SDOf all participants recruited in the study the medianage was years range years and were female participants Among patients with thyroid nodules the median size of the nodules was cm range cm and had a tumor ‰ cm According to thepostoperative pathology patients had PTC of which and patients had central compartment and lateral necklymph nodes metastasis respectively In the test group patients and healthy subjects the median mRNA levelsof the markers were significantly higher in patients thanin healthy controls except for TSHR C CK19 andSurvivin mRNA was not detected in any healthy subject sopositive result of the two markers was defined as detectablemRNA level Tg and Galectin3 mRNA were detected in and healthy subjects respectively Based on theROC curves a cutoff value of ngμg was selected falectin3 with a sensitivity of and a specificity of while a cutoff value of ngμg for Tg giving an optimal sensitivity of and a specificity of The areaunder curve AUC of CK19 Survivin Galectin3 and Tgwere and respectivelyAfter evaluating different combinations of the markers Supporting Information CK19 Survivin and Tgwere selected to form a diagnostic model when positiveresult was recognized when any marker was positive itsThis is an access under the terms of the Creative Commons Attribution License which permits use distribution and reproduction in any medium provided theoriginal work is properly cited The Authors Clinical and Translational Medicine published by John Wiley Sons Australia Ltd on behalf of Shanghai Institute of Clinical BioinformaticsClin Transl Med 202010e142101002ctm2142wileyonlinelibrarycomjournalctm2 of 0c of LETTER TO EDITORF I G U R E Flow chart of the study and diagnostic model establishing A Flow chart of the study B Fluorescence staining of EpCAMCD45 and DAPI after immunomagnetic enrichment of a CTC model PTC cells added into white blood cells separation from ml peripheralblood from a healthy subject CTCs white solid arrow white blood cells white dotted arrow C CK19 Survivin Galectin3 Tg and TSHRmRNA levels of healthy subjects n and patients with PTC n in the test group D HE a and immunohistochemical staining forCK19 b Tg c and Survivin d of a PTC patient enrolled in the test group E Expression of markers in cell lines of PTC by western blotpositive predictive value PPV negative predictive valueNPV sensitivity specificity and AUC value were and respectively and the diagnostic model correctly classified of subjects yielding anoverall accuracy of The expression of selected markers CK19 Survivin and Tg was verified with immunohistochemical analysis in formalinfixed paraffinembeddedpostoperative specimen of PTC patients D andwestern blot in two cell lines of PTC E and thenthese markers were evaluated in the validation groupIn the validation cohort n the median mRNAlevels of CK19 Survivin and were significantly higherin patients with PTC than in patients with benign thyroid nodules P A The clinical sensitivity specificity PPV and NPV of the diagnostic model were and respectively with a diagnostic accuracy of and the AUC for predicting malignancy was CI For detailed performance of individual markers the highest sensitivity of single marker Tg was which was significantlylower than the diagnostic model of 3marker combinationAll patients with positive Survivin n had malignant pathology PPV with a sensitivity of which was consistent with the results of the test groupBIn the diagnostic model based on threeCTCs signatures established in the study has a highdiagnostic performance for patients with suspiciousthyroid nodules multimarker analysissignificantlyimproved sensitivity comparing with single marker 0cLETTER TO EDITOR of F I G U R E Validation of the diagnostic model in a prospective cohort n A CK19 Survivin and Tg mRNA levels of benign andmalignant patients in the validation group B Detailed diagnostic performance of the diagnostic model in the validation groupanalysis Moreover it is discovered in this study thatthe CTCs signature Survivin had a potential value forconfirming PTC diagnosis PPV C O N F L I C T O F I N T E R E S TThe authors have no conflicts of interest to disclosureS TAT E M E N T O F E T H I C SThe study was approved by the ethic committee of CancerHospital Chinese Academy of Medical sciencesAU T H O R S ™ C O N T R I B U T I O N SSiyuan Xu and Jingning Cheng participated in the designof the study data collection and paper writing Bojun Weiand Yang Zhang participated in the data collection andhelped to draft the manuscript Yang Li and Rui Zhangparticipated in the data analysis and validation ZongminZhang Yang Liu and Kai Wang participated in the datacollection and quality control of data Ye Zhang and YingHuang participated in the statistical analysis Xiaolei Wangand Shaoyan Liu participated in the manuscript reviewJie Liu and Zhengang Xu participated in the design of thestudy and helped to revise the manuscript All authors readand approved the final manuscriptSiyuan Xu1Jingning Cheng2Bojun Wei3Yang Zhang4Yang Li5Zongmin Zhang1Yang Liu1Ye Zhang6Rui Zhang7Kai Wang1Xiaolei Wang1Shaoyan Liu1Ying Huang1Zhengang Xu1Jie Liu1 Department of Head and Neck Surgical OncologyNational Cancer CenterNational Clinical Research Centerfor CancerCancer Hospital Chinese Academy of MedicalSciences and Peking Union Medical College Beijing P RChina Department of Otorhinolaryngology ChinaJapanFriendship Hospital Beijing P R China Department of Thyroid and Neck Surgery BeijingChaoyang Hospital Capital Medical University Beijing PR China Department of Endocrinology Peking University FirstHospital Beijing P R China Department of General Surgery Hebei Petro ChinaCentral Hospital Langfang P R China Department of Radiation Oncology National CancerCenterNational Clinical Research Center forCancerCancer Hospital Chinese Academy of MedicalSciences and Peking Union Medical College Beijing P RChina Department of Ultrasound National CancerCenterNational Clinical Research Center forCancerCancer Hospital Chinese Academy of MedicalSciences and Peking Union Medical College Beijing P RChinaCorrespondenceJie Liu and Zhengang Xu Postal address No Panjiayuan Nanli Chaoyang District Beijing PRChinaEmail liujcicamsaccn xuzhg06126com 0c of LETTER TO EDITORSiyuan Xu Jingning Cheng Bojun Wei and Yang Zhangcontributed equally to this workTrial Registration Clinicaltrialsgov IdentifierNCT03772496Funding informationCAMS Innovation Fund for Medical Sciences BeijingHope Run Special Fund of Cancer Foundation of China2016I2m1002 Beijing Hope Run Special Fund of CancerFoundation of China LC2018A26 LC2016A01O RC I DJie Liuorcid0000000339757978R E F E R E N C E S Haugen BR Alexander EK Bible KC et al American Thyroid Association Management Guidelines for adult patients withthyroid nodules and differentiated thyroid cancer the American Thyroid Association Guidelines Task Force on thyroidnodules and differentiated thyroid cancer Thyroid Burman KD Wartofsky L Clinical practice Thyroid nodules NEngl J Med Popoveniuc G Jonklaas J Thyroid nodules Med Clin North Am Cheng SP Lee JJ Lin JL Chuang SM Chien MN Liu CL Characterization of thyroid nodules using the proposed thyroid imagingreporting and data system TIRADS Head Neck Zhang J Liu BJ Xu HX et al Prospective validation ofan ultrasoundbased thyroid imaging reporting and data system TIRADS on thyroid nodules Int J Clin Exp MedS U P P O RT I N G I N F O R M AT I O NAdditional supporting information may be found onlinein the Supporting Information section at the end of the 0c'

Thyroid_Cancer

"At the time of surgery approximately “ of the patients with pancreatic cancer are consideredunresectable because of unexpected liver metastasis peritoneal carcinomatosis or locally advanced disease This leads to futilesurgical treatment with all the associated morbidity mortality and costs More than of all liver metastases develop in thefirst six months postoperatively These subcentimeter liver metastases are most likely already present at the time of diagnosisand have not been identified preoperatively due to the poor sensitivity of routine preoperative contrastenhanced CT CECTMethods The DIAPANC study is a prospective international multicenter diagnostic cohort study investigating diffusionweighted contrastenhanced MRI for the detection of liver metastases in patients with all stages of pancreatic cancerIndeterminate or malignant liver lesions on MRI will be further investigated histopathologically For patients with suspected liverlesions without histopathological proof follow up imaging with paired CT and MRI at and 12months will serve as analternative reference standardDiscussion The DIAPANC trial is expected to report highlevel evidence of the diagnostic accuracy of MRI for the detection ofliver metastases resulting in significant value for clinical decision making guideline development and improved stratification fortreatment strategies and future trials Furthermore DIAPANC will contribute to our knowledge of liver metastases regardingincidence imaging characteristics their number and extent and their change in time with or without treatment It will enhancethe worldwide implementation of MRI and consequently improve personalized treatment of patients with suspectedpancreatic ductal adenocarcinomaTrial registration ClinicalTrialsgov Identifier NCT03469726 Registered on March 19th Retrospectively registeredKeywords Pancreatic cancer Liver metastases MRI Staging Correspondence JohnHermansradboudumcnl G Litjens and D M Rivi¨re contributed equally to this work1Department of Radiology and Nuclear Medicine Radboudumc NijmegenThe NetherlandsFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLitjens BMC Cancer Page of BackgroundPancreatic ductal adenocarcinoma PDAC is one of themost lethal forms of cancer and expected to become thesecond leading cause of cancerrelated deaths before Developments in pancreatic cancer diagnosticssurgical techniques and treatment have hardly improvedthe survival rate in the past years The 5year relativesurvival rate as reported by the American Cancer Societyremains only [ ]Only “ of all patients are eligible for surgery todate the only potential cure [] Approximately “ ofall patients with pancreatic cancer have metastatic diseaseat diagnosis and of all patients have locally advanceddisease with tumor involvement of surrounding vessels orans At the time of surgery approximately “ ofthe patients are considered unresectable because of unexpected liver metastasis peritoneal carcinomatosis or locally advanced disease [“]More than of all liver metastases develop in thefirst six months postoperatively [] These liver metastases are most likely already present at the time of diagnosis and have not been identified preoperatively as theyare too small to be detected by routine preoperativeultrasound and contrastenhanced CT CECT [ ]CECT is highly accurate in assessing the relationship ofthe tumor to critical arterial and venous structures sincetheir involvement can preclude surgical resection HoweverCECT has a poor sensitivity “ for the detection andcharacterization of liver metastases [ “] especiallyfor subcentimeter metastases which are often present inpancreatic cancer [] This leads to futile surgical treatment with all the associated morbidity mortality and costsMoreover patients who were explored with curative intentand were found unresectable due to peritoneal or liver metastases had a worse overall survival compared to patientswith unexpected locally advanced disease []Nowadays diffusionweighted MR imaging DWI appears to be valuable in both detection and characterizationof focal liver lesions with a high sensitivity “ evenfor subcentimeter lesions “ [“] This technique can be used to detect and characterize liver lesionsbased on decreased diffusion of water molecules caused bytumoral hypercellularity and reduced extracellular spaceDWI is especially useful for detecting subcentimeter livermetastasesit is more accurate than conventional T2weighted imaging techniques because signal suppression ofintravascular flow is obtained black blood effect whilemaintaining good residual signal of the liver lesions [] Itis easy to implement and adds very little time to a standardMRI examination However without highquality evidenceof the benefit of MRI the use of MRI as part of the routineworkup is questioned and therefore not implemented Currently most guidelines advise to use MRI as a problemsolving tool in addition to CECT eg when the primarytumor cannot be visualized or in case of undefined liver lesions [“] The American Society of Clinical OncologyASCO leaves the choice of imaging modality in the handsof the physician [] MRI is advised for all patients according to the Japanese guideline however the level of evidenceis low grade C []Most studies that have been performed for liver metasincluding our singletases of PDAC are retrospectivecenter study in patients with potentially resectable pancreatic cancer without liver metastases on CECT [] Inthis study Gadolinium Gd enhanced MRI with DWIdetected synchronous liver metastases in of patientswith potentially resectable pancreatic cancer on CECTwith a sensitivity of DWI showed more lesions thanGdenhanced MRI most of which were particularlysmall mm Correspondingly the only prospectivestudy to our knowledge showed that Gdenhanced MRIespecially DWI depicted small liver metastases in approximately of patients with a potentially resectablepancreatic cancer without liver metastases on CECT[] The reported sensitivity was “ and the specificity “ However due to the relatively low prevalence of patients with liver metastases in their studypopulation in total only patients with liver metastaseswere included in this studyIn the DIAPANC study we will determine the diagnosticaccuracy of Gdenhanced MRI with DWI in the detectionof liver metastases in patients with all stages of PDACMethodsDesigninternationalThe DIAPANC study is a prospectiveinvestigatingmulticenterstudydiffusionweighted Gdenhanced MRI for the detectionof liver metastases in patients with pancreatic cancerdiagnosticcohortThis protocol was written and reported according to theStandard Protocol Items Recommendations for Interventional Trials SPIRIT Guidance and Checklist []Study populationAll patients with suspected pancreatic ductal adenocarcinoma are eligible to be included in this study and will beactively recruited at the outpatient clinic by the treatingphysician Written informed consent will be obtained byone of the members of the research team We will includepatients until patients with liver metastasis are included with a maximum total of patients Exclusioncriteria are age below years previous treatment forpancreatic cancer concomitant malignancies except foradequately treated basocellular carcinoma of the skin subjects with prior malignancies must be diseasefree for atleast years contraindications for MRI or CECT ie untreatable contrast allergy severe renal function impairment not MRI compatible medical implants insufficient 0cLitjens BMC Cancer Page of command of the local language and pregnancy This studyhas been approved by the ethical board of our universitymedical center Approval of the local medical ethicalboard is obliged before the start of inclusion in the participating hospitalsSpecific withdrawal of patientsPatients with adenocarcinoma of the distal common bileduct papilla of Vater or duodenum patients with aneuroendocrine tumor or patients with benign tumorswill be excluded from analysis and followupPrimary outcomeThe sensitivity and specificity of Gdenhanced MRI withDWI for the detection of liver metastases in patientswith pancreatic cancerSecondary outcomesThe secondary outcomes of this study are sensitivityand specificity of CECT for the detection of liver metastases sensitivity and specificity of MRI and CECT forthe prediction of resectability and the effect of the MRIon patient managementData collectionAll patients will be assigned a unique participant codeThe key will be stored separately from the data We planto collect the following baseline data age sex performance status WHO performance score American Societyof Anesthesiologists physical status body mass indexweight loss decreased appetite diabetes mellitus previousliver or pancreatic diseases smoking and alcohol statusand tumor markers CEA and CA19“ using the datamanagement system Castor EDC Castor Electronic DataCapture Ciwit BV Amsterdam The Netherlands Dataon diagnostic procedures like endoscopic imaging and biopsies treatment and clinical followup will be collectedduring the entire study period by the local treating physicians or the trial coordinators using Castor EDC Patientswill be asked to fill in validated quality of life questionnaires EORTC QLQC30 and QLQPAN26 at baselineand after and 12months followupMRI and CTMRI scans will be made on a T scanner with T2 weightedimaging using an intravenous gadoliniumbased contrastagent with a T1 weighted precontrast arterial and portalvenous phase DWI with bvalues of and smm2and with a Magnetic Resonance CholangioPancreatographyMRCP CECT scans are performed with intravenous iodinecontrast agent with a pancreatic phase of the upper abdomen a portal venous phase of the entire abdomen Additionally the chest will be staged using chest CT MRI and CECTwill be performed at baseline and after and 12monthsfollowup the schedule is displayed in a flowchart in Fig Interpretation of MRI and CTAll MRI and CECT scans will initially be evaluated by thelocal radiologist and the findings will be included in theclinical decision making The MRI and CECT scans willalso be independently evaluated by a second radiologistblinded for findings of the first evaluation and the clinicaloutcome If the MRI and CECT of one patient is evaluatedby the same radiologist a minimum interval of weeks willbe used to minimize the risk of recall biasThe MRI and CECT scans will be analyzed for localresectability and suspicious liver lesions Number of liverlesions lesion size liver segment presumed diagnosis ofsuspicious liver lesions indeterminate or malignant andimaging characteristics on MRI will be notedReference standardIndeterminate or malignant liver lesions will be furtherinvestigated histopathologically The first step in obtaining histological proof of suspected liver lesions on CECTandor MRI is transabdominal ultrasound of the liverBiopsy will be performed of visible liver lesions and analyzed with routine histological examination When lesions are not visible or there is no histological proof ofthe visible lesions the next step is surgical explorationlaparoscopic or in borderline resectable pancreatic cancer In case liver lesions are identified a frozensection is performed Hereafter patients are treated according to standard care protocolFor patients with suspected liver lesions without histopathological proof followup imaging with paired CECTand MRI at and months will serve as an alternativereference standard Lesions that are growing or increasingin number over time will be considered metastasesDefinitionsOn MRI liver lesions are defined as malignant on DWIwhen they are moderately hyperintense at b smm2and remains hyperintense at b smm2 A lesion isconsidered benign when it is hyperintense at b smm2and shows a substantial decrease in signal intensity athigher b values b and b smm2 If none ofthe criteria is met a lesion is classified as indeterminateOn CECT liver lesions are defined as malignant if theyare hypodense not showing typical features of a simplecyst fluid attenuation measurements roundoval welldefined borders no contrast enhancement hemangiomalocalization next to vessels peripheral nodular enhancement centripetal fillin or focal fatty infiltration geographic hypodense area angular margins typical locationIf a lesion is showing signs of simple cyst hemangioma or 0cLitjens BMC Cancer Page of Fig Flowchart of study schedule and proceduresfocal fatty infiltration it is defined as benign If a lesion istoo small to characterize it is classified as indeterminateTNM status is classified according to the AmericanJoint Committee on Cancer AJCC 8th edition []Lymph nodes are defined as suspicious ifthey arerounded and ‰¥ mm or if they are notrounded with theshortest axis ‰¥ mmSafety and ethicsThere is a low risk and low burden for patients participating in this study Patients might benefit fromstudy participation due to possible improvement ofdetection of liver metastases The contrast agent usedfor MRI has few known side effects and rarely leadsto a severe allergic reaction [] Extra CECT scansmight be performed in some study patients with theassociated radiation and contrast exposure Patientsdiagnosed with pancreatic cancer have a 5year overall survival of Radiationinduced cancer has a latency yearsTherefore the health risk for this specific oncologicpatient group is almost negligiblesubstantiallyexceedsperiodthat 0cLitjens BMC Cancer Page of MRI can lead to earlier detection of liver metastaseshowever in some patients these lesions might be toosmall to biopsy Consequently we cannot always providethe patient certainty about the nature of the liver lesionsdetected with MRI Furthermore in followup local recurrence or metastases might be detected before a patient has symptoms This may be seen as a disadvantageby some individualsStatisticsSample sizeThe sample size for the study was calculated for the primary endpoint sensitivity and specificity of MRI for thedetection of liver metastasesThe sample size is calculated based on a method forpower calculations for diagnostic studies described by Jones [] Based on literature and our previously performedretrospective study [ “] we estimate the sensitivity ofMRI will be approximately In literature the specificityfor MRI is usually higher than the sensitivity therefore webased our sample size calculation on the sensitivity onlyWith an expected sensitivity of confidence interval of Z and α patients with metastasisare required for analysis Based on literature the expectedpercentage of patients with liver metastases is approximately [ ] With an expected inclusion rate of assuming cannot be analyzed optimally eg becauseno representative liver biopsies could be acquired mortalitybefore first followup or withdrawal we need approximately patients In case the proportion of patients withmetastases is not equal to in our cohort we will include until we reach patients with liver metastasis orup to a maximum total of patientsAnalysisAnalysis will be done using SPSS IBM Corp ArmonkNew York USA Continuous variables will be summarized with standard descriptive statistics including meanstandard deviation median and range Categorical variables will be summarized with frequencies A pvalueless than is considered statistically significantFor the analysis of the diagnostic accuracy sensitivityand specificity a — cross tabulation will be madecomparing MRI and CECT to histopathology and followup Performance of CECT and Gdenhanced MRI withDWI will be compared using McNemar™s test We willreport the changes made in patient management in a descriptive manner Median and 1year survival will be reported Survival endpoints disease free survival andoverall survival will be analyzed using KaplanMeierplots Survival curves are compared using the log ranktest We will compare the results of both readers to determine the interobserver variability A Cohen™s Kappak value of “ is interpreted as excellent “ substantial agreement “ moderate agreement “fair agreement and “ pooragreementWe partly anticipated missing data by introducing thecomposite reference standard of follow up Unfortunatelymissing data still can occur when for instance a patientsuspected of having metastatic disease does not have histopathological confirmation and dies before the compositereference standard follow up could take place If necessaryadditional analysis will be performed to determine the robustness of the results and to deal with missing dataTrial statusThe first patient was included on December 21st Atthe time of protocol submission July 23th active inclusion of patients has started in six centers RadboudUniversity Medical Center Nijmegen the NetherlandsKonstantopouleio General HospitalAthens GreeceMedisch Spectrum Twente Enschede The Netherlandsand Jeroen Bosch Hospital Den Bosch The NetherlandsUniversity Medical Center Groningen Groningen TheNetherlands and University Hospital Ram³n y CajalMadrid Spain and a total of patients have been included Four centers are preparing to start with inclusionInselspital Universit¤tsspital Bern Bern SwitzerlandUCHealth University of Colorado HospitalDenverUnited States of America Azienda Ospedaliera Universitaria Integrata Verona Verona Italy and Policlinico AGemelli Rome Italy Inclusion of patients is expected tobe finished December accuracy ofDiscussionThe purpose of the DIAPANC trial is to investigate thediagnosticcontrastenhanced diffusionweighted MRI in patients with suspected PDAC for thedetection of liver metastases Additionally we will evaluatewhether performing contrastenhanced diffusionweightedMRI will improve the detection of liver metastases compared to CECT by determining the sensitivity and specificity of CECT for the detection of liver metastasesDespite the good diagnostic performance of MRI forliver metastases the benefits of MRI remain unclearmostly because of low level of evidence heterogeneityand bias in the performed studies Two recently published metaanalyses have suggested the results shouldbe confirmed by performing a welldesigned and sufficiently powered study directly comparing liver CT andMRI in the same cohort [ ]A major difficulty in the interpretation of the currentliterature is that most studies are retrospective oftenonly reporting on a subset of patients actually undergoing a resection patients with borderline resectable tumors or patients with indeterminate liver lesions onCECT These patients have a higher probability of 0cLitjens BMC Cancer Page of having liver metastases However in an era of neoadjuvant therapy local ablative therapy for advanced tumorsexpensive targeted therapies and resection of oligometastases MRI may be beneficial to patients with allstages of PDAC Therefore all patients with suspectedPDAC are eligible for inclusion in the DIAPANCMRI field strength T versus T was a significant factor in the heterogeneity between studies that was found ina metaanalysis T MRI had a higher sensitivity anda lower specificity for diagnosing liver metastasiscompared to T MRI sensitivity and specificity[] Because the signaltonoise ratio and thelesiontoliver contrast are higher on T MRI than on T MRI it is reasonable that a T MRI permits a higher lesion detection rate [ ] In the DIAPANC study weplan to perform all MRIs on a T scanner A potentialdownside of a multicenter design is the intervendor variability that could occur when comparing the quantitativeApparent Diffusion Coefficient ADC value this variabilityseems to be more pronounced at T than at T []Availability of MRI is not expected to be an issue asMRI is available in every expert center for pancreaticdiseases However problems with MRI capacity couldarise due to the need for MRI within a short intervalafter CT A time interval of two weeks was chosen toprovide a feasible time frame for MRI to be performedand no intervallesions are expected within this timeinterval []The DIAPANC trial is the first international prospective multicenter cohort study about the diagnostic accuracyof contrastenhanced diffusionweighted MRI On theWorld Health anization trial registry website ICTRPincorporating all inter national trial registries there areonly four other prospective trials registered in this fieldThe first trialis a completed French prospectivemulticenter trial presumably the only one prospective study that has been published [] The studyhas been performed in patients with potentiallyresectable pancreatic cancer on a T scanner usinggadobenate dimeglumine MultiHance as contrastagent The study has been performed to assess thediagnostic performance of diffusionweighted MRIfor the preoperative diagnosis of liver metastasis andthe modification of therapeutic strategy as a consequence ofliver metastasis ondiffusionweighted MRI []the diagnosis ofThe second trial is a British single center observational study with a target sample size of patientswith confirmed or suspected pancreatic cancer referred for pancreaticoduodenectomy and is completed recently The primary outcome of this studyis the proportion of patients correctly identified byMRI to have lymph node peritoneal or liver metastases To our knowledge the results have not beenpublished and there is no information on scan parameters and contrast agent available []The third trial from Australia is the only randomizedcontrolled trial The study has a target sample size of patients and is not yet recruiting The aim of the studyis to compare the 12month recurrence rate in patientswith locally operable pancreatic adenocarcinoma managed with standard preoperative assessment of liver metastases with CECT versus preoperative assessment withliver specific contrast MRI []The fourth trial is a Chinese comparative study and isnot yet recruiting The study aims to compare liver specific contrast MRI and CECT in liver metastasis of pancreatic cancer with a target sample size of patients []The DIAPANC trial hypothesizes a superior value ofMRI for the detection of liver metastases compared toCECT To reliably determine the diagnostic accuracy thegold standard is histopathology of the liver lesions Considering it is not always possible and sometimes even unethical to obtain histopathological proof of every lesionfollowup is used as a reference standard Hence we areable to simultaneously gather information on early localrecurrence or metastases after resection disease progression and therapy response evaluation on MRI and CECTIn conclusion the DIAPANC trial is expected to report highlevel evidence of the diagnostic accuracy ofMRI for the detection of liver metastases compared toCECT resulting in significant value for clinical decisionmaking guideline development and improved stratification for treatment strategies and future trials Furthermore DIAPANC will contribute to our knowledge ofliver metastases regarding incidence imaging characteristics their number and extent and their change in timewith or without treatment When our hypothesis is confirmed it will enhance the worldwide implementation ofMRI and consequently improve personalized treatmentof patients suspected of PDACAbbreviationsADC Apparent Diffusion Coefficient AJCC American Joint Committee onCancer Castor EDC Castor Electronic Data Capture CEA CarcinoembryonicAntigen CECT Contrast Enhanced Computed Tomography CA19“ Carbohydrate Antigen “ DIAPANC Diagnostic accuracy of contrastenhanced diffusionweighted MRI for liver metastases of pancreatic cancerDWI DiffusionWeighted Imaging EORTC European anization forResearch and Treatment EUS Endoscopic Ultrasound FNA Fine NeedleAspiration FNB Fine Needle Biopsy Gd Gadolinium ICTRP InternationalClinical Trials Registry Platform MRCP Magnetic Resonance CholangioPancreatography MRI Magnetic Resonance Imaging PDAC Pancreatic ductaladenocarcinoma SPSS Statistical Package for the Social SciencesTNM Tumor Node Metastasis WHO World Health anization QLQC30 Quality of life questionnaire including questions QLQPAN26 Pancreatic cancer module of quality of life questionnaire including questionsAcknowledgementsWe acknowledge all patients who participated and will participate in thestudy Secondly we acknowledge all participating institutions conduct ofthe study would be impossible without contribution of these institutions 0cLitjens BMC Cancer Page of Authors™ contributionsGL and DR drafted the manuscript of the protocol JH is principalinvestigator of the study and participated in the design of the study MP isthe study sponsor and participated in the design of the study GL DR EGSR LB and CL participated in the design of the study GL primarilycoordinates the study All authors critically reviewed the manuscript andapproved the final manuscript Publications of the study results will be inaccordance with international recognized scientific and ethical standardsconcerning publications and authorship including the UniformRequirements for Manuscripts Submitted to Biomedical Journals establishedby the International Committee of Medical Journal EditorsFundingThe Dutch Cancer Society KWF reviewed and financially funded the DIAPANC study Research Project grant reference number They do notinfluence the data collection interpretation of data the manuscript or thedecision to publishAvailability of data and materialsThe complete dataset will be property of the Sponsor all participatinginstitutions will own the dataset of the included patients from their centerPublic access to the full trial protocol trialrelated documents participantlevel dataset and statistical code may be made available on requestEthics approval and consent to participateThe DIAPANC study will be conducted according to the principles of theDeclaration of Helsinki 64th version October and in accordance withthe Medical Research Involving Human Subjects Act WMO The independent ethics review board region ArnhemNijmegen Nijmegen TheNetherlands has approved the trial protocol NL6047309117 Furthermoresecondary approval for all participating centers from The Netherlands was orwill be individually obtained from all local ethics committees According toDutch law ethical approval by the ethics review board of the study sponsorie initiating center Radboudumc Nijmegen The Netherlands is appropriatefor all Dutch centers For all participating centers outside of The Netherlandsapproval from a local independent ethics review board was or will be obtained The trial is registered in the registry provided by the US National Library of Medicine clinicaltrialsgov with identification number NCT03469726Patients can only participate if written informed consent has been providedProtocol modifications will be communicated to all relevant parties egparticipating centers funder after approval of the ethical committee and willbe updated in the trial registry Possible substudies like Biobank sampleswill be stored at the Radboud Biobank or artificial intelligence analysis areincluded on the informed consent form Patients must give separate consentto participate in these substudies The study will be monitored according tothe guidelines of The Netherlands Federation of University Medical CentresNFU and adverse events related to study procedures will be recordedThere is a study subject insurance for patients that suffer harm from trialparticipationConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Radiology and Nuclear Medicine Radboudumc NijmegenThe Netherlands 2Department of Gastroenterology and HepatologyRadboudumc Nijmegen The Netherlands 3Department of MedicalOncology Radboudumc Nijmegen The Netherlands 4Department ofPathology Radboudumc Nijmegen The Netherlands 5Department ofPathology University Medical Center Utrecht The Netherlands 6Departmentof Surgery Radboudumc Nijmegen The NetherlandsReceived June Accepted July ReferencesSiegel R Ma J Zou Z Jemal A Cancer statistics CA Cancer J Clin“Rahib L Smith BD Aizenberg R Rosenzweig AB Fleshman JM Matrisian LMProjecting Cancer incidence and deaths to the unexpected burden ofthyroid liver and pancreas cancers in the United States Cancer Res “ Willett CG Czito BG Bendell JC Ryan DP Locally advanced pancreaticcancer J Clin Oncol “Raman SP Reddy S Weiss MJ Manos LL Cameron JL Zheng L Impactof the time interval between MDCT imaging and surgery on the accuracyof identifying metastatic disease in patients with pancreatic cancer AJR AmJ Roentgenol 20152041W37“Glant JA Waters JA House MG Zyromski NJ Nakeeb A Pitt HA Doesthe interval from imaging to operation affect the rate of unanticipatedmetastasis encountered during operation for pancreatic adenocarcinomaSurgery “Allen VB Gurusamy KS Takwoingi Y Kalia A Davidson BR Diagnosticaccuracy of laparoscopy following computed tomography CT scanning forassessing the resectability with curative intent in pancreatic andperiampullary cancer Cochrane Database Syst Rev 20167CD009323Van den Broeck A Sergeant G Ectors N Van Steenbergen W Aerts R TopalB Patterns of recurrence after curative resection of pancreatic ductaladenocarcinoma European J Surg Oncol “Haeno H Gonen M Davis MB Herman JM IacobuzioDonahue CA Michor FComputational modeling of pancreatic cancer reveals kinetics of metastasissuggesting optimum treatment strategies Cell ““Holzapfel K ReiserErkan C Fingerle AA Erkan M Eiber MJ Rummeny EJ Comparison of diffusionweighted MR imaging and multidetectorrowCT in the detection of liver metastases in patients operated for pancreaticcancer Abdom Imaging “ Balci NC Semelka RC Radiologic diagnosis and staging of pancreatic ductaladenocarcinoma Eur J Radiol “ Paik KY Choi SH Heo JS Choi DW Analysis of liver metastasis afterresection for pancreatic ductal adenocarcinoma World J GastrointestinalOncol “ Motosugi U Ichikawa T Morisaka H Sou H Muhi A Kimura K et alDetection of pancreatic carcinoma and liver metastases with gadoxeticacidenhanced MR imaging comparison with contrastenhanced multidetector row CT Radiology “Schima W BaSsalamah A Kolblinger C KulinnaCosentini C Puespoek AGotzinger P Pancreatic adenocarcinoma Eur Radiol “ Danet IM Semelka RC Nagase LL Woosely JT Leonardou P Armao D Livermetastases from pancreatic adenocarcinoma MR imaging characteristics JMagnetic Resonance Imaging “Kneuertz PJ Cunningham SC Cameron JL Torrez S Tapazoglou N HermanJM Palliative surgical Management of Patients with Unresectablepancreatic adenocarcinoma trends and lessons learned from a large SingleInstitution Experience J Gastrointest Surg “Eiber M Fingerle AA Brugel M Gaa J Rummeny EJ Holzapfel K Detectionand classification of focal liver lesions in patients with colorectal cancerretrospective comparison of diffusionweighted MR imaging and multisliceCT Eur J Radiol “Lowenthal D Zeile M Lim WY Wybranski C Fischbach F Wieners G et alDetection and characterisation of focal liver lesions in colorectal carcinomapatients comparison of diffusionweighted and GdEOBDTPA enhancedMR imaging Eur Radiol “ Holzapfel K Bruegel M Eiber M Ganter C Schuster T Heinrich P et alCharacterization of small mm focal liver lesions value of respira

Thyroid_Cancer

coronary arterybypass grafting thrombosis fibrin fibrinogen mutationIntroduction Intraoperative thrombosis of saphenous veins SV during harvesting is very rareCase Report We present a case of a 60yearold male patient with multivesselcoronary artery disease and a history of a nonST elevation acute coronary syndromeand type2 diabetes mellitus admitted for coronary artery bypass grafting in whombilateralintraoperative SV thrombosis occurred during graft harvesting Routinethrombophilia screening showed no abnormalities and cancer was excluded Compared with healthy controls we observed prolonged fibrin clot lysis time and increasedthrombin generation reflected by endogenous thrombin potential Scanning electronmicroscopy of the thrombosed material revealed compact and thick fibrin layer on theclot surface with a solid mass of unusually compressed platelets and erythrocytesunderneath The patient was tested for fibrinogen and factor F XIII polymorphismsand was found to be heterozygous for fibrinogen HaeIII 455G A and FXIIIVal34Leu 100G TConclusion fibrinogen HaeIII and FXIII Val34Leu polymorphisms are reflected inreduced clot permeability and susceptibility to lysis and might contribute to intraoperative SV thrombosis during vascular grafting procedures Carriers of those are atrisk of primary venous graft failure after bypass proceduresIntroductionCoronary artery bypass grafting CABG is a method of choicefor revascularization in patients with multivessel disease anddiabetes mellitus DM Although arterial grafts are preferredin selected scenarios the common practice is to use leftinternal thoracic artery LITA to bypass the left anteriordescending artery LAD and to place venous conduits toother target vessels An often chosen vascular graft the greatsaphenous vein SV offers decent durability and is easy toharvest SV graft occlusion may occur in up to of caseswithin the first months and as many as may occludewithin first to weeks1 SV harvesting dramaticallychanges the vein™s environment with disruption of bloodflow in vasa vasorum damage to the adventitia hypoxia andhyponutrition of the vessel wall along with focal endothelialdisruption2 Acute SV graft failure is usually a result of graftthrombosis which among other factors like technical failuregrafttarget vessel disproportion etc may be caused byhypercoagulabilityreceivedMarch acceptedJuly 101055s00401715657ISSN Ge Thieme Verlag KGStuttgart · New York 0ce198Bilateral Saphenous Vein Thrombosis during CABG Mazur et alCase ReportA 60yearold male patient with multivessel coronary arterydisease who suffered from a nonST elevation acute coronarysyndrome NSTEACS month prior to admission a nonsmoker with type2 DM on metformin peptic ulcer diseaseand a history of alcohol abuse was admitted to our institutionfor CABG Just after the NSTEACS a left ventricle LV thrombus was seen on one echocardiographic examination but itwas absent during followup There was no deep venousthrombosis or bleeding diathesis history On admission thepatient was on aspirin mg once daily and enoxaparin mg once daily Routine laboratory tests were withinnormal ranges –ºTable There were no abnormalities onphysical examination apart from obesity body mass index kgm2 when the patient was admitted The lower extremities appeared normal There were no varicose veins nosigns or symptoms of venous insufficiency and the pastmedical history was negative for both personal and familyhistory of chronic venous insufficiency or varicose veins Thepatient was operated on following the standard proceduresDuring LITA harvest a cardiac surgery resident harvested theright SV using the technique The wall of the SV lookedgrossly normal Upon dissection the side branches were tiedoff and clipped and a needle was placed at the distal end whilethe proximal end was still not separated An attempt was madeTable Results of initial and followup laboratory testingVariableCoagulation testsRed blood count 103µLHemoglobin gdLWhite blood count 103µLPlatelet count 103µLAPTT sPT sPT INRPlatelet aggregation mmolL arachidonic acid µmolL ADP Thrombophilia screeningFibrinogen gLAntithrombin III Ddimer µgLantiXa IUmLHomocysteine µmolLProtein C Protein S Factor VIII Leiden c1601G A 0397G AProthrombin cfibrinogen 455G AFactor XIII 100G TLupus anticoagulant ratioLupus anticoagulant ratio APTTAnticardiolipin IgGAnticardiolipin IgMAnti2glycoprotein I IgG antibodyAnti2glycoprotein I IgM antibodyNormal rangesPreoperativePostoperative day Postoperative day “““““““““““““““““““““““““““““““““““““““““““ GG no mutationGG no mutation““ GPL MPL SGU SMU““““““““““““GG no mutationGG no mutationGA heterozygoteGT heterozygote GPL MPL SGU SMUAbbreviations APTT activated partial thromboplastin time GPL IgG phospholipid unit Ig immunoglobulin INR international normalized ratioMPL IgM phospholipid unit PT prothrombin time SGU standard IgG 2 glycoprotein unit SMU standard IgM 2 glycoprotein unitTH Vol No 0cBilateral Saphenous Vein Thrombosis during CABG Mazur et ale199to flush the vein with a solution containing blood mLheparin IU and normal saline mL while the distalend was closed with an atraumatic vascular clamp and veinthrombosis was noted Upon the separation of the distal end aluminal thrombus was visible The left SV was then taken downusing the same protocol by an experienced staff cardiacsurgeon with the same result Presence of a luminal thrombuswas confirmed upon separation of the proximal end Systemicheparin was administered and normal LITA outflow wasconfirmed Concerns regarding safety of cardiopulmonarybypass use were raised due to suspected thrombotic issueand the approach was modified The LITA“LAD anastomosiswas completed offpump on a beating heartThe postoperative course was uneventful On postoperative day the patient received dual antiplatelet therapy withaspirin and clopidogrel and was discharged on day with nosigns of thrombosis or myocardial ischemia Elective angioplasty of nongrafted vessels was scheduled and a completethrombophilia screening was done –ºTable On the and12month followup the patient did wellDiagnostic ApproachBecause a thrombophilia was suspected screening wasinitiated showing no abnormalities –ºTable Cancer was excluded as a cause of thrombosis Positiveantibodies against neutrophil cytoplasm antigens pANCAand cANCA were excluded as a cause of vasculitis We thenproceeded to analyze fibrin phenotype using the previouslydescribed methodology34 Briefly plasma fibrin clot permeability was determined in a hydrostatic pressure systemTubes containing fibrin clots formed from adding mmolLcalcium chloride and UmL human thrombin Sigma tocitrated plasma were connected through plastic tubing to abuffer reservoir M TrisHCl M NaCl pH Thevolume flowing through the gel was measured within minutes A permeation coefficient Ks reflecting poresize was calculated from equation Ks ¼ Q 02 L ηt 02 A 02 Δpwhere Q is the flow rate in time t L is the length of a fibrin gelη is the viscosity of liquid A is the cross section area and Δp isa differential pressure in dynecm2 Lower Ks values indicated reduced permeability Fibrinogen was determined usingthe Clauss method Even though the followup fibrinogenlevel was normal we identified strongly decreased fibrin clotpermeability Ks ¼ 06 02 9cm2 compared withhealthy controls from our previous report n ¼ Ks ¼ 9cm23 samples collected during late follow[“] up appointment on postoperative day Compared withhealthy controls n ¼ we observed prolonged clot lysistime CLT 06 vs 06 minutes and increasedthrombin generation reflected by endogenous thrombinpotential ETP in the studied subject ETP ¼ 06 vs 06 nM 02 min respectively measurement ofthe thrombin generation was done with calibrated automated thrombography thrombinoscope BV Maastricht theNetherlands according to the manufacturer™s instructionsin the 96well plate fluorometer Ascent Reader Thermolabsystems OY Helsinki Finland equipped with the filter set at a temperature of °C Briefly microliters of plateletpoor plasma were diluted with µL of the reagent containing pmolL recombinant tissuefactor micromolar phosphatidylserinephosphatidylcholinephosphatidylethanolamine vesicle and µL of FluCasolution Hepes pH nmolL CaCl2 mgmL bovinealbumin and mmolL ZGlyGlyArg7amino4methylcoumarin Each plasma sample was analyzed in duplicateFor analysis the maximum concentration of thrombin generated was used3Cryosectioned tissue sections were fixed in icecold methanol“acetone mixture peroxidase activity was quenchedwith H2O2 and unspecific background was blocked with bovine albumin BSA Sigma Co St Louis Missouri UnitedStates Primary adequate antibodies against fibrin or tissuefactor TF were applied both Abcam Cambridge UnitedKingdom Primary antibodies were followed by thecorresponding secondary antibodies conjugated with fluorochrome Abcam as previously described5 Images were analyzed using Olympus BX microscope SVs immunostainingrevealed thick layer of fibrin directly on the vessel endothelium–ºFig 1A and high TF –ºFig 1B activity Within the thrombuswe found abundant blood nuclear cells nuclei stained usingDAPI suggesting the presence of proinflammatory monocyteswhich are known source of TF Unfortunately we were not ableto immunostain CD68 due to high unspecific backgroundresulting from large amounts of fibrin The microscopic analysisshowed abundant adventitial vessels –ºFig 1C D Withinalmost every single vessel we found thrombi rich in bothprothrombin –ºFig 1C and TF –ºFig 1DProthrombotic fibrin clot phenotype reflected by reducedKs and prolonged CLT along with enhanced thrombin generation and unusualimages obtained from the immunostaining of the SVs prompted us to perform analysis ofwhole blood clot morphology using scanning electron microscopy SEM as previously described6 After washing thethrombus was fixed with glutaraldehyde phosphatebuffered saline solution Specimens were dehydrated goldcoated and photographed digitally with a JEOL JSM JEOL Tokyo Japan The analysis revealed compact and thickfibrin layer on the clot surface with a solid massof unusually compressed platelets and erythrocytes underneath This observation suggested veryhigh contractileforces during clot formation in a plateletdriven fibrinmediated mechanism of clot contraction and prompted usto study common fibrinogen and factor F XIII polymorphisms The patient was heterozygous for fibrinogen HaeIII455G A and FXIII Val34Leu 100G TDiscussionA dramatic intraoperative SV thrombosis provoked by graftharvesting for CABG lead to change in revascularizationstrategy but its cause remained unknown following thestandard thrombophilia screening The cases of acute SVgraft thrombosis in the perioperative period are very rareand as few as of grafts occlude within first to weeks17TH Vol No 0ce200Bilateral Saphenous Vein Thrombosis during CABG Mazur et alFig Representative images of SV graft immunostaining after massive thrombosis A“D prothrombin stained red TF stained green nucleistained blue using DAPI and scanning electron microscopic images E F of the surface of whole blood clot formed in vitro from citrated bloodobtained from the patient undergoing CABG Box and arrow represent magnification of the fragment in the box Arrows show pertinent stainedfragments see text CABG Coronary artery bypass grafting SV saphenous veins TF tissue factorA normal SV is composed of the intima the media and theadventitia8 The intima is built of the layer of endothelial cellson the luminal side the media consists of smooth musclecells and the adventitia forms the outer part8 In a normalsetting the endothelium is crucial for vein integrity andprevention of thrombosis9 and its focal disruption maypredispose to vessel thrombosis2 SV manipulation andimplantation leads to loss of endothelial integrity and elicitsan inflammatory response with platelet adhesion and leukocyte recruitment Notwithstanding an overt thrombosis isextremely rare in the operating room SV dissection results inblood flow disruption in vasa vasorum and causes adventitial damage hypoxia and vessel wall hyponutrition10 Acuteperioperative saphenous vein graft failure is almost always aresult of graft thrombosis but this very uncommonly occursprior to graft placement Surgical factorslike technicalanastomotic failure or severe disproportion between thetarget vessel and the graft may lead to thrombosis butvessel injury and hypercoagulability are among potentialcauses as well11There was no evident inflammatory process in microscopy inour patient but even if an inflammatory process was presentTH Vol No 0cBilateral Saphenous Vein Thrombosis during CABG Mazur et ale201preoperatively in our patient™s SVs the inflammatory background alone could not explain the dramatic intraoperativethrombosis We hypothesized that increased thrombin generation and prothrombotic fibrin clot phenotype were responsiblefor the clinical presentation Conversion of fibrinogen to fibrinfacilitated by thrombin is a concluding step of coagulation Ithas been shown that fibrin clots with small pores betweentightly packed thin fibrin fibers are relatively lysis resistant12Such clot phenotype has been evidenced in multiple thromboticpathologies such as myocardial infarction6 ischemic stroke13and venous thromboembolism4 The prothrombotic clotphenotype reflected by a tendency to form dense fibrin clotsresistant to lysis has been previously reported in patients withinstent thrombosis14 While routine thrombophilia screeningresults in a high almost detection rate of commonhypercoagulable states15 there are prothrombotic conditionsthat escape routine diagnostic approach The overall microscopic clot appearance and prothrombotic fibrin properties lead tothe discovery of two mutations in our patient that are notroutinely tested during thrombophilia screening namely fibrinogen 455G A and FXIII100G TElevated fibrinogen was postulated as one of the riskfactors for early graft failure after CABG1116 Epidemiologicalstudies have established that elevated fibrinogen is stronglyassociated with cardiovascular diseases17 A metaanalysis of individual records of participants from prospective studies revealed that age and sexadjustedhazard ratio per gL increase in usual fibrinogen level forcoronary heart disease was confidence interval [CI]“ while for stroke the hazard ratio was as high as 95CI “ Risk of coronary disease progression wasalso linked to genetic polymorphisms of the fibrinogen geneDe Maat et al found that A allele of fibrinogen 455G Awas associated with more severe progression of coronarydisease as documented angiographically18 Gu and colleagues in a metaanalysis of studies with patientsfound that A allele of the fibrinogen 455G A is associated with susceptibility to coronary disease and also withischemic stroke odds ratio for stroke ¼ [ CI “]for AA þ GA vs GG19 In a recent study of patients with atrialfibrillation Hu and colleagues found that the A allele of fibrinogen 455G A was a risk factor for cardioembolicstroke probably by elevating the level of plasma fibrinogen20 On the other hand in a metaanalysis of studies involving cases and controls FXIIIVal34Leu polymorphism was shown to be associated withrisk myocardial infarction21 FXIII is crucial to thrombusstabilization and changes of its plasma concentration reflectnonspecifically the extent of thrombosis as shown by Li et alin a study on patients with cerebral venous thrombosis22Interesting associations of FXIII Val34Leu polymorphism andthrombotic disorders have been reported Jung et al reportedin a metaanalysis of studies that FXIII Val34Leu polymorphism is associated with recurrent pregnancy loss23Although no association with the incidence of ischemicstroke was found for this polymorphism24 apparentlywhen the stroke occurs Val34Leu polymorphism of FXIIIaffects the severity of its outcome25 Furthermore Kreutzand colleagues suggested in that FXIII Val34Leu polymorphism may increase risk of recurrent MI and death inpatients with angiographically established coronary arterydisease26 In our group has shown in a study of patients that in patients undergoing CABG FXIII Leu34 alleleis associated with decreased fibrin clot permeability andefficiency of fibrinolysis27ConclusionOur extensive workup showed that fibrinogen HaeIII andFXIII Val34Leu polymorphisms are reflected in reduced clotpermeability and susceptibility to lysis These mutationslikely contributed to intraoperative saphenous graft thrombosis Further studies are needed to elucidate the role ofthese polymorphisms in early graft failure after bypassgrafting procedures however their contributory role seemsevidentFundingThis study was funded by a grant from the JagiellonianUniversity Medical College no KZDS007961 to PMConflict of InterestNone declaredReferences Bourassa MG Fate of venous grafts the past the present and thefuture J Am Coll Cardiol “ Roubos N Rosenfeldt FL Richards SM Conyers RA Davis BBImproved preservation of saphenous vein grafts by the use ofglyceryl trinitrateverapamil solution during harvesting Circulation 19959209II31“II36 Mazur P Sokołowski G HubalewskaDydejczyk A PłaczkiewiczJankowska E Undas A Prothrombotic alterations in plasma fibrinclot properties in thyroid disorders and their posttreatmentmodifications Thromb Res “ Undas A Zawilska K CieslaDul M et al Altered fibrin clotstructurefunction in patients with idiopathic venous thromboembolism and in theirrelatives Blood “ Natorska J Marek G Hlawaty M Sadowski J Tracz W Undas AFibrin presence within aortic valves in patients with aorticstenosis association with in vivo thrombin generation and fibrinclot properties Thromb Haemost “ Undas A Szułdrzynski K Stepien E et al Reduced clot permeability and susceptibility to lysis in patients with acute coronarysyndrome effects of inflammation and oxidative stress Atherosclerosis “ Fitzgibbon GM Kafka HP Leach AJ Keon WJ Hooper GD BurtonJR Coronary bypass graft fate and patient outcome angiographicfollowup of grafts related to survival and reoperation in patients during years J Am Coll Cardiol “ Kim FY Marhefka G Ruggiero NJ Adams S Whellan DJ Saphenous vein graft disease review of pathophysiology preventionand treatment Cardiol Rev “ Allaire E Clowes AW Endothelial cell injury in cardiovascularsurgery the intimal hyperplastic response Ann Thorac Surg“ McGeachie JK Meagher S Prendergast FJ Veintoartery graftsthe longterm development of neointimal hyperplasia and itsTH Vol No 0ce202Bilateral Saphenous Vein Thrombosis during CABG Mazur et alrelationship to vasa vasorum and sympathetic innervation Aust NZ J Surg “ Harskamp RE Lopes RD Baisden CE de Winter RJ Alexander JHSaphenous vein graft failure after coronary artery bypass surgerypathophysiology management and future directions Ann Surg“ Undas A Fibrin clot properties and their modulation in thrombotic disorders Thromb Haemost “ Undas A Podolec P Zawilska K et al Altered fibrin clotstructurefunction in patients with cryptogenic ischemic strokeStroke “ Undas A Zalewski J Krochin M et al Altered plasma fibrin clotproperties are associated with instent thrombosis ArteriosclerThromb Vasc Biol “ GoldmanMazur S Wypasek E Karpiński M Stanisz A Undas AHigh detection rates of antithrombin deficiency and antiphospholipid syndrome in outpatients aged over years using thestandardized protocol for thrombophilia screening Thromb Res“ Moor E Hamsten A Blombäck M Herzfeld I Wiman B Rydén LHaemostatic factors and inhibitors and coronary artery bypassgrafting preoperative alterations and relations to graft occlusionThromb Haemost “ Danesh J Collins R Appleby P Peto R Association of fibrinogen Creactive protein albumin or leukocyte count with coronary heartdisease metaanalyses of prospective studies JAMA “ de Maat MP Kastelein JJ Jukema JW et al 455GA polymorphismof the betafibrinogen gene is associated with the progression ofcoronary atherosclerosis in symptomatic men proposed role foran acutephase reaction pattern of fibrinogen REGRESS groupArterioscler Thromb Vasc Biol “ Gu L Liu W Yan Y et al Influence of the fibrinogen455GApolymorphism on development of ischemic stroke and coronaryheart disease Thromb Res “ Hu X Wang J Li Y et al The fibrinogen gene 455GA polymorphism associated with cardioembolic stroke in atrial fibrillationwith low CHA2DS2VaSc score Sci Rep Jung JH Song GG Kim JH Seo YH Choi SJ Association of factor XIIIVal34Leu polymorphism and coronary artery disease a metaanalysis Cardiol J “ Li B Heldner MR Arnold M et al Coagulation Factor XIIIin Cerebral Venous Thrombosis TH e227“e229 Jung JH Kim JH Song GG Choi SJ Association of the F13A1Val34Leu polymorphism and recurrent pregnancy loss a metaanalysis Eur J Obstet Gynecol Reprod Biol “ Shemirani AH Pongrácz E Antalfi B Adány R Muszbek L FactorXIII A subunit Val34Leu polymorphism in patients sufferingatherothrombotic ischemic stroke Thromb Res “ Shemirani AH Antalfi B Pongrácz E Mezei ZA Bereczky Z Csiki ZFactor XIIIA subunit Val34Leu polymorphism in fatal atherothrombotic ischemic stroke Blood Coagul Fibrinolysis “ Kreutz RP Bitar A Owens J et al Factor XIII Val34Leu polymorphism and recurrent myocardialinfarction in patients withcoronary artery disease J Thromb Thrombolysis “ Stepień E Plicner D Kapelak B Wypasek E Sadowski J UndasA Factor XIII Val34Leu polymorphism as a modulator of fibrinclot permeability and resistance to lysis in patients withsevere coronary artery disease Kardiol Pol 2009678A“TH Vol No 0c'

Thyroid_Cancer

Comparison of different calculationtechniques for absorbed dose assessment inpatient specific peptide receptor radionuclidetherapyDomenico Finocchiaro12 Salvatore Berenato3 Valentina Bertolini1 Gastone Castellani2Nico Lanconelli2 Annibale Versari4 Emiliano Spezi35 Mauro Iori1 Federica FioroniID1Elisa Grassi1 Azienda Unit  Sanitaria Locale di Reggio Emilia”IRCCS Medical Physics Unit Reggio Emilia Italy Department of Physics and Astronomy University of Bologna Bologna Italy Department of MedicalPhysics Velindre Cancer Centre Cardiff United Kingdom Azienda Unit  Sanitaria Locale di Reggio Emilia”IRCCS Nuclear Medicine Unit Reggio Emilia Italy School of Engineering Cardiff University CardiffUnited Kingdom federicafioroniauslreita1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSAbstractCitation Finocchiaro D Berenato S Bertolini VCastellani G Lanconelli N Versari A Comparison of different calculation techniques forabsorbed dose assessment in patient specificpeptide receptor radionuclide therapy e0236466 101371journalpone0236466Editor Choonsik Lee National Institute of HealthUNITED STATESReceived July Accepted July Published August Copyright Finocchiaro This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscriptFunding This work was supported by theEuropean Metrology Programme For InnovationAnd Research EMPIR joint research project15HLT06 Metrology for clinical implementation ofdosimetry in molecular radiotherapyMRTDosimetry which has received funding fromthe European Union The EMPIR initiative is cofunded by the European Union™s Horizon AimThe present work concerns the comparison of the performances of three systems for dosimetry in RPT that use different techniques for absorbed dose calculation anlevel dosimetry voxellevel dose kernel convolution and Monte Carlo simulations The aim was toassess the importance of the choice of the most adequate calculation modality providingrecommendations about the choice of the computation toolMethodsThe performances were evaluated both on phantoms and patients in a multilevel approachDifferent phantoms filled with a 177Luradioactive solution were used a homogeneous cylindrical phantom a phantom with anshaped inserts and two cylindrical phantoms withinserts different for shape and volume A total of patients with NETs treated by PRRTwith 177LuDOTATOC were retrospectively analysedResultsThe comparisons were performed mainly between the mean values of the absorbed dose inthe regions of interest A general better agreement was obtained between Dose kernel convolution and Monte Carlo simulations results rather than between either of these two andanlevel dosimetry both for phantoms and patients Phantoms measurements alsoshowed the discrepancies mainly depend on the geometry of the inserts eg shape and volume For patients differences were more pronounced than phantoms and higher interintrapatient variability was observedPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTresearch and innovation programme and theEMPIR Participating States SB acknowledgesfunding from Cancer Research Wales throughgrant No Competing interests The authors have declaredthat no competing interests existConclusionThis study suggests that voxellevel techniques for dosimetry calculation are potentiallymore accurate and personalized than anlevel methods In particular a voxelconvolution method provides good results in a short time of calculation while Monte Carlo basedcomputation should be conducted with very fast calculation systems for a possible use inclinics despite its intrinsic higher accuracy Attention to the calculation modality is recommended in case of clinical regions of interest with irregular shape and far from sphericalgeometry in which Monte Carlo seems to be more accurate than voxelconvolutionmethodsIntroductionRadiopharmaceutical therapy RPT as defined in ICRP [] is based on the use of specificpharmaceuticals labelled with radionuclides to deliver a lethal dose of radiation to tumourareas Radiopharmaceuticals are specifically designed to have high affinity with given tumoursites so that ionizing radiations such as ps and photons emitted by the isotopes maydeposit energy inside or close to unhealthy tissues saving surrounding healthy tissues Thisapproach produced very encouraging results in the treatment of neuroendocrine tumoursNET in the last decades in particular in therapies which make use of somatostatin analogueslabelled with 90Y or 177Lu [] such as the recently registered Lutathera [] Different responserates and a large interpatient variability of the outcome were however reported by someauthors eg Campana D and Vinjamuri S [ ]The wellestablished experience with external beam radiation therapy EBRT has providedstrong evidence that tumour response and normal an toxicity is related to absorbed dosesFor this reason it was supposed that the treatment outcome correlates with the absorbed dosedelivered to tumours even in RPT [ ] Yet a dosimetry as more accurate and personalized aspossible is needed to this purpose to provide clinicians with reliable resultsDespite the general demand for a more individualized treatment based on pretherapeuticdosimetry study in NET dosimetry is not conducted always in the clinical routine This ismostly because dosimetry is often considered time consuming a lot of time required for imaging expensive costs for every image scan and every measurement and sometimes inaccuratefor the lack of standardization and harmonization mainly At present a standard procedurefor calculating the absorbed dose is not well defined for every kind of radionuclide therapy Inrelation to NET the evidence of prolonged survival has been demonstrated only recently [] ina subgroup of NETDifferent methods have been developed to perform dosimetry since its beginnings Techniques based on standardized reference models were first developed thanks to their simplicityof implementation and have been used for many years These models assume uniform activityie homogeneous uptake in the source regions However evidence indicates that deterministic biological effects including tumour response and normal tissue toxicity may not be wellpredicted by the mean absorbed dose in the region and may be significantly influenced bynonuniform doses [] To take into account this aspect voxelbased techniques were considered similarly to those which have been also used for decades as standard of care in EBRT [] Contrary to what happens for EBRT however in which plenty of software for therapyplanning are available on the market in RPT only few systems which are adequate toPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTdosimetry for Peptide Receptor Radionuclide Therapy PRRT and which can work with multiple 3D imaging have been officially released in the last few years [“] For this reasonmany dosimetry software and tools are in use worldwide but only some of them are commercially available Several of them are homemade tools which were developed before the commercial software were finalized [“] and have been fully customized by clinical users in themeantimeAt present standardization and harmonization of the calculation systems are importantTherefore it is essential to compare the various results obtained with the most advanced existing homemadecommercial software and other less advanced still used worldwide methodsYet both categories should be tested on a larger sample of cases than ever done before Thesetests should provide an example of the most accurate methodology for 3D dosimetry in RPTthanks to the gained experience in the last decades giving recommendations about the appropriate use and the limitations of each methodA few studies presenting some comparisons have already been published [“] Howeverthese works either did not report dosimetry studies performed completely at the voxel level[] or considered a limited number of clinical cases [] or showed a comparison based onthe dose factors and not based on absorbed doses [] Therefore more studies are needed tofully evaluate calculation performances in clinically relevant conditions considering a highnumber of casesIn this context the main objective of the present work is to compare different modalitiesfor absorbed dose calculation to point out the pros and the cons in each modality and to provide recommendations about the choice of the most adequate computation technique for thesingle clinical or research centres approaching the methodology The modalities here considered include the most used techniques in this field worldwide ranging from the less advancedand less personalised to the most accurate and patient specificThe considered modalities listed in growing complexity are an level dosimetry based onstandardized reference models such as OLINDA version [] which has been used fordecades before the recent release of the new updated commercial version OLINDA version [] voxellevel dosimetry based on dose kernel convolution VoxelMed20 [] and voxellevel dosimetry based on Monte Carlo MC simulations RAYDOSE [] OLINDA11 waschosen because it is still widely used for RPT dosimetry VoxelMed20 was chosen because itwas designed to achieve a good compromise between calculation accuracy and easy applicability in clinical practice RAYDOSE was considered because MC techniques are considered toprovide the most accurate approach to dose estimate []The comparison was performed on 3D images of specifically designed phantoms and onmultiple 3D dataset of images of a high number of clinical cases This multiapproach methodbased both on phantoms and patients allowed to investigate the differences of performancebetween the calculation modalities depending on the shape and the volume of the activity distribution and to provide a valuable comparison based on a conspicuous number of clinicalcasesMaterials and methodsThis study involves human participants All participants were enrolled in a clinical trialEUDRACT at Azienda USLIRCCS of Reggio Emilia Italy The study wasapproved by the ethics committee of Azienda USLIRCCS of Reggio Emilia Italy and eachpatient gave written informed consent for the study conductionThe following sections describe in detail the specific phantoms the image set the softwareand the data elaboration approachPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTPreparation of phantomsThree different phantoms filled with 177Lu radiolabelled peptides leftover from the clinicalapplication were used¢ a ˜Cylindrical phantom™ filled with a homogeneous radioactive solution Jaszczak Data Spectrum Corporation USA shown in Fig 1A Details are included in Table ¢ a cylindrical phantom and a set of fillable plastic inserts arranged in two different configurations to originate a couple of ˜Geometrical phantoms™ The inserts different for shapetoroidal pearshaped tubular and ellipsoidal and volume are depicted in Fig 1C Insertstake the name from the shape and the equivalent diameter ie the diameter for a spherewith the same volume as shown in Table Each insert was filled with the same activityconcentration and placed in a nonradioactive water background Details of volume andactivity concentration are shown in Table ¢ an ˜Anthropomorphic phantom™ with an shaped inserts LiquiPhill The Phantom Laboratory Greenwich NY shown in Fig 1B Details are included in Table Every insert wasfilled with an activity concentration typical of real ans in clinical cases and placed in aradioactive water backgroundTo accurately measure the volumes the weight of the phantoms and of the insertsbefore and after refilling was taken with a calibrated scale The density of the waterbasedsolution was of 1gml HCl M was used as a carrier solution to prevent radioactive177Lu deposition on the phantom walls and to guarantee a homogenous radionuclidesolutionEvery phantom was scanned once and the timeactivity curve was generated using the physical decay of the isotope All specific data regarding the volumes of inserts and phantoms andthe activity used are reported in Table Fig CT scans of phantoms used in this study a Cylindrical phantom b Anthropomorphic phantom c Insertswith different shapes placed in the Geometrical phantom101371journalpone0236466g001PLOS ONE 101371journalpone0236466 August PLOS ONE 0cTable Description of phantoms used to test the dosimetry toolsPhantomPhantom volumeInsert nameInsert volumeInsert activity concentration MBqBackground activity concentration MBqComparison of different absorbed dose calculation methods in MRTCylindricalGeometricalmlAnthropomorphicNATo17aTo26E20E30E38To17bP38P39aP39bTu38aTu38bLesionPancreasLeft kidneyRightkidneySpleenLivermlNA101371journalpone0236466t001Clinical trialmlmlNANAThe clinical cases considered in the present work were all extracted from a preexisting clinicalPRRT trial including patients and conducted by Azienda USLIRCCS of Reggio EmiliaItalyAll considered patients were previously enrolled in the trial EUDRACT between and The clinical trial design established that every patient had to besequentially administered with either 177Lu labelled radiopeptides 177LuDOTATOC or 90Ylabelled radiopeptides 90YDOTATOC up to a maximum of infusions or cycles Dosimetry was mandatory in the clinical trial and was to schedule during the first cycle of therapyafter a therapeutic administration of 177LuDOTATOC Each patient underwent SPECTCT scans at h post injection According to the trial design clinical absorbedTable Legend of the insert acronyms for the Geometrical phantomInsert geometryEquivalent diameter mmInsert nameTorusTorusTorusEllipsoidEllipsoidEllipsoidPearPearPearTubeTube101371journalpone0236466t002To17aTo17bTo26E20E30E38P38P39aP39bTu38aTu39bPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTdoses for 177Lu and 90Y labelled radiopeptides for liver spleen and kidneys were calculatedEach an was manually contoured and absorbed doses were calculated in compliance withthe MIRD scheme [] at anlevel from images The number of cycles the isotope and theactivity chosen for every injection were planned by an expert physician on the basis of thedosimetry results The activity prescription had to be determined based on the BiologicalEffective Dose BED delivered to kidneys Kidneys are regarded as the principal ans at riskin PRRT [ ] As suggested by different works [“] in this clinical trial the cumulativedose limit to kidneys was set to Gy of BED for patients with no risk factors hypertensiondiabetes renal failure are considered risk factors for this therapy and at 28Gy for patients withrisk factorsIn the present work absorbed doses to kidneys spleen and liver were calculated to comparethe three dosimetric methodsImage acquisition and reconstructionAll activity measurements were performed with an accurate activity calibrator for 177Lu Aktivimeter Isomed Nuklear Medizintechnik Germany and all image acquisitions were performed through a SPECTCT scanner Symbia T2 Siemens Medical Germany  NaITldetector previously calibrated [] The standard clinical protocol for body studies was usedboth for phantoms and patients with the following SPECT settings MEHR collimators matrix x zoom views x timeview s step and shoot mode degree of rotation ˚ noncircular orbit detector configuration ˚The first CT acquisition per patient was performed with the following parameters 130kVand max mAs using tube current modulation The subsequent CT images were acquiredwith kV and 40mAs for radiation protection safety of patients The CT reconstructed slicethickness was mm and a smooth reconstruction kernel was used B08s Siemens MedicalSolution Germany The higher image quality of the first CT scan is necessary for contouringvolumes of interest more accuratelyThe SPECT projections were reconstructed by an iterative algorithm including CT attenuation correction scatter correction and full collimatordetector response in Siemens ESoftworkstation Syngo MI Application version 32B Siemens Medical Solution Germany withFlash 3D iterative algorithm iterations subsets Gaussian filter cutoff mm mmcubic voxel []All cases of Sample A were rigidly registered to the first CT image of the sequence in Siemens Esoft workstation Images of patients included in Sample B were registered using adeformable multipass algorithm with the Velocity Advanced Imaging workstation Varian Medical Systems Palo Alto USA [] The registration procedures rescaled the originalvoxel size to 39x39x35 mm3The Volumes Of Interest VOI for each phantom and each patient were manually drawnon the reference CT image as recommended by Uribe [] using the VelocityworkstationSoftware for image processing and dosimetry calculationsOLINDA11 OLINDA version [] is an an level dosimetry software based on theMIRD methodology [] for internal dose estimation This is the method adopted in the clinical trial the clinical cases of this work are extracted from Absorbed doses to ans and tolesions can be calculated by using different models in the software human phantom modelsie mathematical representations of the human body to represent ans and whole body andsphere models ie mathematical representations of spheres to represent lesions [ ]PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTUnlike VoxelMed and RAYDOSE OLINDA needs timeintegrated activity A values ofVOIs as input parameters [] which were calculated with VoxelMed20 which will bedescribed in the next section and then inserted in OLINDAOLINDA sphere model commonly used to calculate doses to lesions was used to generatethe results for the inserts placed in the Geometrical phantom and for the dummy lesion housedin the anthropomorphic phantom while OLINDA an model adult male was used for thedummy ans placed in the anthropomorphic phantom Real insert volumes were used forcalculationsThe human models adult male or adult female were used to calculate dosimetry of thecohort of patients Doses were scaled using the true patient weight and the true an massesVoxelMed20 VoxelMed is a homemade software for dose calculation developed atAzienda USLIRCCS research hospital Reggio Emilia Italy It was developed in the MatlabThe Mathworks Natick MA programming environment and designed on the CERR platform wwwcerrinfo It performs voxellevel dosimetry based on the MIRD guidelines []The first version of the software along with the S value matrices for voxel dosimetry used incalculations were described in detail elsewhere []VoxelMed version includes a graphical user interface the possibility to export the resultsof calculations to Microsoft Excel file the visualization of the fitting curves both mono andbiexponential a module for renal BED calculation following the model suggested by StrigariL [] and the possibility to correct activity for partial volume effect PVE as presentedin [] Moreover VoxelMed20 provides the user with the timeintegrated activity A at VOIlevel which can be used for dosimetry with OLINDA version both for ans and lesionsTo calculate the number of disintegrations VoxelMed integrates the timeactivity curvewith the trapezoidal method in the time interval between the first and the last acquisitionBeyond this timeinterval the integration is performed analytically and the timeactivity curveis extrapolated using the effective halflife or the physical halflife it is chosen by the userThe effective halflife of the an or lesion is derived with a biexponential fit of the activitiesin the VOI the physical halflife is known from the selected isotope Timeintegrated activityis calculated in each voxel or in the whole an depending on the modality of dose calculationselected ie voxel level or an levelRAYDOSE RAYDOSE is a software package developed at Cardiff University School ofEngineering Cardiff University UK and designed to carry out 3D patientspecific imagebased dosimetry for RPT RAYDOSE provides personalized 3D dose map performing MonteCarlo simulations on radiation transport based on the Geant4 MC toolkit CERN Switzerland Geant4 is the stateoftheart package for the simulation of the transport of psthrough matter [] RAYDOSE generates voxellevel dose maps using anatomical and physiological data taken from morphologic and functional images []In order to obtain the area under the timeactivity curve RAYDOSE allows to use differentfitting modalities monoexponential decay linear uptake plus monoexponential decay or thetrapezoidal method In this study for the dose calculation of the clinical cases we used thetrapezoidal method at the voxel level up to the last time acquisition point while the timeactivity curve beyond the last scan time was extrapolated from the monoexponential curve fittingof the whole an activities in the VOI For dose calculation in phantoms we used the physical halflife of the isotope to extrapolate the activity from the scan time upwardsData and statistical analysisTwo groups of patients were considered for the purpose of this workPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTTable Demographic and baselines clinical characteristics of all patients�CharacteristicSample A N Sample B N Gender NoMaleFemaleAge yHeight cmWeight kgPrimary tumour site NoIleumPancreasLungThyroidRectumOthers177Lu activity for dosimetry MBq   ± ± ± ± ± ± ± N\\AN\\AN\\A ± � Plusminus values are means ± standard deviation  Injected activity at the first cycle of therapy Dosimetry was performed after the first injection101371journalpone0236466t003A first subgroup of cases named as œSample A was extracted by random samplingfrom the original clinical trial to adequately represent the whole population The number ofcases was calculated safely adopting a margin of error of and a standard deviation of A second independent subgroup of patients named as Sample B was extracted toofrom the original clinical trial similarly to Sample A A sample of cases was considered adequate in relation to the aim of the experiment conducted on Sample BPatient baseline characteristics for Sample A and Sample B are reported in Table The study type the image registration and the software used for the dose calculations of theclinical cases in sample A and sample B are summarised in Table Absorbed doses were calculated separately with OLINDA11 VoxelMed20 and RAYDOSEusing the same set of imagesKidney liver and spleen absorbed doses were calculated for each patient Two differentcomparison studies were performed The first study involved only comparison between VoxelMed and OLINDA based on absorbed dose calculations of patients in Sample A The secondstudy involved comparison between all the three software VoxelMed OLINDA and RAYDOSE based on absorbed dose calculations of patients in Sample B Furthermore in order toreduce the contribution of the fitting of the activitytime curves in the comparison of the software the VoxelMed dosimetry calculations for Sample B were repeated using the same effective halflife applied in RAYDOSE RAYDOSE estimates the effective halflife by fitting theTable Summary of phantom and patient studies performedStudy typePhantomObject of studyImage registrationSoftwareHomogeneous phantomNo registration only scanOLINDA11”VoxelMed”RAYDOSEGeometrical phantomNo registration only scanOLINDA11”VoxelMed”RAYDOSEAnthropomorphic phantomNo registration only scanOLINDA11”VoxelMed”RAYDOSEClinicalSample A patientsRigid registrationOLINDA11”VoxelMedSample B patientsDeformable registrationOLINDA11”VoxelMed VoxelMedλ RD”RAYDOSE101371journalpone0236466t004PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Clinical comparison study workflow Procedure flow of absorbed dose calculation for each patient of sample A top of the image and sample B lowerpart of the image101371journalpone0236466g002an activities against time as previously described Flow chart in Fig illustrates methodology in clinical studyDosevolume histograms DVH were evaluated to compare spatial dose distribution atvoxellevelMean values of absorbed dose were used to compare an level and voxel level techniquesComparison between the different dosimetry methods was statistically evaluated using theLin™s concordance correlation coefficient CCC and the BlandAltman plot [] The CCCsymbolized by ρc allows to evaluate the degree of concordance between two measures whilethe BlandAltman plot is used to analyse the agreement between two quantities The CCC wascalculated using SAS SAS Institute Cary NC USA A value of ρc equal to denotes perfect concordance a value equal to perfect discordance while a value of no correlationResultsPhysical phantom studyThe values of mean absorbed dose for the physical phantoms calculated with OLINDA11VoxelMed and RAYDOSE are reported in Table Visual representation of the same data isprovided in Fig Similar DVH curves were generated with VoxelMed and RAYDOSE both for the Cylindrical phantom Fig and for the other two phantoms Fig Fig shows DHVs only for theinserts in the Geometrical and the Anthropomorphic phantoms with the smallest and the largest relative differences of mean absorbed dose respectivelyClinical studyAbsorbed dose for kidneys liver and spleen of the sample A of patients calculated withOLINDA11 and VoxelMed are shown in Table The absorbed doses to liver and to spleenwere found to be highly correlated while lower correlation was found for kidneys The CCCPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTTable Mean absorbed dose Gy calculated with OLINDA11 VoxelMed and RAYDOSE for all of the three phantoms The absorbed dose calculated withOLINDA11 was performed using either the an model and the Sphere model Absorbed doses to Pancreas Kidneys Spleen and Liver were calculated using the anmodel otherwise the Sphere model was usedPhantomCylindricalGeometricalAnthropomorphicInsert nameOLINDA11VoxelMedRAYDOSENATo17aTo17bTo26E20E30E38P38P39aP39bTu38aTu39bLesionPancreasKidneysSpleenLiver101371journalpone0236466t005[ confidence interval] values were ρc liver [ ] ρc spleen [ ]ρc kidneys [ ] The BlandAltman plot is shown in Fig OLINDA11 VoxelMed VoxelMedλ RD and RAYDOSE calculated mean absorbed dosefor patients of Sample B are shown in Table while the BlandAltman plot is shown in Fig The absorbed doses calculated with VoxelMed and RAYDOSE were highly correlated withρc kidneys [ ] ρc liver [ ] and ρc spleen [ ] andalmost complete agreement were found between VoxelMedλ RD and RAYDOSE withρc kidneys [ ] ρc liver [ ] and ρc spleen [ ]DiscussionIn this study we compared the performances of three tools for dosimetry calculationsOLINDA11 VoxelMed20 and RAYDOSE with the primary aim to evaluate the influence ofthe calculation modality on absorbed dose assessment anlevel based voxellevel dose kernel convolution based and Monte Carlo simulations based respectively The secondary aimwas to give some recommendations about the choice of the adequate technique for dosimetrycalculation to be implemented in a hospital in a research centre or in an academic instituteclinical or research purpose small or large number of patients clinical trials only or standardprocedures This analysis was performed in standard conditions by acquisition and processing of radioactive phantoms provided with inserts of specific volume and geometry and inclinical conditions over a large cohort of patients a selection of clinical cases taken from a clinical trial in which dosimetry had already been calculated Clinical conditions are indeed quitedistant from and more complicated than the standard conditions achievable in a phantom forseveral reasons biological kinetics in place of only physical decay of activity serial acquisitionsof functional images and associated issues related to image registration [] motion of thepatient that creates artefacts in images irregular shape of volumes of interest inhomogeneousactivity distributionPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Comparison of mean absorbed dose Gy calculated using OLINDA11 VoxelMed and RAYDOSE a Homogeneous phantom b Anthropomorphicphantom and cf Geometrical phantom Note the Lesion insert in the Anthropomorphic phantom is missing because beyond the range of dose visualized in thegraph101371journalpone0236466g003Therefore to consider a large sample of clinical cases was of great interest since many studies about methods for dosimetry calculation are based on smaller groups of patients [“]and in a small group the inter patient variability cannot be properly investigatedThe quantitative intercomparison between all the three software was performed betweenthe mean values of absorbed doses In fact OLINDA provides only mean values while RAYDOSE and VoxelMed20 voxelbased tools provide the dose distribution that can be represented with DVHs from which the mean dose values can be derived To compare thetechniques of calculation the relative differences and the correlation between data pairs wereevaluatedFor standard conditions we evaluated discrepancies of calculated absorbed dose in a cylindrical phantom and in differently shaped inserts filled with a homogeneous radioactive solution Table shows the values of absorbed dose obtained with OLINDA11 VoxelMed andRAYDOSE in each of the phantoms These values are also plotted in Fig Lower values ofabsorbed dose were generally calculated using OLINDA in comparison with the dose calculated with other voxel modalities In the case of the cylindrical phantom a good agreementwas obtained between VoxelMed20 and RAYDOSE discrepancy equal to while largerdifference was observed between VoxelMed20 and OLINDA Absorbed dose map provided by VoxelMed and RAYDOSE showed similar spatial distribution close values of standard deviation across voxels around and analogues slope in DVHs Fig PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Comparison of DVHs calculated using VoxelMed continuous line and RAYDOSE dotted line for the Cylindrical phantom101371journalpone0236466g004To compare the calculation techniques in different conditions of volume and geometry theGeometrical phantoms were acquired Relative differences in absorbed dose depend on theshape and on the volume of the inserts smaller is the volume and further from a regular sphereis the shape more the relative difference is higher In the Geometrical phantom the toroidalinserts provided the greatest discordance relative difference with VoxelMed dose rangingfrom to for OLINDA and from to for RAYDOSE while in the otherinserts differences ranged between [ ] for OLINDA11 and [ ] for RAYDOSEOn one hand the insert dose calculations in OLINDA were performed using the spheremodel since OLINDA only allows to perform dosimetry calculation for specified models ieans or spheres This approximation might explain the huge discrepancies obtained withthe voxelbased methods On the other hand a reason for the difference between RAYDOSEand VoxelMed is that the latter applies a mask before the convolution while RAYDOSE doesnot This contribution affects calculations in so far as the geometry and the volume of theinsert may influence the activity distribution and leave empty spaces around or inside theobjects This effect is especially pronounced for example in the case of the toroid The application of a mask also implies the lack of photon cross irradiation contribution between insertswhich has an impact on dose calculation contribution around [] Discrepancies ofPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calc

Thyroid_Cancer

"patients with differentiated thyroid cancer DTC tumor burden of persistent disease PD is avariable that could affect therapy efficiency Our aim was to assess its correlation with the American ThyroidAssociation ATA riskstratification system and its impact on response to initial therapy and outcomeMethods This retrospective cohort study included consecutive DTC patients referred for postoperativeradioiodine RAI treatment Patients were riskstratified using the ATA guidelines according to postoperativedata before RAI treatment Tumor burden of PD was classified into three categories ie very small small andlargevolume PD Very smallvolume PD was defined by the presence of abnormal foci on postRAI scintigraphywith SPECTCT or 18FDG PETCT without identifiable lesions on anatomic imaging Small and largevolume PDwere defined by lesions with a largest size or ‰¥ mm respectivelyResults PD was evidenced in patients Mean followup for patients with PD was ± years Thepercentage of largevolume PD increased with the ATA risk and in low intermediate and highriskpatients respectively p There was a significant trend for a decrease in excellent response rate from thevery small small to largevolume PD groups at “ months after initial therapy and respectively p and at last followup visit and respectively p On multivariate analysis age ‰¥ yearsdistant andor thyroid bed disease smallvolume or largevolume tumor burden and 18FDGpositive PD wereindependent risk factors for indeterminate or incomplete response at last followup visitConclusions The tumor burden of PD correlates with the ATA riskstratification affects the response to initialtherapy and is an independent predictor of residual disease after a mean 7yr followup This variable might betaken into account in addition to the postoperative ATA riskstratification to refine outcome prognostication afterinitial treatmentKeywords Differentiated thyroid cancer Tumor burden Riskstratification Radioiodine 18FDG PETCT Correspondence rciappuccinibaclesseunicancerfr1Department of Nuclear Medicine and Thyroid Unit Fran§ois Baclesse CancerCentre Avenue Gnral Harris F14000 Caen France2INSERM ANTICIPE Caen University Caen FranceFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cCiappuccini BMC Cancer Page of BackgroundIn patients with differentiated thyroid cancer DTCthe riskstratification system described in the American Thyroid Association ATA guidelines is auseful tool to predict the likelihood of postoperativepersistent disease PD the response to initial therapyie surgery ± radioiodine [RAI] treatment and thelongterm outcome [] Several features related to PDare likely to influence the response to treatment andthe longterm prognosis This includes the location ofPD neck lymphnodes [LN] or distant metastases the18FFluorodeoxyglucose 18FDGRAIavidity [] oravidity [] of PD the aggressiveness of pathological variants [] and the degree of celldifferentiation [] thepresence of molecular mutations BRAF TERTp []and the tumor doublingtime [] Alone or in combination with previous characteristics notably RAIaviditythe tumor burden of PD is another variable that canaffect treatment efficiency and prognosis This has beenshown in studiessometimes old and using lowresolution imaging methods focusing on patients withdistant metastases [ ] In the daily practice it is wellknown that microscopic RAIavid lesions are morelikely cured than macroscopic ones eg lung miliary vslung macronodules However no studies have specifiedthe prognostic role of tumor burden estimated usinghighresolution imaging techniques both in the settingof distant metastases and lymphnode diseaseThe aim of the study was to assess the correlationof PD tumor burden with the ATA riskstratification system and its impact on response toinitialtherapy and outcome We hypothesized thatpatients presenting postoperatively a low tumor burden of PD would have better response to initial therapy and better clinical outcomes than patients havinghigh tumor burdenMethodsPatientsThe records of consecutive patients with DTC referred to our institution for postoperative RAI treatment between January and February werereviewed For the purpose of the study patients wereriskstratified according to the ATA guidelinesbased on pathological and surgical data available aftertotalthyroidectomy and before postoperative RAItreatment postoperative risk stratification [] Dataavailable in the preoperative period such as imagingstudies showing distant metastases were also used toinform ATA risk stratification In contrast postoperative serum thyroglobulin Tg level was not used todrive RAI treatment in these patients managed before and no diagnostic RAI scintigraphy was performed before RAI treatmentrhTSHPostoperative RAI treatmentAll patients were administered an RAI regimen ± weeks after total thyroidectomy Patients were prepared after either thyroid hormone withdrawal THWinjections of recombinant humanor after two imthyrotropinThyrogen Genzyme CorpCambridge MA USA as previously described [] TSHlevel was measured the day of RAI treatment and was mUIl in all patients The RAI activity or GBq and the preparation modalities were decided byour multidisciplinary committee All patients underwenta postRAI scintigraphy combining wholebody scanWBS and neck and thorax single photon emissioncomputed tomography with computed tomographySPECTCT A complementary SPECTCT such as abdomen andor pelvis acquisition was performed in caseof equivocal or abnormal RAI foci on WBS Patientswere scanned two or file days following or GBqrespectively Initial therapy was defined as surgery iethyroidectomy ± LN dissection plus first RAI treatmentie postoperative RAI treatmentSerum Tg and antiTg antibodies TgAb assayBlood samples for stimulated serum Tg and TgAb measurements were collected immediately before the RAItreatment Serum Tg measurements were obtained withthe Roche Cobas Tg kit Roche Diagnostics Mannheim Germany with a lower detection limit of ngml and a functional sensitivity of ngml until October and with the Roche Elecsys Tg II kit Roche Diagnostics Mannheim Germany with a lower detectionlimit of ngml and a functional sensitivity of ngml thereafter TgAb was measured using quantitativeimmunoassay methods Roche Diagnostics MannheimGermany TgAb positivity was defined by the cutoffsprovided by the manufacturerPathologyPathological variants were defined according to the WorldHealth anization classification [] Poorly differentiated carcinoma widely invasive follicular carcinomaH¼rthle cell carcinoma and among PTC variants tall cellcolumnar cell diffuse sclerosing and solid variants wereconsidered as aggressive pathological subtypes [] Tumorextent was specified according to the TNM []Tumor burden of persistent diseaseAs previously described [] PD was defined as evidenceof tumor in the thyroid bed LN or distant metastasesafter completion ofinitial therapy Confirmation wasachieved either by pathology or by complementary imaging modalities neck ultrasound examination [US]postRAI scintigraphy 18FDG positron emission tomography [PETCT] CT scan or MRI and followup 0cCiappuccini BMC Cancer Page of The tumor burden of PD was classified into three categoriesie very small small and largevolume PDVery smallvolume PD was defined by the presence ofabnormal foci on posttherapeutic RAI scintigraphy withSPECTCT or 18FDG PETCT without identifiable lesions on anatomic imaging neck ultrasound CT scan orMRI Small or largevolume PD were defined by thepresence of metastatic lesions with a largest size or ‰¥ mm respectively regardless of RAI or 18FDGuptake Examples of patients with very small small orlargevolume PD are presented in Fig RAI and 18FDG uptake in persistent diseaseThe RAI or 18FDG uptake profile was defined at time ofPD diagnosis PD was considered RAIpositive RAI ifat least one metastatic lesion showed RAI uptake andRAInegative RAI otherwise Similarly PD was defined18FDGpositive 18FDG if at least one metastatic lesionpresented significant 18FDG uptake and 18FDGnegative18FDG otherwiseClinical outcome assessmentAs previously described [] clinical assessment ofpatients with a negative postRAI scintigraphy wasscheduled at three months with serum TSH Tg andTgAb measurements while on levothyroxine LT4treatment When the Tg level at three months was ngml in the absence of TgAb the disease status wasassessed at “ months by serum rhTSHstimulated Tgassay and neck US and in recent years by Tg II assayon LT4 and neck US If there was an excellent responselevel ngmlat “ months according to the ATA criteria iestimulatedTgor nonstimulatedTglevel ngml without TgAb and negative neck USpatients were followed up on an annual basis For anything other than an excellent response imaging modalities such as CT scan of the neck and thorax 18FDGPETCT or MRI were performed In case of a secondRAI regimen given “ months after the first RAI therapy for RAIavid PD postRAI scintigraphy with SPECTCT was also used to assess initial treatment responseResponses to initial therapy as assessed at “ monthsand status at lastvisit were categorized as excellent response indeterminate response biochemical incompleteresponse or structural incomplete response according tothe ATA guidelines []Data analysisQuantitative data are presented in mean ± standard deviation SD except for Tg levels which are presented inmedian range Patients™ characteristics were comparedusing Chisquare or Fisher™s exact test the Wilcoxontest or the KruskalWallis test as appropriate TheCochranArmitage trend test was used to examineproportions of excellent response over the differentsubgroups in the following order verysmall small andlargevolume PD The analysis of diseasespecific survival and progressionfree survival was performed usingthe Cox regression model The analysis of prognosticfactors was performed using logistic regression Statistical significance was defined as p All tests wereFig Examples of very small small and large tumor burden in patients with persistent disease PD On the left side a 43yearold female patientwith a 40mm PTC at lowrisk after initial surgery T2NxMx and very smallvolume PD ac posttherapeutic 131I WBS showed a solitary bonyfocus on the right hip a arrow Fused transaxial image of 131I SPECTCT b arrow confirmed the bony uptake and hybrid CT c arrow did notdisplay any bone abnormality On the middle part a 74yearold female patient with a 40mm PTC at lowrisk after initial surgery T2N0Mx andsmallvolume PD df posttherapeutic 131I WBS showed pulmonary metastases d red and black arrows Fused transaxial image e red arrowand hybrid CT scan f red arrow depicted RAIavid lung micronodules ef mm On the right side an 88yearold female patient with a 40mmPTC tall cell variant at highrisk after initial surgery T2N1bM1 and largevolume PD gi no abnormal RAI uptake on posttherapeutic 131I WBSwith SPECTCT whereas 18FDG PETCT showed pulmonary and mediastinal metastases g Maximum intensity image arrows Fused transaxialimage h arrow and hybrid CT scan i arrow showed high 18FDG uptake SUVmax by an 18mm lung nodule 0cCiappuccini BMC Cancer Page of twosided SAS statistical software SAS InstituteInc Cary NC USA was used for data analysisstratification Patients™ characteristics are reported inTable ResultsCharacteristics of patientsThe study group included papillary thyroid cancers PTC follicular thyroid cancers FTC and poorlydifferentiated thyroid cancers PDTCThere were women and men The mean agewas ± years Three hundred and seventytwo patients were prepared with rhTSH stimulation Eightytwopatients presented positive TgAb at the time of postoperative RAI treatment In the postoperative setting priorto RAI administration patients were at lowriskLR at intermediaterisk IR and athighrisk HRaccording to the ATA riskPersistent disease and tumor burdenOverall PD was detected in patientsTheir characteristics in terms of ATA risk RAI preparation modality PD sites and RAI or 18FDG uptake arepresented in Table Of patients had very smallvolumelargevolume smallvolume and PDFigure shows two points First the rate of PDincreased from in LR patients and in IR to in HR patients p Second the percentage of patients with largevolume PD increased with risk stratification from LRIR to HR patients and respectively p Table Characteristics of patients according to the ATA riskstratification system in the postoperative settingMean age ± SD yrsSex ratio FemaleMean tumor size ± SD mmHistologyPTCFTCPDTCAggressive pathological subtypesNoYesExtrathyroidal extensionMinimalGrossT status TNM T1a T1bT2T3a T3bT4a T4bN status TNM NxN0N1a N1bM status TNM M0M1Positive TgAb levelStimulated Tg level at RAI treatment rangeaaIn patients without positive TgAb levelLRn ± ± IRn ± ± “ “HRn ± ± “p 0cCiappuccini BMC Cancer Page of Table Characteristics of patients with persistent disease according to the tumor burdenVery smallvolumePD n SmallvolumePD n LargevolumePD n Postoperative ATA riskLRIRHRPreparation modalityTHWrhTSHPD siteLNLN DMDMTB diseaseTB disease DMRAI and 18FDG statusRAI18FDG or NPRAI18FDGRAIˆ’18FDGRAIˆ’18FDGRAIˆ’18FDG NPa21 RAI18FDG NP and one RAI18FDGb15 RAI18FDG NP and two RAI18FDGc10 RAI18FDG NP and six RAI18FDG 22a 17b 16c pFig Tumor burden in patients with persistent disease correlation to the ATA riskstratification system The figure first shows that the rateof PD increased from in LR patients in IR to in HR patients p Second the percentage of patients with largevolume PDincreased with risk stratification from LR IR to HR patients and respectively p 0cCiappuccini BMC Cancer Page of Table Characteristics of patients with persistent diseaseaccording to the ATA riskstratification systemLRn IRn HRn pPD tumor burdenVery smallvolume SmallvolumeLargevolume The distribution of very small small andlargevolume PD in LR IR and HR patients is presented in Table Outcome of patients with persistent diseaseTreatment modalities within the first year of management and during the remaining followup are detailed inTable Mean followup for patients with PD was ± years and was similar between the three groups of tumorburden p Of the patients with PD at “months after initial therapy had excellent response indeterminate response biochemical incomplete response and structuralincomplete response At last followup visit the figureswere and respectively The outcome in each of the tumor burden groupsis presented in Table There was a significant trend fora decrease in excellent response rate from the verysmall small to the largevolume PD groups at “months after initial therapy and respectivelyp and at last followup visit and respectively p Fig Among the patients died related to DTCduring followup Seven were in the largevolume PDgroup and one in the smallvolume PD group All hadstructural incomplete response at “ months after initial therapy with 18FDGpositive diseaseFigures and show diseasespecific survival DSSand progressionfree survivalPFS according to theATA riskstratification 18FDG status and tumor burdenSignificant differences in DSS were observed for bothATA riskstratification and 18FDG status but not fortumor burden Patients with 18FDGpositive disease hadshorter PFS Hazard Ratio 95CI “ thanthose with 18FDGnegative disease Also IR HazardRatio 95CI “ and HR patients HazardRatio 95CI “ had shorter PFS than LRpatients Finally patients with small Hazard Ratio 95CI “ and largevolume PD Hazard Ratio 95CI “ had shorter PFS than those withverysmall volume PDPrognostic factor analysis in patients with persistentdiseaseMultivariate analysis controlling for age sex postoperative ATA riskstratificationaggressive pathologicalTable Treatment modalities and outcome of patients with PD at “ months after initial therapy and at last followup visitaccording to tumor burdenVery smallvolumePD n “ months after initial therapySmallvolume PDn Largevolume PDn pVery smallvolumePD n At last followup visitSmallLargevolume PDvolume PDn n p a Treatment modalities at “ months after initial therapy treatments given within the first year of followup treatment modalities at last followup visittreatments given after the first year during followupb Local treatment of DM external radiation beam therapy surgery or radiofrequencyAbbreviations PD Persistent disease RAI Radioiodine DM Distant metastasesTreatment modalitiesaRAINeck surgeryNeck external radiationbeam therapyLocal treatment of DMbTyrosinekinase inhibitorsChemotherapyOutcomeExcellent response Indeterminate responseBiochemical incompleteresponseStructural incompleteresponse 0cCiappuccini BMC Cancer Page of Fig Excellent response rate according to tumor burden “ months after initial therapy a and at last followup visit b in patients withpersistent disease There is a significant trend for a decrease in excellent response rate from the very small small to the largevolume PD groupsat “ months after initial therapy and respectively p and at last followup visit and respectively p subtypes site of PD tumor burden of PD and RAI or18FDG uptake showed age ‰¥ years Odds ratio [OR] p distant andor thyroid bed disease OR p smallvolume OR p andlargevolume tumor burden OR p and18FDGpositive disease OR p to be independent risk factors for indeterminate biochemical orstructuralincomplete response at last followup visitTable DiscussionThis study confirms that the incidence of PD aftertotal thyroidectomy and postoperative RAI treatmentis limited in LR patients as compared to IR or HR patients Moreover it demonstrates thatthe tumor burden of PD is correlated to postoperativeriskstratification with very smallvolume lesions preferentially observed in LR patients and small and largevolume in IR or HR patients Most importantly tumorburden of PD is shown as an independent predictor ofresponse to initial therapy and to outcome These findings confirm that tumor burden of PD is a variablewhich might be taken into account to refine outcomeprognosticationTumor burden covers a large range of locoregionalandor distant metastases from a unique microscopic lesion to multiple macroscopic ones sometimes clinicallyevident Also tumor burden encompasses structural egvisible on conventionalfunctionalradiology andorlesions eg visible on RAI scintigraphy or 18FDG PETCT The diagnostic performances of imaging methodsand consequently the concept of tumor burden havedramatically evolved in the last decades The detectionof small LN disease has been improved by the combination of highresolution neck US postRAI SPECTCTand 18FDG PETCT imaging Regarding distant metastases although postRAI WBS still remains the referencefor detecting lung miliary disease the routine use ofdiagnostic CT scan and MRI now enables the detectionof infracentimetric lung bone or brain lesionsIn the past tumor burden of PD as a potential indicator of successful treatment and prognosis was assessedusing different approaches In a study on DTC patients with lung metastases diagnosed from to multivariate analysis showed that lung nodules visible on XRay vs those not visible RAIrefractory lunglesions and multiple metastatic sites were associatedwith poor survival [] In Gustave Roussy™s experienceoverall survival was reported in DTC patients withdistant metastases lung bone or other sites diagnosedbetween and [] Tumor extent was classifiedinto three categories according to both postRAI planarscintigraphy and Xrays Category consisted in lesionsvisible on postRAIscan but with normal Xraycategory in metastatic lesions cm on Xrays andcategory in lesions cm regardless of RAI avidityOverall metastases were RAIavid in of patientsmore frequently in patients years than 0cCiappuccini BMC Cancer Page of Fig Diseasespecific survival in the patients with PD according to ATA riskstratification a 18FDG status b and tumor burden cyears Multivariate analysis demonstrated that female sex young age years well differentiatedtumor RAI avidity and limited extent category wereindependent predictors ofrecentlyRobenshtok reported the outcome of patientssurvival Morewith RAIavid bone metastasis without structural correlate on CT scan or MRI among DTC patients withbone metastases between and [] After afollowup period of years all patients were alive nonehad evidence of structural bone metastases and none 0cCiappuccini BMC Cancer Page of Fig Progressionfree survival in the patients with PD according to ATA riskstratification a 18FDG status b and tumor burden chad experienced skeletalrelated events confirming theexcellent prognosis after RAI treatmentIn DTC patients with persistent nodal disease there isalso indirect evidence supporting that tumor burden affects treatment response and outcome In a recent retrospective study Lamartina reported the outcome of patients without distant metastases who underwenta first neck reoperation for nodal persistentrecurrentdisease [] Male sex aggressive histology and the presence of more than LN metastases at reoperation wereshown to be independent risk factors of secondary relapse following complete response achieved with first 0cCiappuccini BMC Cancer Page of Table Risk factors for indeterminate biochemical or structural incomplete response at last followup visitVariableAge years ‰¥ SexFemaleMaleInitial ATA riskstratificationLRIRHRAggressive histological subtypesNoYesSite of PDLN onlyDM andor TB disease with or without LNTumor burden of PDVery smallvolumeSmallvolume mmLargevolume ‰¥ mmRAI and 18FDG status of PDRAI18FDG or NPRAIˆ’18FDG or NPRAI or RAI18FDGPatients at risk nInitial modelOR CIp valueFinal modelOR CIp value““““““““““““““““reoperation Conversely the excellent outcome of microscopic nodal involvement detected on SPECTCT at RAIablation was demonstrated by a study from Schmidt [] Of patients with RAIavid LN metastasesat ablation only three still showed nodes with significantuptake on a diagnostic RAI scintigraphy at monthsThe LN successfully treated by RAI were less than cmexcept in one patient whereas those still visible at months were above cm confirming that RAI is highlymore efficientin microscopic than in macroscopiclesionsIn the present study multivariate analysis showed thatage over years distant andor thyroid bed diseasesmall or largevolume tumor burden and 18FDGpositive disease were independent risk factors for indeterminate or incomplete response at last followup visit Incontrast ATA risk stratification and aggressive pathological subtypes did not emerge from multivariate analysis possibly because of the number of patients thenumber of variables tested and confounding variablesHoweverand progressionfreethe diseasespecificsurvival curves confirmed the high prognostic value ofthe ATA riskstratification In practice data supportsthat LR patients have a better outcome than the IR andHR groups not only because PD is uncommon in thosepatients but also because the excellent response rate ishigher in very smallvolume than in small or largevolume lesions We suggest that tumor burden usingthis threeclass discrimination could be implemented inthe assessment of patients with structural incomplete response to help refining the risk prediction This variablecould also be incorporated with the other risk predictorssuch as RAI or 18FDG uptake molecular profile tumorhistology degree of cell differentiation and Tg level andtumor volume doubling time to further improve riskestimatesAlthough retrospective the present study presents several strengths including the large cohort of consecutivepatients and the significant followup Patients diagnosedbetween and were uniformly evaluated usingmodern imaging studiesincluding postRAI scintigraphy with neck and thorax SPECTCT [] and 18FDG 0cCiappuccini BMC Cancer Page of PETCT with a dedicated headandneck acquisition [] Tumor burden was assessed combining functionaland anatomic imaging as adapted from previous papersof our group [ ] One can argue that it would havebeen even more pertinent to assess tumor burden withquantitative values rather than with a threeclass discrimination ie very small small and largevolumeActually a quantitative volumetric assessment is notfeasible because of the RAIavid nodal or metastatic lesions without structural correlate Also a quantitativeassessment based on RAI or 18FDG uptake is notpossible either because of RAIrefractory or nonhypermetabolic lesions Nevertheless we believe that ourdefinition is simple to use in routine practice and easilyreproducibleConclusionsThe tumor burden of PD correlates with the postoperative ATA riskstratification affects the response to initialtherapy and is an independent predictor of residual disease after a mean 7yr followup This variable might betaken into account in addition to the postoperative ATAriskstratification to refine outcome prognostication afterinitial treatmentAbbreviationsATA American thyroid association DM Distant metastasesDTC Differentiated thyroid cancer 18FDG 18FfluorodeoxyglucoseFTC Follicular thyroid cancers HR Highrisk IR Intermediaterisk LN Lymphnodes LR Lowrisk MRI Magnetic resonance imaging NP Not performedOR Odds ratio PD Persistent disease PDTC Poorlydifferentiated thyroidcancers PETCT Positron emission tomography with computed tomographyPTC Papillary thyroid cancers RAI Radioiodine rhTSH Recombinant humanthyrotropin SPECTCT Single photon emission computed tomography withcomputed tomography Tg Thyroglobulin TgAb AntiTg antibodiesTHW Thyroid hormone withdrawal TB Thyroid bed US Ultrasoundexamination WBS Wholebody scanAcknowledgmentsWe are indebted to Gee Knight for the reviewing of the manuscriptAuthors™ contributionsRC and SB conceived the study and its design RC ALC VSR CL VLH DV EBand SB performed data acquisition and analysis NH performed the statisticalanalysis RC and SB drafted the manuscript All authors read and approvedthe final manuscriptFundingNot applicableAvailability of data and materialsThe datasets used and analysed during the current study are available fromthe corresponding author on reasonable requestEthics approval and consent to participateAll procedures were in accordance with the ethical standards of theinstitutional committee and with the Helsinki declaration and its lateramendments Baclesse Cancer Centre has licensed from the FrenchCommission for Data Protection and Liberties CNIL MR004 ref v0This study was approved by the institutional review board of Baclesse hospital and all subjects gave written informed consentConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Nuclear Medicine and Thyroid Unit Fran§ois Baclesse CancerCentre Avenue Gnral Harris F14000 Caen France 2INSERM ANTICIPE Caen University Caen France 3CETAPS EA Rouen UniversityRouen France 4Department of Head and Neck Surgery Fran§ois BaclesseCancer Centre Caen France 5Department of Pathology Fran§ois BaclesseCancer Centre Caen France 6Department of Oncology Fran§ois BaclesseCancer Centre Caen France 7Department of Cancer Biology and GeneticsFran§ois Baclesse Cancer Centre Caen France 8Department of Head andNeck Surgery University Hospital Caen FranceReceived February Accepted August ReferencesHaugen BR Alexander EK Bible KC Doherty GM Mandel SJ Nikiforov YEPacini F Randolph GW Sawka AM Schlumberger M Schuff KG Sherman SISosa JA Steward DL Tuttle RM Wartofsky L American ThyroidAssociation management guidelines for adult patients with thyroid nodulesand differentiated thyroid Cancer the American Thyroid Associationguidelines task force on thyroid nodules and differentiated thyroid CancerThyroid “Durante C Haddy N Baudin E Leboulleux S Hartl D Travagli JP Caillou BRicard M Lumbroso JD De Vathaire F Schlumberger M Longtermoutcome of patients with distant metastases from papillary andfollicular thyroid carcinoma benefits and limits of radioiodine therapy J ClinEndocrinol Metab “Robbins RJ Wan Q Grewal RK Reibke R Gonen M Strauss HW Tuttle RMDrucker W Larson SM Realtime prognosis for metastatic thyroid carcinomabased on [18F]fluoro2deoxyDglucosepositron emission tomographyscanning J Clin Endocrinol Metab “ Michels JJ Jacques M HenryAmar M Bardet S Prevalence and prognosticsignificance of tall cell variant of papillary thyroid carcinoma Hum Pathol“de la Fouchardiere C DecaussinPetrucci M Berthiller J Descotes F Lopez JLifante JC Peix JL Giraud Delahaye A Masson S BournaudSalinas CBorson CF Predictive factors of outcome in poorly differentiated thyroidcarcinomas Eur J Cancer “ Melo M Gaspar da Rocha A Batista R Vinagre J Martins MJ Costa G RibeiroC Carrilho F Leite V Lobo C CameselleTeijeiro JM Cavadas B Pereira LSobrinhoSimoes M Soares P Gaspar da Rocha A Batista R Vinagre JMartins MJ Costa G Ribeiro C Carrilho F Leite V Lobo C CameselleTeijeiroJM Cavadas B Pereira L SobrinhoSimoes M Soares P TERT BRAF andNRAS in primary thyroid Cancer and metastatic disease J Clin EndocrinolMetab “Sabra MM Sherman EJ Tuttle RM Tumor volume doubling time ofpulmonary metastases predicts overall survival and can guide the initiationof multikinase inhibitor therapy in patients with metastatic follicular cellderived thyroid carcinoma Cancer “Casara D Rubello D Saladini G Masarotto G Favero A Girelli ME BusnardoB Different features of pulmonary metastases in differentiated thyroidcancer natural history and multivariate statistical analysis of prognosticvariables J Nucl Med “Ciappuccini R Hardouin J Heutte N Vaur D Quak E Rame JP Blanchard Dde Raucourt D Bardet S Stimulated thyroglobulin level at ablation indifferentiated thyroid cancer the impact of treatment preparationmodalities and tumor burden Eur J Endocrinol “Lloyd RV Osamura RY Kl¶ppel G Rosai J editors WHO classification oftumours of endocrine ans 4th edition Lyon International Agency forResearch on Cancer Brierley JD Gospodarowicz MK Wittekind C TNM classification of malignanttumours 8th edition Oxford Wiley Blackwell Ciappuccini R Heutte N Trzepla G Rame JP Vaur D Aide N BardetS Postablation I scintigraphy with neck and thorax SPECTCTand stimulated serum thyroglobulin level predict the outcome ofpatients with differentiated thyroid cancer Eur J Endocrinol “ 0cCiappuccini BMC Cancer Page of Robenshtok E Farooki A Grewal RK Tuttle RM Natural history of smallradioiodineavid bone metastases that have no structural correlate onimaging studies Endocrine “Lamartina L Bet I Mirghani H Al Ghuzlan A Berdelou A Bidault FDeandreis D Baudin E Travagli JP Schlumberger M Hartl DM Leboulleux SSurgery for neck recurrence of differentiated thyroid Cancer outcomes andrisk factors J Clin Endocrinol Metab “Schmidt D Linke R Uder M Kuwert T Five months' followup of patientswith and without iodinepositive lymph node metastases of thyroidcarcinoma as disclosed by 131ISPECTCT at the first radioablation Eur JNucl Med Mol Imaging

Thyroid_Cancer

Genomic characterization of malignant progressionin neoplastic pancreatic cystsMicha«l No«et alIntraductal papillary mucinous neoplasms IPMNs and mucinous cystic neoplasms MCNsare noninvasive neoplasms that are often observed in association with invasive pancreaticcancers but their origins and evolutionary relationships are poorly understood In this studywe analyze samples from IPMNs MCNs and small associated invasive carcinomas from patients using whole exome or targeted sequencing Using evolutionary analyses weestablish that both IPMNs and MCNs are direct precursors to pancreatic cancer Mutations inSMAD4 and TGFBR2 are frequently restricted to invasive carcinoma while RNF43 alterationsare largely in noninvasive lesions Genomic analyses suggest an average window of overthree years between the development of highgrade dysplasia and pancreatic cancer Takentogether these data establish noninvasive IPMNs and MCNs as origins of invasive pancreatic canceridentifying potential drivers of invasion highlighting the complex clonaldynamics prior to malignant transformation and providing opportunities for early detectionand interventionA list of authors and their affiliations appears at the end of the paperNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178Pancreatic cancer is a deadly disease with a dismal prognosisthat is predicted to soon be the second leading cause ofcancer death in the United States1 However like otherepithelial malignancies pancreatic cancer arises from noninvasiveprecancerous lesions that are curable if detected and treated earlyenough Although the majority of pancreatic cancers are believedto originate in microscopic precancerous lesions pancreaticintraepithelial neoplasia or PanIN a significant minority arise inassociation with larger cystic neoplasms that can be detectedusing currently available imaging technologies2 These neoplasmswhich includeintraductal papillary mucinous neoplasmsIPMNs and mucinous cystic neoplasms MCNs are frequentlydiagnosed incidentally on abdominalidentifying acohort of atrisk patients with an important opportunity forprevention of invasive pancreatic cancer2 However preventionmust be balanced with potential overtreatment of lowrisk lesionsas pancreatic resection carries significant morbidity and evenoccasional mortality3 There is a critical need to understand themolecular alterations that are associated with the development ofinvasive cancer as these represent potential biomarkers to identify cysts at high risk for progression to carcinoma and thusrequiring clinical interventionimagingAlthough genomic analyses have been performed on hundredsof invasive pancreatic cancers relatively few noninvasive neoplasms have been analyzed comprehensively Whole exome andtargeted sequencing of small cohorts of IPMNs and MCNs haverevealed driver genes characteristic of each type of cystic neoplasm4“ while targeted analyses in larger cohorts have confirmed the prevalence of specific driver gene mutations thatcorrelate with grade of dysplasia or histological subtype7 Thesestudies have confirmed that hotspot mutations in the oncogenesKRAS and GNAS occur in lowgrade lesions while mutations inother driver genesincluding CDKN2A TP53 RNF43 andSMAD4 occur with increasing prevalence in lesions with highgrade dysplasia or associated invasive carcinoma8 Targeted nextgeneration sequencing has been used to analyze pancreatic drivergenes in different regions of IPMNs revealing a surprising degreeof intratumoral genetic heterogeneity even with respect to wellcharacterized driver gene mutations9“ However the aboveanalyses were based on studies of either single regions from eachneoplasm or a limited number of genes from multiple regionsand did not provide an analysis of the evolutionary relationshipbetween different regions of pancreatic cysts and associatedcancers These limitations highlight the need for comprehensivegenomic analysis of these cysts and associated invasive cancers tounderstand the molecular alterations that underlie the transitionto invasive carcinomaIn this study we perform whole exome sequencing of IPMNsand MCNs and their associated invasive carcinomas Importantlywe focus our study on small invasive carcinomas cm inorder to more precisely analyze the genetic alterations that occurat malignanttransformation in pancreatic tumorigenesis Inaddition in a subset of our samples we perform deep targetednext generation sequencing on a larger set of additional tissuesamples in order to assess mutated loci through entire neoplasmsincluding areas of lowgrade dysplasia highgrade dysplasia andinvasive carcinoma These analyses reveal important features ofpancreatic tumorigenesisincluding evolutionary relationshipsbetween different regions within cystic neoplasms as well asmolecular alterations that may drive the transition from a noninvasive precursor lesion to invasive cancerResultsOverall approach In order to dissect the molecular relationshipsbetween noninvasive dysplastic lesions and invasive pancreaticcancers we performed whole exome sequencing of neoplastictissue samples from patients with small invasive carcinomas cm associated with neoplastic pancreatic cysts including patients with IPMNs and patients with MCNs Supplementary Data Whole exome sequencing was performed onone sample from the noninvasive component with highgradedysplasia and one sample from the invasive cancer in each caseand for three cases an additional noninvasive sample with lowgrade dysplasia was also analyzed by whole exome sequencingMatched normalsamples were analyzed by whole exomesequencing in each case to exclude germline variants and toidentify somatic mutations Whole exome sequencing was performed with an average total coverage of — distinct coverageof — generating TB of sequencing data SupplementaryData In addition to whole exome analyses we performed targetednext generation sequencing of microdissected tissue samplesfrom seven of the above cases six IPMNs and one MCN Forthese targeted analyses we performed laser capture microdissection to isolate neoplastic cells from every available tissue block ofthe noninvasive cyst and cancer specimens Separate sampleswere microdissected based on grade of dysplasia cell morphollocation This resulted in “ogy architecture and spatialadditional samples per case The targeted panel analyses includedall mutated loci identified in the whole exome sequencing of theseseven cases as well as the entire coding regions of wellcharacterized pancreatic driver genes Supplementary Data The targeted sequencing had an average coverage of —distinct coverage of — Supplementary Data We developed an integrated mutation calling pipeline torigorously assess mutations in all sample types in our analyses inorder to confidently identify even subclonal alterations in sampleswith low neoplastic purity see œMethods Fig Supplementary Data In addition we utilized both on target and off targetreads to examine focal copy number changes as well as loss ofheterozygosity throughout the genome Fig SupplementaryData and From our whole exome sequencing analyses weidentified an average of somatic mutations in samples fromnoninvasive components range “ and an average of somatic mutations in invasive carcinoma samples range“Fig 1a Supplementary Data An average of somatic mutations were shared between the noninvasive andinvasive components while somatic mutations were unique tosamples from noninvasive components and somatic mutationswere unique to samples from invasive cancer Fig 1a We alsoidentified an average of five shared copy number alterationsbetween noninvasive and invasive components as well as anaverage of one copy number alteration unique to samples frominvasive cancer A similar mean proportion of somatic mutationsand copy number alterations were unique to invasive samples for somatic mutations for copy number alterationsAnalysis of our combined whole exome and targeted sequencing data provided multiple insights into IPMN and MCNtumorigenesis In every analyzed case there were multiple sharedmutations between the noninvasive and invasive componentsThese included both driver and passenger mutations indicatingthat they shared a common phylogenetic ancestor Fig 1a b Inaddition accumulation of unique mutations in both noninvasiveand invasive components demonstrated independent evolutionafter the divergence of the subclone that gave rise to the invasiveFig Supplementary Figs S1“S18 Analysis ofcanceradditional adjacent lowgrade or highgrade samples from thesame lesions revealed a subset of shared mutations suggestingthat these dysplastic lesions preceded the development of theinvasive carcinoma Fig Supplementary Figs S1 S2 S3 S5and S16 Evolutionary analyses showed a branched phylogeny inNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178asnoitatuMCancerization onlyDuctal cancer onlyMucinous cancer onlySharedIPMN onlyMCN onlyPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMbKRASTP53CDKN2ASMAD4RNF43GLI3GNASMUC16PTPRTTGFBR2ATMCNTN5PIK3CAALKAPCRYR2STK11CTNNB1ERBB4EGFRSF3B1NOTCH1KEAP1ERBB2MYCRETTSC2PrecursorSharedCancerFig Somatic mutations identified in matched noninvasive and invasive cancer samples a In each patient sample multiple mutations were sharedbetween the noninvasive and invasive cancer samples gray In addition some mutations were limited to the noninvasive bluegreen while others werelimited to the cancer redpink Darker colors indicate alterations that were likely restricted to one component but where sequencing coverage in thesecond component was limited The proportions of shared and distinct mutations varied between different lesions b Somatic mutations in the mostfrequently mutated genes are categorized as shared between noninvasive and cancer gray limited to noninvasive blue or limited to cancer redMutations in some genes such as KRAS were always shared while others were enriched in samples from noninvasive RNF43 or cancer SMAD4MutationsMTP1MTP2MTP3MTP4MTP5MTP6MTP7MTP8MTP9MTP11MTP13MTP14MTP18MTP19MTP23MTP24MTP26MTP30chr1chr2chr3chr4chr5chr6chr7chr8chr9chr10chr11chr12chr13chr14chr15chr16chr17chr18chr19chr20chr21chr22Copy number log2ˆ’ˆ’ˆ’SamplesLG IPMNHG IPMNLG MCNHG MCNDuctal cancerMucinous cancerCancerizationFig Copy number alterations identified in matched noninvasive and invasive cancer samples Chromosomal gains red and losses blue are shownfor each chromosome in each patient with noninvasive samples on the left and cancer samples on the rightNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178MTP19HEbefore LCMafter LCMMTP8KRASG12DKRASG12RKRASQ61Hˆ’1225380275_TGKRASQ61Hˆ’1225380275_TAKRASG12VTMEM56ˆ’RWDD3c5662TC [SP]KCNA5A451AC17orf98T142MDUSP27T652TADAMTS12P429LCOL12A1R933HSCAF8G961VTMEM246A372AMCM10E613KPNLIPRP1T465TC13orf35L33LSMAD6L179LGRIN2AS397SAP1G1T798TTP53E171FCHO1c22471GC [SP]LRFN1S632SLTBP4P243LLOHchr17001ˆ’ABRF645SALPK1R873RLOHchrX275ˆ’DELchr9218ˆ’2377CDKN2ABSND861NSUPT6HI104TPLEKHF1D258DLOHchr221716ˆ’LTBRR425GACOT11R306LOR6N1Y120YENPP5Y341YITGB3C562BTKP116TSLC1A7G465DFGF3R104QZBED4P1100LSIM1R192CPHC1S627CMS4A1T41TLOHchr712725ˆ’SH2D5R199WRNPEPL1G413VGAL3ST2P85PJADE2N352SHHLA1A97VTMEM141Q61QBPTFR296HKLHL22R603RGALEQ261LANKS4BQ368RKRI1S326SOR8I2C240CLRTM2G319VLOHchr97123ˆ’LAMB3R887LSPTAN1I1484FTP53R175HTissue sourceLG IPMNHG IPMNMUCINOUS CANCERDUCTAL CANCERCANCERIZATIONSequencingTargeted SeqWhole Exome SeqDriver MutationHotspotInactivating_OtherDELLOH µm µmMPT19 LG IPMN µm µm µmMPT19 HG IPMN µm_________ µm µm µmMPT19 ductal cancer µm µm µmMPT19 cancerization T2 T1 µm µmMPT8 LG IPMN µm µm µmMPT8 HG IPMN µm__ µm µm µmMPT8 mucinous cancer T1 T2 TTC39AN83ILRRC8DI741MOBSCNR2837QRGS7A195AOR14A2A156ACCDC13S534SEGFLAMG553VITGA1V27LBDP1N622NKIAA0319V824FRIMS1R211QPRDM13A303VIFNGR1M1PHACTR2A348PGPR85F208FGNED83HHTR7T240MGRM5T946KKRASG12DUNC13CN497SBNIP2D58DCCDC33D354NCLDN9P187PNOTUMR308CPIEZO2R2407QC19orf24G52AMUC16P9201QCCNE1R85WGNASR201HPHF21BA116TTNS1R894QNDUFAF6L320ISLC15A1E26KCLUHR433PRR23AA151TBRD3R313HUSP34R3136HRHOAK27ESIM1A654TNRG1A540VVRK3S129NCCDC166A84TST3GAL4V165MTSPAN17P63PMAD1L1S327SC17orf47E142GCHERPL47ITRPC5E418DZFP37R18WGNASD464NMED12E1497KBAG2T93TRNF43R371CACNG2V255MMAGEB18A63VBRINP3A748APDE1BE401EQRICH2R160RRNF43P370fsINTS1G527VMYOM3G152SPRRT3P407STGFBR2R553HPDZD2I810IFAM120BR646CEPHB4V682MNOBOXR302CCEP164R897QMAB21L1T171MZCCHC14T80MCD68L295LGAS2L2S728LRUNDC3AE49EPHBI122VDNMT1N109NZNF865A721ACASS4S376SKCNJ6R322SLC5A4L390PLRRC8CR355HNPR1R439HPPP1CBV263LBIRC6E4424VIL1Q468QMOGAT3cˆ’2359GAUSP20A675TMEX3BS256YMUC13T30AZBTB10R456QSLC26A3V54IC9orf84E8DPPP1R36R303CPLEKHA6Q171SSH3P639PDSCAML1G1252DSLCO1C1R639KENDOVA2VCOL9A1A680TMC2RF228LBPIFB2A427SLOHchr812546ˆ’DELCDKN2ARNF43P660fsRNF43C471fsRNF43E39fsRNF43M1_G4delRNF43S321RNF43P231LFig Somatic mutations identified in MTP19 and MTP8 in targeted and whole exome sequencing We show mutations identified in each sampleincluding lowgrade IPMN light blue highgrade IPMN dark blue ductal cancer red mucinous cancer pink and cancerization purple The type ofsequencing analysis targeted or whole exome performed for each sample is indicated in a track on the bottom Representative images of neoplastic tissuestained by hematoxylin and eosin as well as isolated regions before and after laser capture microdissection are shown for MTP19 and MTP8NATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178MTP2MTP8MTP3MTP19MTP1MTP5MTP24LG IPMNHG IPMNLG MCNHG MCNDuctal cancerMucinous cancerCancerizationMutationsFig Evolutionary reconstruction of samples analyzed by whole exome and targeted sequencing In all cases noninvasive samples bluegreenprecede invasive samples redpink in the evolutionary history In MTP5 different invasive cancer samples are placed in different regions of thephylogeny highlighting multiple independent invasion events in this lesion In MTP19 a sample of cancerization purple has descended from invasivecancer sampleseach case with multiple clonally related but distinct dysplasticsamples from each neoplasm Fig Importantly removal ofdriver gene mutations from these analyses did not significantlyalter the resulting phylogenies indicating that the evolutionaryrelationships are supported by mutations in addition to driveralterations which might be shared by chance Thus the inferredevolutionary relationships between IPMNMCNs and cancersamples are robust as the probability of sharing a nonhotspotmutation due to chance alone is vanishingly small in million while the mean number of shared point mutations was the vast majority of which did not occur in hotspotsAnother potentialexplanation forshared mutationsthis sample impurityinnoninvasive and invasive samples is the presence of a smallnumber of cancer cells contamination in IPMNMCN samplesOur detailed pathological characterization and macro or microdissection minimized the risk ofInaddition variant allele frequencies VAFs of shared mutationsin IPMNMCN and cancer samples can also help to evaluate thelikelihood of such contamination In mutations shared betweenthe IPMNMCN and cancer the mean VAF in the noninvasivesamples was with only of shared mutations having aVAF below in the noninvasive samples These high VAFsindicate that the shared mutations were not the result of a smallnumber of cancer cells contaminating the noninvasive samplesOverall these data provide evolutionary evidence that IPMNs andMCNs were precursors to invasive pancreatic cancer with lowgrade regions usually preceding highgrade regions and ultimatelyresulting in invasive carcinomaDriver genes of IPMNMCN tumorigenesis Through wholeexome and targeted sequencing analyses of IPMNsMCNs andassociated invasive carcinomas we confirmed the high prevalenceof mutations in previously identified pancreatic driver genesincluding mutations of KRAS of cases GNAS CDKN2A TP53 SMAD4 and TGFBR2 Fig 1b Somatic mutations were also identified in RNF43 which has been previously highlighted for its role as a driver inmucinproducing pancreatic cysts4 Somatic mutations wereobserved at low prevalence in key positions in the PI3K PIK3CATSC2 and WNT APC CTNNA2 CTNNB1 signaling pathwaysas well as in STK11 Fig 1b Supplementary Data Alteration ofthese genes and pathways has been previously reported in afraction of IPMNs471314 The two MCNs analyzed were similarto the IPMNs in the cohort with hotspot mutations in KRAShomozygous deletion of CDKN2A and inactivating mutations inRNF43 among others but as expected these MCNs did not haveGNAS alterations Supplementary Figs S3 S76In addition to driver genes previously reported in IPMNs ourdata provide an opportunity to discover novel drivers of IPMNtumorigenesis We identified somatic mutations in the DNAdamage response gene ATM in of lesions including onenonsense mutation Fig 1b Supplementary Data In additionwe identified alterations in Hedgehog pathway member GLI3 in of cases Fig 1b Supplementary Data We alsoidentified somatic mutations in a previously described hotspot inSF3B1 which encodes a protein critical for RNA splicing Fig 1bSupplementary Data Amplifications of the wellcharacterizeddriver genes ERBB2 and MYC were each observed in a single caseand have not been previously reported in IPMNs SupplementaryData Other altered genes with a previously unknown role inIPMN tumorigenesis include MUC16 four cases PTPRT fourcases and CNTN5 three cases Fig 1b Intriguingly in onecase an STK11 mutation was found in combination with biallelicloss”theATM lossand cancerspecific biallelic KEAP1NATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178combination ofreported in lung cancers Supplementary Fig S315these three mutations has previously beenAlthough KRAS mutations occur in the majority of IPMNs andare thought to initiate tumorigenesis in these lesions two IPMNslacked mutations in this gene One case contained a hotspotmutation in codon of GNAS another potential initiator ofIPMN tumorigenesis as well as alterations in TP53 and RNF43Supplementary Fig S15 The other case lacked mutations in anyof the frequently altered pancreatic driver genes but containedhotspot mutations in both CTNNB1 S45P and SF3B1 H662QSupplementary Fig S10 These cases highlight alternativepathways of initiation and progression in IPMNs lacking KRASmutationsOrder of genetic alterations in IPMNMCN tumorigenesis Ourmultiregion sequencing approach of IPMNsMCNs and associated invasive carcinomas provided insights into the order ofspecific genetic alterations in pancreatic tumorigenesis In ofthe cases at least one somatic mutation in the initiating drivergenes KRAS and GNAS was shared between the noninvasivecomponent and associated invasive cancer with the remainingcase lacking mutations in these genes Somatic mutations in TP53and CDKN2A were also shared in the noninvasive componentand associated invasive cancer in the majority of cases In contrast SMAD4 had alterations confined to the invasive carcinomain three cases and was shared between noninvasive and invasivesamples in four cases Fig 1b The majority of SMAD4 alterations in all sample types were biallelic including in noninvasive samples and in invasive samples SupplementaryData Alteration of TGFBR2 which functions in the samesignaling pathway as SMAD4 was also restricted to the cancer inone case Fig 1b The other genes with mutations restricted tothe invasive cancer CDKN2A CNTN5 PIK3CA KEAP1 andRET only had this pattern in a single sample Fig 1bOur study also identified driver mutations in subclones ofnoninvasive neoplasms that diverged from and were not presentin invasive cancer These included hotspots mutations in wellcharacterized oncogenes and inactivating mutations in tumorsuppressor geneseg PIK3CA pE545K CTNNB1 pS45FSMAD4 pE33fs and multiple inactivating RNF43 mutations inpatient MTP3 Supplementary Fig S3 Mutations in RNF43were a particularly striking finding in these cases as somenoninvasive components contained several different RNF43mutations each limited to a small number of sections and noneinvolving the invasive cancer Fig Supplementary Fig S3 Inaddition to heterogeneity in RNF43 in early lesions we alsoidentified two cases with multiple mutationsin KRAS inprecursor lesions of which only one was present in the invasivecancer For example in MTP19 KRAS pG12V was present in themajority of IPMN samples as well as all the invasive cancersamples but there were an additional four other KRAS mutationsall occurring in hotspot positions that were present in a smallnumber of sectionsin lowgrade IPMN samples Fig Intriguingly these three lowgrade IPMN regions shared nomutations with the invasive cancertheyrepresented genetically independent clonessuggesting thatNotably while there were often many differences in thesomatic point mutations identified in the matched noninvasiveand invasive samples the copy number profiles were quite similarbetween IPMNMCNs and invasive cancers Fig Supplementary Data and the proportion of copy number alterationsunique to cancer samples was similar to that observed forsomatic mutations While homozygous deletion of some genesoccurred in the invasive cancer but notthe noninvasivecomponent such as CDKN2A in MTP8 Fig analyses ofchromosomal gains and losses through assessment of allelicimbalance revealed that an average of of the genome wassimilar in copy number in matched noninvasive and invasivesamples Fig Supplementary Data Insights into pancreatic neoplasia revealed by sequencing Thesamples analyzed by targeted sequencing were characterizedmorphologically and meticulously isolated using laser capturemicrodissection Even with this process we identified samples intwo cases that were characterized morphologically as IPMNs butthrough genomic and evolutionary analyses were determined tobe identical to or descendants of the associated invasive cancersFor example in MTP19 some of the samples originally identifiedmorphologically as noninvasive IPMN and T1 sharedall the mutations present in the invasive cancer sample T2 andcontained additional mutations suggesting that these samplesdescended from the cancer Figs Evolutionary analysessuggested that some of the morphologically identified IPMNsamples in this case as well as MTP1 actually representedintraductal spread of invasive carcinoma also referred to ascancerization of the ducts In these cases after invading thestroma the carcinoma invaded back into and colonized the cystsuch that it was morphologically indistinguishable from IPMNwith highgrade dysplasiaIn one case MTP5 we also identified an interesting pattern ofmultifocal invasion of the carcinoma In this case we analyzedfive different samples from invasive cancer”three samples wereisolated from a mucinous carcinoma and two samples wereisolated from a ductal carcinoma Based on evolutionary analysesof the patterns of shared and distinct mutations in the cancersand IPMNs we conclude thatthere were multiple separateinvasion events in this lesion as represented by the mutationsshared between the invasive cancers and noninvasive componentsas well as those that were unique to the specific invasive cancersFig Supplementary Fig S5As our study represents the largest cohort of comprehensivelysequenced IPMNsMCNs we also analyzed mutational signaturesin our dataset Intriguingly our data contrast somewhat with themutational signatures previously reported in pancreatic ductaladenocarcinoma PDAC1617 Like PDAC the most prominentmutational signature was associated with age Signature 1Awhich was identified in almost every case SupplementaryFig S19 However we also identified signatures associated withAPOBEC enzymes four cases smoking three cases andmismatch repair deficiency cases Although smoking isconsidered a risk factor for pancreatic cancer until now themutational signature associated with smoking has not beenreported in pancreatic neoplasia18Evolutionary timeline of highgrade IPMN to PDAC To estimate the time between the development of highgrade IPMN andPDAC we evaluated Bayesian hierarchical models for the numberof acquired mutations under a range of possible mutation ratesThese models estimate the time interval between a founder cell ofa PDAC and the ancestral precursor cell in the associated highgrade IPMN assuming that mutation rates and cell division timesare constantseeœMethods We performed this analysis on the paired WES datafrom of our cases Supplementary Fig S20 We excludedMTP19 because our evolutionary analyses demonstrated intraductal spread of invasive carcinoma and as such we lacked WESdata from an IPMN sample in this case In the analyzed casesthe average median time to progression from IPMN to PDAC was years but the models showed a bimodal distribution Thismedian time was nearly years for patients but nearly yearsthis period of developmentthroughoutNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178for patients with more than acquired mutations highlightingpotential variability in progression time between patients Forexample in patient MTP1 most models suggested an average of years between the development of the IPMN and the PDAC CI “ years In contrast for patient MTP2 with additional mutations acquired in the PDACthe transitionappears to have been slower with an average estimate of years CI “ years from the Bayesian models Overall theseanalyses suggest that for most patients there is a significantwindow of time between development of highgrade dysplasiaand pancreatic cancer providing an opportunity for surveillanceand interventionDiscussionThis study representsthe largest dataset of whole exomesequencing of IPMNs and MCNs to date Importantly our dataestablished that both IPMNs and MCNs are direct precursors ofinvasive pancreatic cancer Fig This conclusion has beenpreviously suggested by the morphological relationship betweenthe noninvasive neoplasms and invasive cancer on traditionalhistologic sections as well as shared driver gene mutations intargeted sequencing studies679“ However the presence ofmany shared driver and passenger mutations clearly demonstrated the common origin of IPMNsMCNs and invasive pancreatic cancers in our study and evolutionary analyses revealedthat dysplastic lesions precede invasive cancers Evolutionaryanalyses suggested that highgrade noninvasive lesions occur over years before invasive carcinoma providing a window ofopportunity for early detection and interventionIn this study we identified somatic mutations in driver genesthat had not been previously implicated in IPMNsMCNs Forexample we identified alterations in the DNA damage responsegene ATM in of the analyzed cases Germline mutations inATM have been recently reported in patients that developedIPMNs highlighting the potential importance of this gene inIPMN risk19 In addition mucinous colloid carcinomas aresignificantly more common than typical ductal carcinomas inpatients with germline ATM mutations further highlighting thelink between mutations in this gene and IPMNs20 Although theATM gene is large potentially increasing the likelihood of passenger or artifactual mutations even larger genes such as TTNhad a lower mutation prevalence suggesting that at least some ofthe alterations identified in ATM are likely to be bona fidesomatic mutations Somatic mutations in GLI3 which encodes acomponent of the Hedgehog signaling pathway were identified in of cases Somatic mutations in GLI3 were recently reportedin a distinct morphological variant of pancreatic carcinomaundifferentiated carcinoma with osteoclastlike giant cells aswell as at a low prevalence in sporadic PDAC suggesting that theimportance of GLI3 mutations and its signaling pathway inpancreatic tumorigenesis may extend beyond IPMNsMCNs21“The hotspot mutations in SF3B1 which encodes a protein criticalfor RNA splicing are also potential drivers in the IPMN pathwayHowever somatic mutations in this gene have been reported in avariety of other neoplasms including hematologic malignanciesand uveal melanoma24“We highlight somatic alteration of the SMAD4 pathway as aputative driver of progression to invasive cancer as mutations inSMAD4 or TGFBR2 occurred only in invasive cancer samples in of the cases analyzed SMAD4 was the only gene with cancerspecific mutations in more than one case highlighting thepotentially unique role this gene plays in pancreatic carcinogenesis This role has been previously suggested by next generationsequencing of highgrade PanINs showing an absence of SMAD4mutations in precancerous lesions as well as cancerspecificSMAD4 mutationsreported in a paired PanINcarcinomaanalyses2728 Loss of SMAD4 expression limited to invasivecarcinomas has been reported in MCN and IPMNassociatedinvasive cancers and targeted sequencing of a small number ofIPMNs and matched cancers identified a single case with aSMAD4 mutation occurring only in the cancer72829 In our datathere were also four cases where mutations in SMAD4 wereshared between noninvasive and invasive cancer samples and twowhere SMAD4 mutations were limited to the noninvasive component Although our wholeexome approach could not detect alltypes of SMAD4 alterations such as rearrangements or epigeneticchanges the majority of SMAD4 mutations observed in bothnoninvasive and invasive components affected both alleles Takentogether this suggests that the role of SMAD4 mutations may notbe universal and may depend on other factors including cellintrinsic such as somatic mutations in other driver genes andcell extrinsic such as stromal and immune microenvironmentmechanismsAlthough some of our cases had SMAD4 mutations limited tothe invasive cancer most of the IPMNMCNassociated cancerslacked driver gene alterations that were associated with invasivedisease suggesting that malignant progression is not universallydriven by point mutations Previous studies have specificallydemonstrated the importance of copy number alterations andchromosomal rearrangements in pancreatic tumorigenesis30 Wedid not identify large differences in the copy number profilesbetween noninvasive components and associated invasive cancers suggesting that global genomic instability may be importantas an early feature of tumorigenesis but is not likely to drivemalignant transformation in many casesOur study also revealed prevalent genetic heterogeneity indriver gene mutations in early lesions demonstrating morecomplex processes than previously suggested by traditional lineartumorigenesis models Similar to our recently reported polyclonalorigin of IPMNs12 we identified multiple independent clonesinitiated by distinct KRAS mutations in two cases in the currentstudy In addition our study identified multiple distinct inactivating mutations in RNF43 limited to unique tumor subclones apattern previously observed by our group and not shared by othergenes in our whole exome sequencing analyses1231 In most casesRNF43 mutations were enriched in noninvasive components andabsent from the associated invasive cancers More generally weobserved multiple instances of clear driver mutations includinghotspot mutations in oncogenes as well as inactivating mutationsin tumor suppressor genes that were limited to the noninvasivecomponents and not present in the associated invasive cancerThus these mutations occur and clonally expand in the IPMN orMCN but are not present in the subclone that subsequentlyinvades Such independent evolution of premalignant lesions hasbeen observed in other an sites and does not diminish theconclusion of our evolutionary analyses that IPMNsMCNs aretheseprecursors ofobservations suggest unique selective processes at different timepoints in tumorigenesis such that mutations selected in theprecancerous lesion are not selected for or are even selectedagainst in the invasive cancerinvasive pancreatic cancer32“ RatherIn addition to these observations about clonal evolution innoninvasive lesions our data also provide genetic evidence formultiple underappreciated processes in pancreatic neoplasiaFirst we provide genetic evidence for intraductal spread ofinvasive carci

Thyroid_Cancer

" formulated a traditional Chinese medicine TCM prescription Hanshiyi Formula HSYF which was approved and promoted by the Wuhan Municipal Health Commission for treating mild and moderate coronavirus disease COVID19 We aimed to evaluate the effect of HSYF on the progression to severe disease in mild and moderate COVID19 patients We conducted a retrospective cohort study of patients with mild and moderate COVID19 in a quarantine station in Wuchang District Wuhan Using the realtime Internet information collection application and Centers for Disease Control for the Wuchang District patient data were collected through patient selfreports and followups HSYF intervention was defined as the exposure The primary outcome was the proportion of patients who progressed to a severe disease status and a stratification analysis was performed Univariate and multivariate regression analyses were performed to identify influencing factors that may affect the outcome Further we used pr sity score matching PSM to assess the effect of HSYF intervention on the conversion of mild and moderate to a severe disease status Totally mild and moderate COVID19 patients were enrolled including HSYF users exposed group and nonusers control group No cases in the exposed group and P cases in the control group progressed to severe disease and the difference between the two groups exposed groupcontrol group was cid0 [ confidence interval CI cid0 cid0 ] Univariate regression analysis revealed sex male age fever cough and fatigue as risk factors for progression to severe disease After PSM none of the HSYF users and P non users transitioned to severe disease and the difference between the two groups exposed groupcontrol group was cid0 [ CI cid0 cid0 ] Multivariate regression analysis revealed that sex male [OR CI “ P ] and age years [odds ratio OR CI “ P ] were independent risk factors for conversion to severe disease Therefore HSYF can significantly reduce the progression to severe disease in patients with mild and moderate COVID19 which may effectively prevent and treat the disease However further larger clinical studies are required to verify our results Corresponding authors Email addresses tina_yai126com J Tian yanshiyan0927sinacom S Yan wanghan4313163com H Wang novelzhangsinacom Y Zhang luoyuorz163com Y Zheng drwhrfoxmailcom H Wu leexiuyang126com X Li zhengzhigaozezheng163com Z Gao aiyanke163com Y Ai christiana55555163com X Gou tcmxpzl126com L Zhang hely3699163com L He lfm565sohucom F Lian 13601180524139com B Liu tongxiaolinvip163com X Tong Jiaxing Tian Shiyan Yan Han Wang and Ying Zhang contributed equally to this study 101016jphrs2020105127 Received April Received in revised form August Accepted August PharmacologicalResearch1612020105127Availableonline10August2020104366182020ElsevierLtdAllrightsreserved 0cJ Tian Introduction At the end of December a series of coronavirus disease COVID19 cases emerged in Wuhan Hubei Province China and the number of diagnosed cases have rapidly increased since then [] In the early stage of the epidemic a large number of patients with mild and moderate COVID19 were diagnosed and treated in quarantine stations Identifying effective treatments for early intervention can improve the prognosis of patients with mild and moderate COVID19 which can also help control the epidemic progression and reduce the medical burden Wuchang District is located at the center of Wuhan with a high population density It was one of the most severely affected areas with many confirmed COVID19 cases For a large number of patients with mild and moderate COVID19 in the quarantine station the Wuhan Municipal Health Commission adopted a combination of Chinese herbal medicines and used Internet data to tackle the pandemic Our study group formulated a traditional Chinese medicine TCM prescription named Hanshiyi Formula HSYF to prevent the exacerbation of the epidemic This was approved and promoted by the Wuhan Municipal Health Commission for the treatment of mild and moderate COVID19 patients in quarantine stations [] Data was collected from the realtime Internet information collection application Yuge medical system which was convenient for physicians to communicate with isolated patients in real time dynamically monitor changes in the patient™s condition observe the efficacy of HSYF in mild and moderate COVID19 patients and adjust the treatment plan if necessary In guiding the treatment of mild and moderate COVID19 patients we found that after HSYF intervention patients with mild and moderate disease experienced rapid resolution of symptoms and fewer patients transitioned to a severe disease status Therefore we compared the clinical data of mild and moderate COVID19 patients treated with HSYF with patients who were diagnosed but were not treated with HSYF Data were collected from the database of Wuhan Wuchang District Disease Control Center CDC as well as from the realtime Internet information collection application to evaluate the effect of HSYF on the proportion of COVID19 patients who progressed from a mild and moderate to severe disease status Methods Study design and participants In this retrospective cohort study conducted in the Wuchang District Wuhan data were collected from mild and moderate COVID19 patients in quarantine stations in Wuchang District Wuhan before March The exposed group included patients who were administered HSYF for more than days If there is no adverse effect or disease progression HSYF can be taken continuously until recovery The control group included patients who were diagnosed with COVID19 but were not administered HSYF including decoction granules etc at the same time Data on age sex disease duration medical history initial symptoms concomitant medication and outcomes of the two groups were collected and the difference in the proportion of mild to moderate COVID19 patients who transitioned to a severe status were analyzed Fig The diagnosis of COVID19 patients was made by the designated tertiary hospital in Wuchang District Wuhan The diagnostic criteria was according to the Diagnosis and Treatment Guideline for COVID19 released by the National Health Commission of the People™s Republic of China [] The study included patients who met the criteria of mild and moderate COVID19 the mild COVID19 patients usually presented mild clinical symptoms with no radiological manifestations of pneumonia the moderate COVID19 patients had fever and respiratory symptoms with radiological manifestations of pneumonia Severe and critical patients were excluded This study was approved by the institutional ethics board of Hubei Provincial Hospital of Traditional Chinese Medicine No HBZY2020 C0101 and was registered with chictrcn ChiCTR2000029601 Data collection Data from the exposure group was first provided by the patients who scanned the QR code on the medication box and entered data on the real time Internet information collection application After downloading and sorting the data if there were mistakes or lack of relevant information a medical staff member followed up the patients by telephone and if necessary contacted the staff of the community health service station to verify the information to ensure accuracy and completeness of the data Data from the control group were provided by the Wuhan Wuchang District CDC database Data of mild and moderate COVID19 patients diagnosed at the same time were screened and downloaded and a telephone followup was conducted by a medical staff member to ensure accuracy and completeness of the data All data were collected using a standard electronic database In order to ensure the accuracy of the data and avoid bias data were verified by two researches A and B and a third researcher C resolved any dispute between the first two researchers The results of the study were analyzed and reported in accordance with the STROBE guidelines Fig Flow chart of the observational research PharmacologicalResearch16120201051272 0cJ Tian Table Demographic and Patient Characteristics All patients Exposed group Control group Age Mean ± SD 014yr 1549yr 5064yr ‰¥65yr ‰¤48yr ＞48yr Sex Female Medical history Medical history Hypertension Coronary heart disease Diabetes Bronchial asthma Chronic obstructive pulmonary disease Hyperlipidemia Fatty liver Gallbladder disease Thyroid disease Stroke Chronic glomerulonephritis Cancer Hepatitis Tuberculosis Other diseases Initial symptoms Initial symptoms Fever Cough Diarrhea Fatigue Conjunctivitis Other symptoms Concomitant medication Antiviral treatment Oseltamivir Lopinavir Aciclovir Ribavirin Arbidol Other antiviral drugs Antibiotics Antibiotics Amoxicillin Cephalosporin Levofloxacin Moxifloxacin Clarithromycin Other antibiotics Chinese patent medicine Chinese patent medicine Lianhua Qingwen capsule Xiaochaihu granule Shuanghuanglian oral solution Huoxiang Zhengqi preparation Cough syrup Banlangen preparation Ganmao Qingre granule Other Chinese patent medicines ± ± ± Statistics Z10789 X210724 X2 X20817 X21796 X23145 X21788 X21023 X23933 X2＜ X2 X20318 X20186 X24649 X22578 X20812 X22638 X2＜ X25259 X20567 X2 X2 X2 X2 X2 X21049 X2 X20091 X27352 X2 X2 X2 X20250 X2 X21648 X20218 X27552 X210734 X20257 X22752 X27191 X23935 X24895 X2 X211160 X210579 X2 X27812 Pvalue ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ Outcomes Statistical analysis The primary outcome of this study was the proportion of mild and moderate COVID19 patients who progressed to a severe disease status We also analysed factors that may affect the outcome including the patient™s age sex disease duration medical history initial symptoms concomitant medication and grouping Pr sity score matching PSM was used to further assess the effect of HSYF intervention on the conversion from mild and moderate COVID19 to a severe status Statistical analyses were conducted using SPSS software SPSS Inc Chicago IL United States Twosided tests were used and Pvalues were considered statistically significant Numerical variables are summarized as mean ±SD The data of the categorical variables are described as numbers and percentages The ttestWilcoxon ranksum test was used to compare the ages between the two groups The chisquare testFisher™s exact test was used to compare the sex medical history initial symptoms and concomitant PharmacologicalResearch16120201051273 0cJ Tian medication between the two groups to calculate the rate difference and confidence interval CI Using the proportion of patients who transitioned to a severe status as the dependent variable the effects of age sex disease duration medical history initial symptoms concomitant antiviral drugs antibiotics Chinese patent medicines and grouping were analyzed by univariate and multivariate logistic regression For factors with a statistical significance in the univariate regression analysis a logistic regression was performed to match the two groups of patients by pr sity score in a ratio and the difference in the primary outcome between the two groups was evaluated Results Demographic and patient characteristics By March we enrolled mild and moderate COVID19 patients from quarantine stations in the Wuchang District After further screening patients who refused followup patients who were not diagnosed and two patients who took other types of TCM prescriptions were excluded Finally patients were included in our study cohort including HSYF users and nonusers including decoction granules etcAmong all patients enrolled in this study were men and were women The median age of the patients was years old The median age of the exposed group was significantly lower than that of the control group P We found that of patients had a medical history which included hypertension fatty liver and diabetes A significantly higher proportion of patients in the exposed group had a history of bronchial asthma vs P and thyroid disease vs P compared with the control group while the proportion of patients with hyperlipidemia vs P was higher in the control group than the exposed group Among all patients had experienced initial COVID19 symptoms The number of patients with diarrhea in the exposed group was significantly higher than that in the control group vs respectively P and the number of patients with fever vs P cough vs P and fatigue vs P was significantly lower in the exposed group than in the control group The patients were administered multiple medications during the treatment There were no differences in the number of patients receiving antiviral drugs antibiotics and Chinese patent medicines between the two groups In terms of antiviral treatment ribavirin vs P and arbidol vs P were the most commonly administered medications in the exposed group while oseltamivir vs P and acyclovir vs P were the most commonly administered medication in the control group In terms of antibiotics amoxicillin vs P and moxifloxacin vs P were most commonly used in the exposed group while clarithromycin vs P were most commonly used in the control group In terms of Chinese patent medicines the patients in the exposed group were usually administered Xiaochaihu granules vs P Shuanghuanglian oral solution vs P cough syrup vs P Banlangen preparation vs P and Ganmao Qingre granules vs P while the patients in the control group were administered Lianhua Qingwen capsule vs P and Huoxiang Zhengqi preparation vs P as shown in Table The majority of onset times in the two groups were from January to February vs The proportion to severe status Our primary outcome was the proportion of COVID19 patients who progressed from mild and moderate COVID19 to a severe disease status There were no cases of progression to severe disease in the exposed group while cases P in the control group progressed to severe disease The difference between the two groups in terms of progression to severe disease exposed groupcontrol group was cid0 [ CI cid0 cid0 ] Stratification analysis showed that excluding diarrhea there were significant differences between the two groups in terms of sex age years medical history initial symptoms fever cough diarrhea fatigue concomitant medication etc P Table Efficacy evaluation The proportion to severe status The proportion to severe status Sex Male Female Age Age‰¤48yr Age＞48yr Underlying disease Yes No Initial symptoms Yes No Fever Yes No Cough Yes No Diarrhea Yes No Fatigue Yes No Chinese patent medicine Yes No All patients Exposed group Control group Statistics X2 X2 X28004 X29521 X2 X29251 X2 X2 X2 X27842 X26575 X211744 X2 X21915 X2 X2 X28085 Pvalue ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ ＜ PharmacologicalResearch16120201051274 0cFactors OR95 CI Univariate analysis Pvalue Table Univariate and multivariate regression analysis J Tian therefore these factors were accounted for in the subsequent analysis Table The univariate logistic regression analysis showed that sex male age years fever cough and fatigue were risk factors for progression to a severe status We further performed a multivariate logistic regression analysis to adjust for the above risk factors The results showed that after adjusting for all factors sex male OR CI “ P and age years OR CI “ P were independent risk factors for progression to a severe disease status Table Considering the difference in sample size between the two groups and the imbalance in confounding factors the risk factors analyzed in the univariate logistic regression analysis were used as variables for PSM caliper 025σ and the number of patients in both groups were Among them no patients in the exposed group progressed to severe disease and seven patients in the control group progressed to severe disease P The difference between the two groups in terms of progression to severe disease exposed groupcontrol group was cid0 [ CI cid0 cid0 ] Table Discussion Age The global prevalence of COVID19 has brought tremendous medical pressure worldwide According to data from Johns Hopkins University in the United States by April the number of COVID19 cases exceeded million globally Furthermore the number of diagnosed cases in the United States were over and that in Italy and Spain were over with the number of newly diagnosed cases still rising [] With a large number of people in close contact with mildmoderate COVID19 patients all countries have adopted community control and home quarantine measures to prevent the spread of the epidemic However during home quarantine problems such as crossinfection and cluster outbreaks in the community have emerged due to the lack of strict community supervision as well as the need to purchase necessities and travel for hospital consultations In addition mild and moderate COVID19 patients who were under home quarantine lacked effective intervention strategies without the guidance from physicians which may have resulted in problems such as the progression from mild and moderate to severe disease [] In such patients an effective largescale community intervention strategy could reduce this conversion to severe disease and reduce the number of severe and critical COVID19 patients thus significantly improving and controlling the COVID19 epidemic For mild and moderate COVID19 patients antiviral antibiotic and symptomatic supportive treatments are most commonly used However most of the antiviral drugs used to treat COVID19 currently are based on previous treatments for severe acute respiratory syndrome SARS Middle East respiratory syndrome and influenza A and uncertainties on the efficacy and side effects of these drugs remain problematic One case report stated that a patient was cured with remdesivir [] In another clinical study lopinavirritonavir had no clinical benefit on the treatment of patients with severe COVID19 and adverse gastrointestinal events were identified [] Furthermore Abidol and lopinavirritonavir did not improve COVID19 symptoms or shorten the conversion of the viral nucleic acid to a negative result in a clinical study of patients [] The adverse reactions and side effects of related antiviral drugs such as hypocalcemia hemolytic anemia hypomagnesemia as well as those that emerged during the treatment of SARS by ribavirin cannot be ignored [] Hydroxychloroquine and chloroquine were urgently approved by the Food and Drug Administration to treat COVID19 but chloroquine drugs hydroxychloroquine and chloroquine phosphate can cause adverse reactions such as dizziness headache vertigo loss of appetite and nausea [] Our study showed that under the conditions of home quarantine TCM is effective in reducing the progression of mild and moderate COVID19 to severe disease which can serve as a reference for the Multivariate analysis Pvalue ＜ ＜ OR95 CI continued on next page SexRef female Medical history Ref no Fever Cough Diarrhea Fatigue PharmacologicalResearch16120201051275 0cJ Tian Table continued Factors Univariate analysis Pvalue OR95 CI Multivariate analysis Pvalue Antiviral treatment Ref no Antibiotics Ref no Chinese patent medicine Ref no Grouping Ref control group OR95 CI \u3000 The model includes age and sex The model includes age gender and medical history The model includes age sex medical history and initial symptoms The model includes age gender medical history initial symptoms and antiviral drugs The model includes age gender medical history initial symptoms antiviral drugs and antibiotics The model includes age gender medical history initial symptoms antiviral drugs antibiotics and Chinese patent medicine The model includes age gender medical history initial symptoms antiviral drugs antibiotics Chinese patent medicine and grouping prevention of the disease As a key point of COVID19 prognosis there have only been a few studies assessing the proportion of mild and moderate COVID19 patients who have progressed to a severe disease status currently In attempting to prevent and control the epidemic in China TCM has been widely used Some studies revealed that TCM can reduce the clinical symptoms of fever and cough in mild and moderate patients [] and improve relevant imaging indexes [] However these studies usually had small sample sizes and did not assess the proportion of mild and moderate patients who progressed to a severe disease status In this retrospective cohort study the results showed that the main influencing factors for the progression of mild and moderate COVID19 to severe disease included sex male age fever cough and fatigue which are consistent with other studies [“] After excluding the effects of the above factors we found that the HSYF intervention can effectively reduce the conversion of mild and moderate COVID19 to severe disease providing evidence on the effectiveness of TCM Thus TCM can be an option in addition to antibiotic therapy antiviral treatment and supportive oxygen therapy for the treatment of mild and moderate COVID19 patients HSYF administered in the study mainly contains the following Chinese materia medica Ma Huang Ephedrae Herba Shi Gao Gypsum fibrosum Xing Ren Armeniacae Semen Qiang Huo Notopterygii Rhizoma seu Radix Ting Li Zi LepidiiDescurainiae Semen Guan Zhong Cyrtomii Rhizoma Di Long Pheretima Xu Chang Qing Cynanchi paniculati Radix Huo Xiang Pogostemonis Herba Pei Lan Eupatorii Herba Cang Zhu Atractylodis Rhizoma Yun Ling Poria Sheng Bai Zhu Atractylodis macrocephalae Rhizoma Jiao Shan Zha Crataegi Fructus Jiao Shen Qu Massa medicate fermentata Jiao Mai Ya Hordei Fructus germinatus Hou Po Magnoliae officinalis Cortex Jiao Bing Lang Arecae Semen Wei Cao Guo Tsaoko Fructus and Sheng Jiang Zingiberis Rhizoma recens In TCM theory these prescriptions can improve the patients™ healthy qi and dispel evil factors Ma Huang Ephedrae Herba Shi Gao Gypsum fibrosum Xing Ren Armeniacae Semen Ting Li Zi LepidiiDescurainiae Semen focus on improving asthma symptoms in the lung Yun Ling Poria Sheng Bai Zhu Atractylodis macrocephalae Rhizoma Jiao Shan Zha Crataegi Fructus Jiao Shen Qu Massa medicate fermentata Jiao Mai Ya Hordei Fructus germinatus Hou Po Magnoliae officinalis Cortex Jiao Bing Lang Arecae Semen and Wei Cao Guo Tsaoko Fructus help strengthen the healthy qi Huo Xiang Pogostemonis Herba and Pei Lan Eupatorii Herba can dispel the evil factors Studies have shown that many Chinese components of HSYF can exert antiviral effects and improve respiratory symptoms In terms of the antiviral components methyl ephedrine Lephedrine and Dpseudoephedrine are present in Ma Huang Ephedrae Herba which can significantly inhibit the in vitro proliferation of influenza A H1N1 virus [] Patchouli alcohol in Huo Xiang Pogostemonis Herba can effectively inhibit the replication of H1N1 virus [] Hou Po Magnoliae officinalis Cortex can alter the cell cycle and promote H1N1 infected cells into the S phase [] the magnolol extract can inhibit the secretion of CD44 and CD54 reduce the levels of inflammatory factors IL1β IL6 and TNFα and relieve inflammation [] The active ingredients of Wei Cao Guo Tsaoko Fructus inhibit the binding region of severe acute respiratory syndrome coronavirus SARSCoV2 Sprotein to human ACE2 and control the replication of SARSCoV2 in the human body [] In addition to its antiviral effect Ma Huang Ephedrae Herba is a widely used Chinese herbal medicine for respiratory diseases and it also stimulates β receptors of the bronchial smooth muscle expands the bronchus exerts antiinflammatory effects [] and can stimulate central nervous system excitability and regulate body temperature [] This plays an important role in improving the symptoms of COVID19 including fever cough shortness of breath and other respiratory symptoms The clinical benefits and risks need to be balanced for the treatment of mild and moderate COVID19 The lack of understanding on the efficacy side effects and other risks associated with current antiviral treatments make it difficult to be used as a conventional intervention for mild and Table Pr sity score matching PSM Variables Mean ± SD MedianQ1Q3 Age SexRef female Fever Cough Fatigue Before PSM Exposed group n ± Control group n ± Pvalue ＜ ＜ ＜ ＜ After PSM Exposed group n ± Control group n ± Pvalue PharmacologicalResearch16120201051276 0cAppendix A Supplementary data J Tian moderate COVID19 patients However the results of our study provide evidence on the effectiveness of TCM for the treatment of mild and moderate COVID19 TCM treatment has the advantage of being widely used and inexpensive making it convenient for mild and moderate COVID19 patients isolated in the largescale community to quickly recover Furthermore our study used a realtime Internet information collection application to collect data which enabled physicians to communicate with the patients in real time and dynamically monitor changes in the patient™s condition Thus selfquarantined patients did not need to go to the hospital for a consultation which prevented cross infection Although the results of our study are significant and provides insight on the use of TCM treatment for mild and moderate COVID19 there are still some limitations Firstly our study was a retrospective study rather than a prospective randomized clinical trial RCT due to the current situation In the future we aim to conduct RCTs and experiments to further verify the efficacy and mechanism of HSYF Secondly as a retrospective cohort study some baseline factors between the exposed group and the control group were not completely accounted for and the age of the exposed group was significantly younger than that of the control group which may be related to our data collection method Using a realtime Internet information collection application is a more acceptable method in the younger population and imbalances in baseline data such as sex age and medical history could have introduced bias which may have affected the results of the study Nevertheless we have corrected these possible influencing factors during our analysis In order to improve the reliability of the study results a cohort study with a larger sample size or RCTs is necessary Conclusion [] National Health Commission of the People™s Republic of China National HSYF can significantly reduce the progression of mild and moderate COVID19 to a severe disease status which has a positive effect on the prevention and treatment of the disease However future clinical studies with a larger sample size are needed to further verify our results Funding This work was funded by the Special Project for Emergency of the Ministry of Science and Technology 2020YFC0845000 and the Traditional Chinese Medicine Special Project for COVID19 Emergency of National Administration of Traditional Chinese Medicine2020ZYLCYJ041 Declaration of Competing Interest Authors declare no competing interests Jiangsu Kanion Pharmaceutical Co Ltd provided the medications for the study while they did not participate in research design data collection data analysis data interpretation nor article writing Acknowledgements We acknowledge all volunteers and healthcare workers in the Wuchang community health service station Hubei Provincial Hospital of Traditional Chinese Medicine and Jiangsu Kanion Pharmaceutical Co Ltd Supplementary material related to this article can be found in the online version at 101016jphrs2020105127 References [] H Lu CW Stratton YW Tang Outbreak of pneumonia of unknown etiology in Wuhan China the mystery and the miracle J Med Virol “ [] Medical treatment group of Wuhan COVID prevention and control headquarters Notice Concerning the Recommendation of the Prescription of Traditional Chinese Medicine in the Treatment of COVID19 February [] XL Tong XY Li LH Zhao Discussion on traditional chinese medicine prevention and treatment strategies of coronavirus disease COVID19 from the perspective of œColddampness pestilence J Tradit Chin Med “ Administration of Traditional Chinese Medicine of the People™s Republic of China Guidance for Corona Virus Disease Prevention Control Diagnosis and Management People™s Medical Publishing House March [] World health anization Coronavirus disease COVID19 Situation dashboard whosprinklrcom [] A Kimball KM Hatfield M Arons

Thyroid_Cancer

thyroid stimulating hormone highlightspleiotropic effects and inverse association withthyroid cancerWei Zhouet alThyroid stimulating hormone TSH is critical for normal development and metabolism Tobetter understand the genetic contribution to TSH levels we conduct a GWAS metaanalysisat million genetic markers in up to individuals and identify genomewidesignificant loci for TSH of which are previously unreported Functional experiments showthat the thyroglobulin proteinaltering variants P118L and G67S impact thyroglobulin secretion Phenomewide association analysis in the UK Biobank demonstrates the pleiotropiceffects of TSHassociated variants and a polygenic score for higher TSH levels is associatedwith a reduced risk of thyroid cancer in the UK Biobank and three other independent studiesTwosample Mendelian randomization using TSH index variants as instrumental variablessuggests a protective effect of higher TSH levels indicating lower thyroid function on risk ofthyroid cancer and goiter Our findings highlight the pleiotropic effects of TSHassociatedvariants on thyroid function and growth of malignant and benign thyroid tumorsA list of authors and their affiliations appears at the end of the paperNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zNormal thyroid function is essential for proper growth anddevelopment and for metabolic functions Approximately million people in the United States are affected bythyroid disorders and of the population is expected todevelop thyroid conditions over their life span1 Thyroidstimulating hormone TSH is secreted by the pituitary glandand stimulates the growth of the thyroid gland and its synthesisand secretion of thyroid hormones These include thyroxine T4most of which is converted to its more bioactive form ²triiodothyronine T3 TSH levels are negatively regulated by T3and T4 and lower or higher levels than the reference rangerespectively usually suggest that the thyroid gland is overactive asin primary hyperthyroidism or underactive as in primary hypothyroidism The complex inverse relationship between TSH andthyroid hormones means TSH is a more sensitive marker ofthyroid status a feature that has been used to identify individualswith thyroid dysfunction2Thyroid disorders affect multiple ans and are associatedwith a range of clinical consequences including an increased riskof metabolic disorders and cardiovascular mortality3“ Over thepast few decades a steady increase of the incidence rates of nonmedullary thyroid cancer henceforth referred as thyroid cancerhas been observed in most areas of the world including in Europe7 Previous studies have led to inconsistent s on therelationship between TSH levels and thyroid cancer risk8 Severalstudies have observed an association between low TSH levelswhich can occasionally occur as a consequence of autonomousthyroid nodules and an increased risk of thyroid cancer8“ Incontrast several studies have indicated that TSH promotes thegrowth of thyroid cancers814“ which has led to the recommendation to lower TSH levels among people with thyroid cancerto reduce the risk of cancer recurrence Two initial genomewideassociation studies GWAS identified five significant loci forthyroid cancer in Europeans and the risk alleles of all five locihave been associated with decreased TSH levels1718 In contrast amore recent GWAS identified five additional loci associated withthyroid cancer none of which were even nominally associatedwith TSH19 A recenttwosample Mendelian randomizationstudy suggested a causal inverse association between TSH levelsand overall cancer risk including thyroid cancer20 Additionalstudies are needed to clarify the role of TSH and TSHassociatedvariants in thyroid cancerTwin studies have shown TSH levels are moderately heritablewith estimates up to Previous TSH GWAS studies haveidentified independent TSHassociated loci172223 accountingfor of TSH variance thus leaving a large proportion of theTSH heritability unexplained2324 With the goal of identifying themissing genetic components for TSH to further understand itsunderlying genetic architecture and impact on thyroid cancer weperform a GWAS metaanalysisfor TSH levels on thepopulationbased NordTr¸ndelag Health Study HUNT studyN Michigan Genomics Initiative26 MGI N consortium up to N and the ThyroidOmics samples23To investigate the genetic relationship between TSH andthyroid cancer and other human diseases we examine phenomewide associations in the UK Biobank UKBB27 for TSHassociated index variants We also conduct phenomewide association tests for the polygenic scores PGS of TSH in the UKBBand the FinnGen study We observe an association between highTSH PGS and low thyroid cancer risk and replicate that observation in two other study populations from Columbus USA andIceland19 To evaluate the potential causality of TSH on thyroidcancer we perform a twosample Mendelian Randomizationanalysis using the TSHassociated top association signals asinstrumental variables and the thyroid cancer GWAS results on individuals cases and controls from ametaanalysis of UKBB2728 MGI26 and results from a previousmetaanalysis for thyroid cancer based on a Icelandic data setfrom deCODE referred to as deCODE in this manuscript aswell as four other case“control data sets with European ancestryas reported in Gudmundsson et al19ResultsDiscovery of genetic loci for TSH We identified loci associated with TSH Table Supplementary Data and andSupplementary Fig in our metaanalysis of the HUNT studyN the MGI biobank N and the ThyroidOmics consortium23 up to N Twentyeight of the loci have not been previously reported for TSH172223Table To identify secondary independent association signalswe performed stepwise conditional analysis within each locususing GCTACOJO29 based on GWAS summary statistics fromthe metaanalysis of HUNT MGI and ThyroidOmics and thelinkage disequilibrium LD correlation between variants estimated in HUNT We observed additional associations in novelTSH locus B4GALNT3 and previously known TSH lociTable and Supplementary Data In total independent topvariants have been identified at the loci explaining ofthe variance of TSH levelsDespite having only moderate effect sizes top variants inseveral novel TSH loci point to nearby genes with a known orsuspected link to thyroid function Table and SupplementaryFig An intronic variant rs10186921 in the thyroid adenomaassociated gene THADA was identified to be associated with TSHTHADA has been identified as a somatic mutatedrearrangedgene in papillary thyroid cancer30 and observed to be truncated inthyroid adenoma31 Although THADA is known to play a role incold adaptation obesity and type diabetes its role in thyroidvariantfunction remainsrs145153320 in gene B4GALNT3 is associated with TSHminor allele frequency in HUNT MAFHUNT effectsizeHUNT standard deviation SD confidence intervalCI “ SD PvalueHUNT — ˆ’ and is times more frequent in the Norwegian HUNT samples than inother nonFinnish Europeans34 The WNK1B4GALNT3 genefusion has been identified in papillary thyroid carcinoma35elusive3233 A rare missenseTwo novel independent rare coding variants with effect sizeslarger than one SD were identified in the known TSH locus TSHRwhich encodes the TSH receptor Both variants were only observedin HUNT The rare missense variant TSHR pR609Q rs139352934MAFHUNT effect sizeHUNT SD CI “SD is the most significant variant in the locus PvalueHUNT — ˆ’ followed by pA553T rs121908872 MAFHUNT CI “ SDPvalueHUNT — ˆ’ TSHR pR609Q rs139352934is times more frequent in HUNT than in other nonFinnishEuropeans34 TSHR pR609Q has been reported to aggregate in afamily with nonautoimmune isolated hyperthyrotropinemia36 andTSHR pA553T has been previously detected in a family withcongenital hypothyroidism37sizeHUNT SDeffectAs singlevariant association tests may lack power for rarevariants MAF ‰ and to search for genes with multiple rareproteinaltering variants we performed exomewide genebasedSKATO38 tests as implemented in SAIGEGENE39 to identifyrare coding variants associated with TSH We grouped missenseand stopgain variants with MAF ‰ and imputation qualityscore ‰¥ within each gene and tested genes with at leasttwo variants This analysis identified two genes TSHR andB4GALNT3 as significantly associated with TSH Pvalue —ˆ’ Supplementary Table and Supplementary Fig RareNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zeulavPytienegoreteHcnoitceriDNPESbtceffEˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’E“E“Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’Eˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’aqerFIRMCNKDCIFRMfroCPAHTKOBADAHTLXSASRECCFGEVDEDPPPLSNTCOLNDLCGARPCOMSTCADXNSSRFSTAEYTNLAGBTNLAGBCELOCCDCCKENSAHPDFNZBHSBARHOPAGNGCNILCSACGRDCNILPBPSHRPPPGNGIRMTNWmaertsnwoDicnegretniicnegretnIiicnortn_ANRcnicnortnIicnegretnIicnortnIicnortnIicnortnIicnegretnIicnortnIicnortnIicnortnIicnegretnIicnegretnIicnortnIicnortnIicnortnIicnortnIsuomynonysnoNicnegretnIsuomynonysnoNicnortnIsuomynonysnoNicnortnIiicnortn_ANRcnicnegretnIicnortnIicnegretnIicnegretnIGAAACTGGGAGCAATCGGATCCCTAGCGTCAGGGACTAAGCTGCATACGCTTTCGCTACTsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsrsisylanaatemscimOdioryhTIGMTNUHseneGtseraeNyrogetaCtlAfeRDIsrdliubnoitisoPemosomorhCxednIsucoLscimOdoryhTidnaIGMTNUHfosisylanaatemehtnidefiitnediHSThtiwdetaicossaicoltnednepednilevoninhtiwstnairavdaeLelbaTtesatadigndnopserrocehtnignissimsitnairavehtfisadetoNlyevitcepseriscmOdoryhTidnaIGMTNUHstesatadliaudvdniinieelllaetanretlaehtfoHSTnonoitceridslevelHSTfoDSfotinuehtnieelllaetanretlaehtottcepserhtiwdetropererasezistceffEbtceffEctesatadlsisyanaatemdenbmociehtnieelllaetanretlaehtottcepserhtiwdetropereraisecneuqerFaNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zvariants in both genes associated with TSH were also identifiedfrom singlevariant analysis After conditioning on the two rarevariants in TSHR that were genomewide significant in the singlevariantandrs139352934 the gene TSHR was still exomewide significantwith Pvalue — ˆ’ while B4GALNT3 was no longersignificantly associated with TSH with Pvalue afterconditioning on the top variant rs145153320Pvalue — ˆ’rs121908872analysisFinemapping for potentially causal variants among TSH lociTo identify potentially causal variants at TSH loci we conductedfinemapping using SuSiE40 which estimates the number ofcausal variants and obtains credible sets of variants with cumulative posterior probability through Iterative BayesianStepwise Selection41 Supplementary Data The LD matrixused in SuSiE was calculated based on HUNT We identified eightindependent causal variants at the TSHR locus by finemappingusing SuSiE40 and seven independent association signals by thestepwise conditional analysis Supplementary Data suggestingallelic heterogeneity at the TSHR locusIn addition finemapping by SuSiE40 and stepwise conditionalanalysis identified two association signals in the locus of thethyroglobulin gene TG TG encodes a highly specializedhomodimeric multidomain glycoprotein for thyroid hormonebiosynthesis27 it is the most highly expressed gene in the thyroidgland and its protein product represents roughly half the proteinof the entire thyroid gland4243 The TG locus has been reportedin a recent TSH GWAS23 The credible set for each causalassociation contains one missense variant that is in strong LDwith the most strongly associated intronic variant Supplementary Table and Supplementary Fig In the HUNT study themissense variant TG pG67S rs116340633 MAF effectsize SD CI “ SD Pvalue — ˆ’is in strong LD r2 with the most strongly associatedvariant rs117074997 intronic At the other association signalmissense variant TG pP118L rs114322847 MAF effectsize SD CI “ SD Pvalue — ˆ’is in strong LD r2 with the most strongly associatedvariant rs118039499 intronic Supplementary Table andSupplementary Fig TG pP118L has been previously detectedamong familial cases with congenital hypothyroidism44 TG pP118L rs114233847 is significantly associated with nontoxicnodular goiter odds ratio OR CI “Pvalue — ˆ’ in the UKBB2728 while the association ofTG pG67S rs116340633 with nontoxic nodular goiter isless significant OR CI Pvalue —ˆ’ TG pP118L has been previously detected in patients withsporadic congenital hypothyroidism in a Finnish cohort44Functional followup of missense variants in the gene TG Weperformed sitedirected mutagenesis studies to investigate theimpact on the protein expression of TG of the two independentmissense variants both located in the highly conserved Tg1domain of unclear function The protein encoded by the humanTG is conserved in mice with nearly perfect conservation of allcritical amino acid residues including those that maintain theprotein structure and hormone synthesis45 A cDNA encodingwildtype mouse Tg mTgWT expressed in 293T cells hasnormal synthesis and secretion of thyroid hormones46 We thenintroduced the observed human TG variants rs116340633 andrs114322847 into the mTg cDNA 293T cells were eitheruntransfected or transfected with pcDNA31 in which a cytomegalovirus promoter drives expression of mTgWT or the pP118L or pG67S Tg variants mature Tg numbering Then weexamined the intracellular vs secreted levels of the mTgWT andthese two human Tg variants TgpP118L and TgpG67STransfected cells were incubated overnight and the culturemedium and cell lysates were analyzed by SDSpolyacrylamide gelelectrophoresis PAGE and immunoblotting with antiTg antibody The experiment was independently repeated three timesand the results analyzed in a manner that is independent oftransfection efficiency On average of the total expressedWT form of mTg was recovered in the media and extracellular intracellular MC ratio of mTg was as expected between and the TgP118L variant showed a significant reduction in the MC ratio Pvalue and the TgG67S variant also showed asignificant reduction in the MC ratio Pvalue Fig Compared with the WTPrioritization of TSH genes pathways and tissues To furtherunderstand the biology underlying TSH associations we prioritized associated genes tissues and cell types in which TSH genesare likely to be highly expressed using Datadriven ExpressionPrioritized Integration for Complex Traits DEPICT47 based on loci with TSH association Pvalue cutoff — ˆ’ andclumped based on LD in HUNT As expected the membranesand thyroid gland are the most strongly associated tissues followed by tissues from the digestive system ileum gastrointestinaltract pancreas and colon respiratory system lung and accessory ans for eyes conjunctiva eyelids and anterior eyealthough none of the tissues reached the Bonferroni significantthreshold Pvalue or have false discovery rate FDR Supplementary Data Based on functional similarity toother genes among TSH loci genes at the TSHassociated lociwere prioritized by DEPICT with FDR ‰ SupplementaryData among which the prioritized genes ZFP36L2B4GALNT3 PPP1R3B FAM109A GNG12 GADD45A BMP2VEGFC LPP and MAL2 were at the novel TSH loci identified inour metaanalysis Table In addition among reconstituted gene sets gene sets were enriched among TSH lociwith FDR The most significantly enriched one is the CTSDPPI subnetwork followed by gene sets for regulation of phosphorylation Supplementary Data Pleiotropic effects of TSH loci To explore the pleiotropic effectsof the TSH loci we examined associations of the nonhumanleukocyte antigen HLA independent TSH top variants with human diseases PheCodes272848 and continuoustraits httpwwwnealelabisukbiobank in the UKBB variants and rs121908872 are not available in the UKBB Dueto the strong associations between HLA variants and autoimmune diseases49 we excluded two HLA variants associatedwith TSH rs1265091 and rs3104389 in the analysis for pleioPvalue — ˆ’tropic effects We identified significantpleiotropic association for out of nonHLA variants across disease phenotypes Supplementary Fig 5a and Supplementary Data including thyroid disorders diabetes cardiovasculardisease digestive system disorders asthma and cataractsIn addition nonHLA variants were significantly associatedPvalue — ˆ’ with one or more quantitativetraitsincluding body mass index lung function measurements metricsof bone density spherical powermeridian measurements andblood cell counts Supplementary Fig 5b and SupplementaryData TSHincreasing alleles at one or more loci were associated with an increased risk of cardiovascular disease smallerbody size reduced bone mineral density decreased lung functionand an increased risk of hypothyroidism and a decreased risk ofgoiter These results are generally consistent with previous studies23 We also examined the associations between the TSH indexin the ThyroidOmicsvariantsand freethyroxinelevelsNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zaTg kDabcUntransfectedmTgWTmTgP118LmTgG67SMCMCMCMCMCMCMCTg kDaTg kDaddnab nillubogoryhTeUA ytisnetniCellMediaWTG67SP118Loitar CMFig Both the TGP118L and TGG67S point mutants exhibit a secretion defect a“c Three independent replicate experiments Western blotting ofTG in 293T cells that were either untransfected a no detectable bands or transfected with constructs encoding mouse TG wild type WT or P118L 67S point mutants in the pcDNA31 background in which the CMV promoter drives the respective cDNA expression Serumfree media M werecollected overnight and the cells C were lysed Equal volumes of media and cells were analyzed by SDSPAGE electrotransfer to nitrocellulose andimmunoblotting with antiTgspecific antibodies Full scans of western blotting are presented in Supplementary Fig From scanning densitometryd shows the content of thyroglobulin and its variants intracellularly and in the secretion e The extracellular intracellular MC ratio of each constructd e Three independent replicate experiments All boxplots in d and e indicate median center line 25th and 75th percentiles bounds of box andminimum and maximum whiskersWTG67SP118Lconsortium23 Out of TSHassociated variantsfor whichassociation results with free thyroxine were available have TSHlowering alleles associated with higher free thyroxinelevels Supplementary Data Pvaluebinomial — ˆ’We further examined the association with thyroid cancer forTSH index variants We metaanalyzed UKBB2728 and a previousmetaanalysis of deCODE and four other case“control data sets19for thyroid cancer in thyroid cancer cases and controls and examined out of TSH nonHLA index variantsthat are available in the metaanalysis for thyroid cancerSupplementary Data The TSHincreasing alleles of outof TSHassociated variants were associated withreduced thyroid cancerrisk Supplementary Data andSupplementary Fig 6a and 6b Pvaluebinomial — ˆ’Eighteen out of the TSHassociated variants tested wereat least nominally associated with thyroid cancer P Pvaluebinomial — ˆ’ For out of the TSHassociatedvariants the TSHincreasing alleles were associated with reducedthyroid cancer risk Pvaluebinomial — ˆ’ SupplementaryData and Supplementary Fig 6c d Moreover when weexamined alleles that predisposed to thyroid cancer17“ out of had a consistent direction of effect towards lower TSH Pvaluebinomial Ofriskalleles that were at least nominally associated with TSH levelP all six variants were associated with lower TSHPvaluebinomial Supplementary Data and Supplementary Fig thyroidcancerthesixAssociations of polygenic scores of TSH with other phenotypesAlthough individual TSH variants may exhibit pleiotropic effectsit is also possible that the cumulative effects of TSHmodifyinggenetic variants may lead to disease Therefore we constructedcodesICDPGS from the independent nonHLA TSH top variantsrs1265091 and rs3104389 are HLA variants and rs121908872and were not in UKBB and examined their association with the human diseases constructed from International Classification of Diseasesin theUKBB272848 As in the pleiotropy analysis we excluded the twoHLA variants in the PGS calculation to study the cumulativegenetic effects of TSHassociated variants in nonHLA regionswith human diseases The TSH PGS was significantly associatedwith phenotypes Pvalue — ˆ’ Bonferroni correctionfor phenotypes including an increased hypothyroidism riskand decreased risk of goiter thyrotoxicosis and hyperhidrosisSupplementary Data and Supplementary Fig We alsoevaluated the phenotypic variance Nagelkerke™s r250 explainedby TSH PGS for phenotypes in the UKBB that have at least cases in unrelated white British samples Supplementary Data The phenotypes with highest r2 were nontoxicnodular goiter r2 secondary hypothyroidism r2 and thyrotoxicosis with or without goiter r2 InFinnGen we also observed that high TSH PGS was associatedwith high risk of hypothyroidism and low risk of goiter HighTSH PGS in FinnGen was marginally associated with an increasein risk of depression OR per SD of TSH PGS CI “ Pvalue — ˆ’ and a reduced risk of pregnancyhypertension OR per SD of TSH PGS CI “ Pvalue — ˆ’ The phenomewide associationresults for the TSH PGS in FinnGen are shown in SupplementaryData and Supplementary Fig Depressive symptomsand hypertension during pregnancy have been observed to beclinically associated with hypothyroidism and thyroid dysfunction51“ respectively However their genetic associations havenot been extensively studiedNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zaUKBBrecnac doryhit f oecneaverPlQuintiles of TSH PGSbdeCODEirecnac doryht fo ecneaverPlQuintiles of TSH PGSrecnac doryhit fo oitar sdrecnac doryht fo oitar sddOQuintiles of TSH PGSQuintiles of TSH PGScFinnGenirecnac doryht fo ecneaverPlirecnac doryht fo oitar sddOQuintiles of TSH PGSQuintiles of TSH PGSFig The risk of thyroid cancer is lower for individuals with genetically predicted higher TSH levels Plots of thyroid cancer prevalence by quintiles ofTSH PGS left and odds ratio of thyroid cancer in relation to the lowest quintile right in data sets UKBB a N case N control deCODEb N case N control and FinnGen c N case N control N sample size N case sample size of cases N controlsample size of controls Error bars represent confidence intervalsthyroid cancer CI “In the UKBB TSH PGS was significantly associated with aOR per SD ofdecreased risk ofPvalue — ˆ’TSH PGSSupplementary Data and Fig Compared with the rest ofthethyroid cancer ofindividuals with TSH PGS in the lowest quintile was “ and the OR for thyroid cancer of individuals withTSH PGS in the highest quintile was “ suggestingthe OR CIsamplesforthe protective effects of TSHincreasing genetic variants onthyroid cancer riskWe successfully replicated the association between high TSHPGS and low thyroid cancer risk in study populations fromColumbus USA19 OR CI “ Pvalue — ˆ’ and deCODE19 OR CI “Pvalue — ˆ’ We also observed the association inFinnGen OR CI “ Pvalue — ˆ’NATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s4146702017718zalthough the evidence was much less strong The Columbusstudy19 is a case“control study of thyroid cancer cases and controls with much higher thyroid cancer prevalence thanthe three populationbased biobanks UKBB3031 cases and controls FinnGen cases and controls anddeCode19 cases and controls In Fig theprevalence of thyroid cancer left panel and OR of thyroid cancerright panel are plotted against the TSH PGS for the threepopulationbased cohorts results for Columbus are provided inSupplementary Fig Similar plots of hypothyroidism andgoiter are plotted for UKBB and FinnGen in SupplementaryFigs and Mendelian randomization for TSH thyroid cancer and goiterWe investigated a possible causal effect of TSH on thyroid cancerusing twosample Mendelian randomization Ninetyfour nonHLA genetic variants for TSH identified by our metaanalysis ofHUNT MGI and ThyroidOmics were used as instrumentalvariables Fstatistic for all single nucleotide polymorphismsSNPs rs1265091 and rs3104389 are HLA variants andthe summary statistics for thyroid cancer were not available forrs121908872 rs4571283 and To avoid sampleoverlap for the TSH and thyroid cancer GWASs we used effectson TSH estimated by metaanalyzing HUNT and ThyroidOmicsto construct the instrumental variable for TSH levels and wemetaanalyzed MGI deCODE and UKBB for thyroid cancer Wefound that a one SD increase in TSH SD mUL wasassociated with a decreased risk of thyroid cancer inversevariance weighted OR CI “ MREgger intercept Pvalue Sensitivity analyses using the penalizedweighted median method the weighted median method and theweighted mode method including all variants are presented inFig 3a and Supplementary Data Similar results were observedbetween methods with the exception of the weighted modewhich was strongly attenuated To reduce the possibility that theresults were influenced by occult thyroid dysfunction typicallyoccurring in older age we repeated the analysis using SNPTSHeffect estimates obtained among those younger than years ofage at the time of TSH measurement Supplementary Data Similar results were observed except for the weighted modewhich was again attenuated towards the null OR CI“ Supplementary Data Furthermorethere wasstrong evidence of heterogeneity suggesting some instrumentswere invalid Nevertheless when repeating the main analysisusing MRPRESSO which excluded nine variants due to thedetection of specific horizontal pleiotropic outlier variants54 thecausal association was similar MRPRESSO outlier corrected OR CIs “ Fig 3a and Supplementary Data Finally using only the proteincoding nonsynonymous variant pP118L in the TG gene Fstatistic we observed a protective effect of increased TSH on thyroid cancer Wald ratio OR CI “ To investigate if TSH may also influencethe risk of benign thyroid growth disorders we similarly performed atwosample Mendelian Randomization analysisbetween TSH and goiter The effects on TSH were estimated by ametaanalysis of HUNT and ThyroidOmics SupplementaryData and and the GWAS results for goiter from the UKBBwere used2728 A SD increase in TSH SD mULwas associated with a decreased risk of goiter inversevariance weighted OR CI “ MREgger interceptPvalue Fig 3b and Supplementary Data DiscussionMetaanalysis of the HUNT study the MGI biobank and theThyroidOmics consortium for TSH on up to individualsidentified TSH loci of which are previously unreported AllTSH loci reported by previous GWAS studies172223 are replicated in our metaanalysis Several novel loci pointed to nearbygenes with a known or suspected link to thyroid functionAdditional independent signals were identified among several locibased on GWAS results in the metaanalysis and LD informationin the HUNT study including two rare variants rs546738875 andrs145153320 at the B4GALNT3 locus and two rare missensevariants TSHR pA553T rs121908872 and TSHR pR609Qrs139352934 which have been observed to be associated withcongenital hypothyroidism in previous family studies3637 TSHRpR609Q rs139352934 is the most strongly associated with TSHin the TSH receptor gene TSHR with an effect size greater thanone standard deviation of TSH mUL As these rare variants were only imputed in HUNT not in MGI or ThyroidOmicsfurther followup to verify the associations is needed As individual GWAS was conducted on inversenormal transformed TSHlevels before metaanalysis it is challenging to convert the effectsizes reported by our metaanalysis to actual scales of TSH levelsFinemapping for potential causal variants among TSH locidetected two independent missense variants in the TG gene TGpG67S and pP118L The two variants have a similar frequency but pP118L shows stronger evidence for an associationwith goiter and with thyroid cancer Functional experimentsdemonstrated each of these defects in the TG gene pP118L andpG67S respectively causes defective secretion of TG Furtherstudies are needed to investigate how the proteinaltering variantsimpact TSH levelsAs expected membranes and thyroid gland were identified asthe tissue in which genes from TSHassociated loci are most likelyto be highly expressed Genes ZFP36L2 B4GALNT3 PPP1R3BFAM109A GNG12 GADD45A BMP2 VEGFC LPP and MAL2were prioritized as functional candidates among the novel TSHassociated loci based on functional similarity to other genes at allTSH loci using DEPICT47A PheWAS ofthe TSHassociated variants in the UKBBdemonstrated that TSHincreasing alleles are associated with anincreased risk of cardiovascular disease smaller body sizereduced bone mineral density decreased lung function and anincreased risk of hypothyroidism but were favorably associatedwith a decreased risk of goiter Our results suggest that thesevariants have pleiotropic effects although they tend to affect TSHthrough actions in the thyroid glandPhenomewide association tests in the UKBB and FinnGen forthe TSH PGS showed that genetically predicted increased TSH isassociated with a high risk of hypothyroidism and a low risk ofgoiter thyrotoxicosis hyperhidrosis and thyroid cancer Twosample Mendelian randomization analyses suggested that lowerTSH causes an increased risk of thyroid cancer and goiter This isan unexpected direction given that TSH promotes the growth ofthyroid cancers814“ Nonethelessit has previously beenspeculated that lower TSH levels may lead to less differentiationof the thyroid epithelium which could predispose to malignanttransformation17 Alternatively our genetic instrument for TSHmay represent a thyroid gland phenotype that influences bothTSH through the negative feedback of thyroid hormones on thepituitary gland and thyroid growth increasing the risk of thyroidcancer and goiter Supplementary Fig In that scenario theeffect on thyroid cancer would not be downstream of TSH andaltering TSH levels eg by medication would not be expected toalter thyroid cancer risk Tissue enrichment analyses of genes atTSHassociated loci and the observation that TSHloweringalleles were generally associated with higher free thyroxine levelsSupplementary Data suggest that most TSHassociated variants act primarily on the thyroid gland where effects on boththyroid function and growth have previously been described forNATURE COMMUNICATIONS 101038s4146702017718z wwwnaturecomnaturecommunications 0carecnac doryhit no tceff ePNSbretiog no tceffe PNSNATURE COMMUNICATIONS 101038s4146702017718z““““MethodInverse variance weightedMR EggerMRPRESSOPenalized weighted medianWeighted medianWeighted modeSNP effect on TSHMethodInverse variance weightedMR EggerMRˆ’PRESSOPenalized weighted medianWeighted medianWeighted modeSNP effect on TSHFig Twosample Mendelian randomization analysis for casual associations between TSH and thyroid cancer and between TSH and goiter a Twosample MR between TSH and thyroid cancer based on summary statistics from the metaanalysis of HUNT and ThyroidOmics n for TSH andfrom the metaanalysis of UKBB2728 MGI26 deCODE and four other case“control data sets with European ancestry as reported in Gudmundsson et al19for thyroid cancer association casescontrols b TSH and goiter based on summary statistics from the same TSH metaanalysis asabove and from the GWAS of UKBB2728 for goiter association N case N control The crosshairs on the plots represent the confidence intervals for each SNPTSH or SNPoutcome association The variant on the top left corner is the rare nonsynonymous variant B4GALNT3 pR724W rs145153320 N sample size N case sample size of cases N control sample size of controlsTSHR mutations55 Nonetheless the Mendelian randomizationfindings were robust to most of the sensitivity analyses performedto detect and correctfor pleiotropy Restricting the geneticinstrument to TSHlowering variants associated with lower freethyroxine levels could have helped resolve the causal question butthose variants were too few to yield meaningful estimat

Thyroid_Cancer

"coronavirus disease COVID19 caused by severe acute respiratory syndrome coronavirus SARSCoV2 emerged in China Currently it is breaking out globally and posing a serious threat to public health The typically clinical characteristics of COVID19 patients were fever and respiratory symptoms and a proportion of patients were accompanied by extrapulmonary symptoms including cardiac injury kidney injury liver injury digestive tract injury and neurological symptoms Angiotensin converting enzyme ACE2 has been proven to be a major receptor for SARSCoV2 and could mediate virus entry into cells And transmembrane protease serine TMPRSS2 could cleave the spike S protein of SARSCoV2 which facilitates the fusion of SARSCoV2 and cellular membranes The mRNA expressions of both ACE2 and TMPRSS2 were observed in the heart digestive tract liver kidney brain and other ans SARSCoV2 may have a capacity to infect extrapulmonary ans due to the expressions of ACE2 and TMPRSS2 in the cells and tissues of these ans It seems that there is a potential involvement of ACE2 and TMPRSS2 expressions in the virus infection of extrapulmonary ans and the manifestation of symptoms related to these ans in patients with COVID19 Here we revealed the expressions of ACE2 and TMPRSS2 in extrapulmonary ans and we also summarized the clinical manifestation and the management of extrapulmonary complications in patients with COVID19 Introduction Since the late a novel coronavirus officially named as severe acute respiratory syndrome coronavirus SARSCoV2 was identified as the pathogen to cause pneumonia [] As a member of the Betacoronavirus genus SARSCoV2 has genomic nucleotides identity with human severe acute respiratory syndrome coronavirus SARSCoV and shares amino acid sequence identity with SARSCoV [] The World Health anization WHO named the disease caused by SARSCoV2 as coronavirus disease COVID19 Until April the virus has swept through countries more than million cases with COVID19 have been confirmed and more than cases died which has been posing significant threats to public health SARSCoV2 can cause respiratory diseases and may lead to acute respiratory distress syndrome ARDS multiple an failure and even death in severe cases [] In addition to typical symptoms such as cough and fever some patients developed the symptoms in multiple systems such as cardiovascular system digestive system and Abbreviations ACE2 Angiotensin converting enzyme AKI Acute kidney injury ALI Acute liver injury ALP Alkaline phosphatase ALS Artificial liver system ALT Alanine aminotransferase AMI Acute myocardial infarction ARDS Acute respiratory distress syndrome AST Aspartate aminotransferase AT2 Alveolar cells BUN Blood urea nitrogen CCLE Cancer Cell Line Encyclopedia CNS Central nervous system COVID19 Coronavirus disease GEO Gene Expression Omnibus GGT Gammaglutamyltransferase GI Gastrointestinal injury GTEx GenotypeTissue Expression ICU Intensive care unit MCS Mechanical circulatory support NP Nucleoprotein PCI Percutaneous coronary intervention SARSCoV2 Severe acute respiratory syndrome coronavirus Scr Serum creatinine ScRNASeq Single cell RNA sequencing STEMI STelevation myocardial infarction TBIL Total bilirubin TEM Transmission electronic microscope TMPRSS2 Transmembrane protease serine VV venousvenous WHO World Health anization Corresponding authors at Department of Infectious Disease and Institute of Hepatology Qingdao Municipal Hospital Qingdao University Digestive Disease Key Laboratory of Qingdao Qingdao China Email addresses xinyongning9812163com Y Xin zlk0823163com L Zhuang 101016jbiopha2020110678 Received June Received in revised form August Accepted August BiomedicinePharmacotherapy1312020110678Availableonline24August2020075333222020TheAuthorsPublishedbyElsevierMassonSASThisisan accessundertheCCBYNCNDlicensehttpcreativecommonslicensesbyncnd40 0cexpressed not only in the cells and tissues of lung but also in extrapulmonary ans [“] Fig In this section the expression levels of ACE2 and TMPRSS2 in extrapulmonary ans including heart kidney liver digestive tract brain and other ans were reviewed Heart M Dong nervous system in the early stages of COVID19 which brings more challenges to the timely diagnosis of patients [] Angiotensin converting enzyme ACE2 as a metalloproteinase is a carboxyterminal dipeptidyl peptidase [] The primary physiological role of ACE2 is involved in the regulation of vasoconstriction and blood pressure [“] Transmembrane protease serine type2 TMPRSS2 belonging to the type II transmembrane serine protease family could cleave the coronavirus spike S protein [“] It was demonstrated that ACE2 and TMPRSS2 were crucial for the entry of SARSCoV and SARSCoV2 into the host cells [] Cell entry of SARSCoV2 depends on binding of the S protein to the specific cellular receptor and S protein priming by host cell proteases As shown in Fig each S protein of SARSCoV2 consists of two subunits a globular S1 domain at the Nterminal region and the membraneproximal S2 domain SARSCoV2 utilizes receptorbinding domain within the S1 domain to bind to the cellular receptor ACE2 which could trigger the effects of TMPRSS2 on the cleavage of protein S at the S1 and S2 sites and priming cell membrane fusion for viral entry [] As receptors and mediators of virus entry are important for determining viral host and an the route of SARSCoV2 infection and the infected an may depend on the expression and distribution of ACE2 and TMPRSS2 [] Studies have shown that ACE2 and TMPRSS2 are expressed not only in lung tissues but also in extrapulmonary ans including heart kidney liver colon esophagus brain gallbladder and testis suggesting that SARSCoV2 may also affect extrapulmonary ans [“] In this review the distributions of ACE2 and TMPRSS2 in extrapulmonary ans and the characteristics and clinical managements of extrapulmonary an injury caused by SARSCoV2 were summarized We believe that this will be important in understanding on the infection of extrapulmonary ans in patients with COVID19 The mRNA expressions of ACE2 and TMPRSS2 in extrapulmonary ans The mRNA expressions of ACE2 in different human ans were analyzed and the results showed that ACE2 was expressed in the heart [] Furthermore Chen analyzed the feature of ACE2 expressions among cardiac cell types and found that ACE2 was specifically expressed in pericyte [] Moreover RNA sequencing from patients with failing hearts and normal donors revealed that myocardial ACE2 expressions were significantly increased in patients with heart failure which was further validated at the protein level by proteomics profiling from heart failure and normal donors [] Another study also showed that the expression of ACE2 in heart tissues of patients with underlying heart disease was higher than that in normal heart tissues [] These two studies suggested that the expression of ACE2 in heart tissue of patients with underlying heart disease was higher than that in normal heart tissue Guo et al analyzed the mRNA expression of TMPRSS2 from the GenotypeTissue Expression GTEx database and the results showed that TMPRSS2 is also expressed in the heart [] By singlecell RNA sequencing scRNASeq to profile the gene expression landscapes of cardiac cells from human embryos Qi revealed that the cardiomyocytes from the heart contain ACE2expressed cells and TMPRSS2expressed cells and the cardiovascular progenitor cells and cells TMPRSS2expressed cells respectively [] These data showed that both ACE2 and TMPRSS2 were expressed in the heart contain ACE2expressed Kidney Studying the viral susceptibility of extrapulmonary ans is important for a deeper understanding for the pathogenesis of SARSCoV infection Studies have shown that ACE2 and TMPRSS2 were Expression analysis from the GTEx database showed that kidney displayed the fifth high expression of ACE2 [] To investigate the expression of ACE2 in kidney Lin analyzed the public singlecell transcriptome dataset of normal kidneys from healthy donors the Fig Entry of SARSCoV2 into host cells SARSCoV2 infected the host cells by the spike protein of the virus and the functions of ACE2 and TMPRSS2 in host cells BiomedicinePharmacotherapy13120201106782 0cM Dong Fig Tissue distributions of ACE2 and TMPRSS2 in human A“B the schematic diagram of the expressions of ACE2 A and TMPRSS2 B in multiple human tissues The colour strength is corresponding to the gene expression level ACE2 and TMPRSS2 were expressed in the brain and heart ACE2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while TMPRSS2 is expressed in the hepatocytes and cholangiocytes ACE2 and TMPRSS2 were highly expressed in kidney and intestinal epithelial cells Both ACE2 and TMPRSS2 were also expressed in the esophagus stomach nose testis pancreas breast prostate and thyroid results showed that the ACE2 was distributed across multiple cell types and was mostly enriched in proximal tubule cells [] Fan et al confirmed the specific ACE2 expression in tubular cells from the Gene Expression Omnibus GEO dataset while it was not observed in immune cells and glomerular parietal epithelial cells RNA and protein expression data of ACE2 in different human tissues and cancer cell lines were obtained from three online datasets including the Cancer Cell Line Encyclopedia CCLE GTEx database and the Human Protein Atlas dataset and the results indicated that both mRNA and protein expression levels of ACE2 were relatively high in kidney cells especially in renal tubular cells [] Meanwhile Suryawanshi analyzed the data of kidney tissues in scRNASeq datasets and found that either proximal tubular cells or tubular progenitor cells in the kidney coexpressed ACE2 and TMPRSS2 [] The data of the scRNAseq from GEO dataset GSE134355 showed that ACE2 and TMPRSS2 expression levels were high in nephron epithelial cells epithelial cells endothelial cells and mesangial cells of the kidney [] Recently Pan also found that the TMPRSS2 gene was coexpressed with ACE2 in kidney podocytes [] These data showed that both ACE2 and TMPRSS2 were highly expressed in tissues and cells of kidney Liver Chai et al analyzed the scRNAseq data from GEO database GSE124395 to evaluate ACE2 gene expression in liver the results showed that ACE2 was highly expressed in cholangiocytes which level was about times higher than that in hepatocytes [] The GTEx database also showed that both ACE2 and TMPRSS2 were expressed in the liver [] Zhou identified that TMPRSS2 is highly expressed in hepatocytes from Human Cell Atlas database [] Recently Wen indicated that ACE2 and TMPRSS2 are specifically coexpression in TROP2 liver progenitors of human liver tissue using scRNA sequencing [] These data indicate that ACE2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while TMPRSS2 is expressed in hepatocytes Digestive tract A previous study showed that ACE2 could be found in the upper esophagus and it could be detected in stratified epithelial cells and absorptive enterocytes of the ileum and colon [] Quantitative mRNA expression profiling of ACE2 across human tissues by Harmer showed that ACE2 was expressed at a high level in gastrointestinal tissues [] Zhang et al analyzed datasets with singlecell transcriptomes of esophagus gastric ileum colon and lung and the data showed that ACE2 was not only highly expressed in the type II alveolar cells AT2 of lung but also in the stratified epithelial cells ileum absorptive enterocytes cells and colon enterocytes [] Similarly the immunofluorescent staining of esophagus stomach duodenum and rectum showed that ACE2 was stained mainly in the cytoplasm of gastrointestinal epithelial cells [] Besides the scRNAseq data showed that ACE2 was significantly elevated in the proximal and distal enterocytes [] Guo et al suggested that TMPRSS2 was highly expressed in almost all ans of the digestive tract including colon stomach small intestine and esophagus [] Using published scRNAseq data and seven inhouse normal colon samples Lee reported that the coexpressions of ACE2 and TMPRSS2 transcripts were mainly observed in the small intestine and colon [] The highest expressions of TMPRSS2 and ACE2 were found in enterocytes among the intestinal cell types []These data showed that TMPRSS2 and AEC2 are highly expressed in the digestive tract Nervous system Analysis using the GTEx database showed that both TMPRSS2 and ACE2 are expressed at relatively low levels in the brain cortex [] Chen found that ACE2 was relatively highly expressed in some important brain areas such as the substantia nigra and brain ventricles using seven brain transcriptome databases [] ACE2 was expressed at high level in the piriform cortex of human brain and its expression could also be detected in many neurons including both excitatory and inhibitory neurons and some nonneuron cells including astrocytes and oligodendrocytes in human middle temporal gyrus and posterior cingulate cortex [] Qi analyzed the scRNAseq data of substantia nigra BiomedicinePharmacotherapy13120201106783 0cClinical classification of acute cardiac injury NonICUcases ICU cases NonICUcases ICUcases Nonsevere cases Severecases1965 Recoveredcases Died cases NonICUcases ICUcases Survivor cases Nonsurvivor cases Chen [] Hong [] Zhou [] China Korea China M Dong and cortex of brain from GEO database the results showed that both ACE2 and TMPRSS2 were expressed in the oligodendrocyte precursor cells and the astrocytes of the substantia nigra and cortex [] There are limited reports on the expressions of ACE2 and TMPRSS2 in peripheral nervous system Brann analyzed the ACE2 and TMPRSS2 expression in different cell type from human scRNAseq dataset GSE139522 and found that neither olfactory sensory neurons nor olfactory bulb neurons expressed these two genes while ACE2 and TMPRSS2 were expressed in the nonneuronal cells including the sustentacular cells and olfactory bulb pericytes [] These data showed that ACE2 and TMPRSS2 could also be coexpressed in the nervous system Other ans or tissues Table Characteristics of acute cardiac injury after SARScid0 CoV2 infection Study Basic heart disease Acute cardiac injury Country Subject China Wang [] China NA Huang [] Li [] China Moreover ACE2 and TMPRSS2 were also reported to be coexpressed in some other ans [] It has been revealed that both ACE2 and TMPRSS2 are expressed in testis by scRNA sequencing and expression profile analysis indicating that testicular cells might be the potential targets of SARSCoV2 Another report revealed that multiple kinds of cells in the nose including nasal brushing epithelial cells nasal turbinate epithelial cells and nasal airway epithelial cells contained ACE2expressed and TMPRSS2expressed cell clusters [] Moreover ACE2 and TMPRSS2 were also expressed in pancreas breast prostate and thyroid [] and these ans might also be the targets of SARSCov2 Infection of SARSCoV2 and extrapulmonary an injury of patients with COVID19 SARSCoV2 infection and cardiac injury Recently autopsy analysis by Fox revealed that the histopathology of the heart was consistent with the typical pattern of viral myocarditis [] SARSCoV2 RNA was detected in the cardiac tissues of the patients with COVID19 [] These data suggested that SARSCoV2 may directly infect heart The epidemiology of COVID19 reported that cardiac injury was one of the most severe an damages [] The clinical manifestations of cardiac injury in COVID19 patients are complex and could present with heart failure arrhythmias or acute myocardial infarction AMI [ ] Inciardi reported the first case who had the symptom of heart failure at first and later the patient was positive for SARSCoV2 using nucleic acid test [] Cardiac injury is a common symptom in patients with COVID19 Shi reported that patients with COVID19 had cardiac injury [] Moreover there were patients with acute cardiac injury in a cohort including COVID19 patients and of patients with acute cardiac injury in the intensive care unit ICU [] Furthermore a study by Wang showed that there were patients with acute cardiac injury and patients presented with arrhythmia of COVID19 patients while acute cardiac injury was observed in of patients with CIVID19 in the ICU [] These cases suggested that SARSCoV2 may cause serious heart damage which should be widespreadly concerned Furthermore acute cardiac injury is more prevalent in severe cases with COVID19 [“] Table And it has been reported that COVID19 patients with cardiac injury had higher mortality than those without cardiac injury [] In this review we also summarized the possible relationship between basic heart disease and further cardiac injury [“] Table In a cohort of COVID19 patients from Renmin Hospital of Wuhan University China Shi demonstrated that cardiac injury occurred in patients during hospitalization of which had basic heart disease including coronary heart disease and chronic heart failure And only patients with basic heart disease of COVID19 patients without cardiac injury [] Similarly Liu suggested that patients with basic heart disease in COVID19 patients had Table Comorbidity with cardiac injury in COVID19 patients with basic heart disease Subjects with COVID19 Proportion of basic heart disease Patients with Cardiacinjury Study Shi [] Liu [] Xu [] Ma [] Guo [] Patients with basic heart disease With cardiac injury Without cardiac injury cardiac injury compared with patients with basic cardiovascular diseases of COVID19 patients without cardiac injury [] Other studies also indicated that the patients with basic cardiovascular disease are more likely to present heat injury in COVID19 patients [ ] In view of the points above COVID19 patients with underlying cardiac conditions seem to have higher rates of cardiac injury SARSCoV2 infection and kidney injury Recently autopsy analysis on six COVID19 patients showed that varying degrees of acute tubular necrosis were observed in all the renal specimens Nucleoprotein NP antigens and NP positive inclusion body of SARSCoV2 could be seen in kidney tissues from all the samples Moreover viruslike ps were seen in kidney tissues by transmission electronic microscope TEM [] Su analyzed kidney abnormalities in autopsies of patients with COVID19 and found that diffuse proximal tubular damage with the loss of brush border were BiomedicinePharmacotherapy13120201106784 0c SARSCoV2 infection and liver injury M Dong observed Further investigation showed that diffuse necrosis can be seen under the light microscope and electron microscopic examination also showed the clusters of coronavirus ps with distinctive spikes in the tubular epithelium and podocytes [] It was reported that both NP antigens and RNA of SARSCoV2 were detected in urine of COVID19 patients [] These data coincide with the finding of the SARSCoV2 invasion in kidney Collectively SARSCoV2 could directly infect human renal tubules and lead to kidney damage Recent studies have shown that the incidence of acute kidney injury AKI in COVID19 patients ranged from “ and higher frequency of renal function damage with elevated blood urea nitrogen BUN or serum creatinine Scr was observed in COVID19 patients [ ““] Table A study of patients with COVID19 indicated that levels of BUN and Scr were increased in and patients with COVID19 respectively And routine urine tests were performed on patients among which patients were positive for urinary protein and patients were positive for hematuria [] Another study also showed that about patients with COVID19 had abnormal renal function [] Moreover COVID19 patients with more severe disease progression have higher rates of AKI Huang and colleagues reported that of patients with AKI in the ICU were observed and none of the patients who did not require care in the ICU suffered AKI [] Xu found that the fatality rate was obviously higher in COVID19 patients with AKI than those without renal injury [] Furthermore in another study investigating patients with COVID19 at hospital admission more severe patients had higher rates of AKI and the Cox regression analysis also suggested that COVID19 patients who developed AKI had a significantly higher mortality risk [] Therefore AKI is more prevalent in severe cases with COVID19 An autopsy report of a 50yearold patient with COVID19 showed moderate microvesicular steatosis and mild lobular activity in liver tissues [] Moreover Zhao used human liver ductal anoids as a tool to investigate the SARSCoV2 infection and the tissue damage induced by SARSCoV2 ex vivo and the results showed that the expression of SARSCoV2 NP was easily detected in the patchy areas of the hepatic duct indicating that liver ductal anoids were susceptible to SARSCoV2 infection [] In addition SARSCoV2 infection could disrupt the barrier and bile acid transporting functions of cholangiocytes which indicated that SARSCoV2 might directly induce cholangiocyte injury and consequently bile acid accumulation [] In view of the points above liver damage in the COVID19 patients might be directly caused by the viral infection Abnormal liver functions were frequently reported in COVID19 patients [] Epidemiologic studies showed that almost half of the patients had differing degrees of liver damage [“ “] Table Chen reported that out of patients had elevated alanine aminotransferase ALT patients had elevated aspartate aminotransferase AST and had elevated total bilirubin TBIL in Wuhan Jinyintan Hospital Wuhan China [] Similarly a nationwide study involving patients with COVID19 in China showed that more than of patients had elevated ALT and AST and of patients had elevated TBIL [] It was revealed that the levels of direct bilirubin indirect bilirubin ALT alkaline phosphatase ALP and gammaglutamyltransferase GGT were significantly higher in males than that in females with COVID19 and multivariate logistic regression analysis showed that male was an important independent risk factor for predicting acute liver injury ALI in COVID19 patients [] These data indicated that male patients with COVID19 may be more susceptible to liver injury Furthermore Table Characteristics of acute kidney injury after SARScid0 CoV2 infection Study Chen Wang Huang Guan Xu Preexisting kidney conditions NA NA NA [] [] [] [] [] Country China China China China China Subject Li [] Chen [] Hong [] Cheng [] Xiao [] Richardson [] Wan Li Qian Pei [] [] [] [] China China Korea China China America China China China China NA NA NA NA NA NA Scr Serum creatinine BUN Blood urea nitrogen AKI Acute kidney injury Abnormal renal functional indices Scr BUN NA Scr Scr Scr Scr BUN NA Scr BUN Scr NA NA NA Scr BUN Scr NA AKI NA Clinical classification of AKI NA NonICU cases ICU cases ICU cases Nonsevere cases Severe cases Mild cases Severe cases Critical ill cases Nonsevere cases Severe cases Recovered cases Died cases NonICU cases ICU cases NA Nonsevere cases Severe cases Cured cases In hospital cases Died cases Mild cases Severe cases Nonsevere cases Severe cases NA Moderate cases Severe cases Critically ill cases BiomedicinePharmacotherapy13120201106785 0cM Dong Table Characteristics of liver injury after SARScid0 CoV2 infection Study Country Subject China China China China China China China Korea America China China Chen [] Wang Huang Guan [] [] [] Xu Li [] [] Chen Hong [] [] Richardson et Wan Li al [] [] [] Qian [] China NA Patients with preexisting liverconditions NA NA NA Patients with abnormal liver functional indices ALT AST TBIL NA AST AST ALT TBIL ALT AST ALT AST TBIL ALT AST ALT AST TBIL AST ALT AST ALT AST TBIL ALT AST Abnormal liver functional indices in the Nonsevere patients AST ALT NA NA TBIL NA Abnormal liver functional indices in the severe patients ALT NA TBIL NA AST NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Nonsevere patients include patients without ICU care and recovered patients Severe patients include patients with ICU care and death ALT alanine aminotransferase AST aspartate aminotransferase TBIL total bilirubin multiple studies found that AST ALT and TBIL were significantly higher in patients treated in the ICU than that in nonICU patients [] Li suggested that among the patients with abnormal liver function moderate and severe types of patients were more likely to have liver injury and respectively [] Fu analyzed the relationship between ALI and mortality risk in COVID19 patients and the results showed that ALI is more common in the critically ill patients and ALI at the early stage increased death risk of COVID19 patients [] Together abnormal liver functions might be associated with the severity of patients with COVID19 SARSCoV2 infection and digestive tract injury Epithelial cells of the esophagus stomach duodenum and rectum in one COVID19 patient tested positive for SARSCoV2 RNA and the staining of viral NP was also visualized in the cytoplasm of epithelial cells in stomach duodenum and rectum [] Moreover minimally invasive autopsies were performed on three patients died of COVID19 and the results showed that some epithelial cells of the gastrointestinal mucosa were degenerated necrotic and detached [] These studies strongly supported that SARSCoV2 may directly infect the epithelial cells of digestive tract Table SARScid0 CoV2 detection in gastrointestinal specimens Study Subject [] [] Xiao Zhang Tan [] Xing Young Holshue Lescure Tang Wang Xu [] [] [] [] [] [] [] Gastrointestinal samples Stool Anal swabs Rectal swab Stool Stool Stool Stool Stool Stool Rectal swabs Tested positive in gastrointestinal specimens The positive time in gastrointestinal specimens days NA 6cid0 1cid0 5cid0 NA 3cid0 Positive time for Gastrointestinal samples after respiratory samples were negative days NA NA 8cid0 NA NA NA NA NA 2cid0 BiomedicinePharmacotherapy13120201106786 0cM Dong Multiple studies have identified that the SARSCoV2 RNA was detected in anal swabs [] rectal swabs [] and stool specimens [ ] of COVID19 patients It has been demonstrated that SARSCoV2 RNA could be detected in feces from more than half of COVID19 patients [] In another study Xing reported that SARSCoV2 RNA was detected in the feces of three pediatric cases with COVID19 in Qingdao China and the persistence of SARSCoV2 in the digestive tract lasted for 6cid0 days [] The possibility of fecaloral transmission of SARSCoV2 infection needs to be taken into account Furthermore as shown in Table long duration of SARSCoV2 detection in digestive tract by RTPCR has been reported and viral RNA remained detectable in the digestive tract for 2cid0 days after nucleic acid turned negative in respiratory samples [“] The studies suggested that SARSCoV2 could be detected from respiratory tract specimens during the early period to digestive tract specimens during the late period and viral nucleic acid tests in both the respiratory and digestive tract are necessary to confirm the complete clearance of virus Some COVID19 patients presented gastrointestinal symptoms such as diarrhea nausea vomiting and abdominal pain [] Holshue reported the first case of COVID19 patient in the USA which had nausea and vomiting before admission [] Multiple studies found that gastrointestinal symptoms including diarrhea “ nausea and vomiting “ and abdominal pain “ were common at presentation in COVID19 patients [ ““] Table In a cohort of patients with COVID19 in Wuhan China gastrointestinal symptoms were described in up to [] Moreover Sun showed that critically ill patients with COVID19 had gastrointestinal injury GI during hospital stay and the survival curves showed that the mortalities of patients with GI was greater than that of patients without GI [] Jin also found that the rate of the severe type was markedly higher in COVID19 patients with GI symptoms than that in those without GI symptoms [] These data suggested that GI is one of the common extrapulmonary an injuries in COVID19 patients and may be related to the severity of the disease On the other hand many studies showed that patients with COVID19 could present initially with the typical gastrointestinal sympto

Thyroid_Cancer

" The advent of new cancer therapies alongside expected growth and ageing of the population better survival rates and associated costs of care is uncovering aneed to more clearly define and integrate supportive care services across the whole spectrum of the disease The current focus of cancer care is on initialdiagnosis and treatment and end of life care The Multinational Association of Supportive Care in Cancer defines supportive care as ˜the prevention andmanagement of the adverse effects of cancer and its treatment™ This encompasses the entire cancer journey and necessitates involvement and integration ofmost clinical specialties Optimal supportive care can assist in accurate diagnosis and management and ultimately improve outcomes A national strategy toimplement supportive care is needed to acknowledge evolving oncology practice changing disease patterns and the changing patient demographic“ The Royal College of Radiologists Published by Elsevier Ltd All rights reservedKey words Beyond cancer chronic cancer definition living with supportive careStatement of Search Strategies UsedA series of searches were constructed and carried out viaPubMed EMBASE and MEDLINE This generally consisted ofusing phrase searching due to the specificity of the subjectOnce concepts were established the authors used Booleanoperators to combine the concepts together and retrieve themost relevant papers Once a set of results were retrieved theauthors scanned each of the s using and titlefields to identify key papers Fulltext access to papers weresourced via the Christie Library and Knowledge ServiceIntroductionSupportive Care Makes Excellent Cancer Care PossibleMultinational Association of Supportive Care in CancerwwwmasccAdvances in diagnosis surgery radiotherapy and newdrugs have led to improvements in cancer survival PeopleAuthor for correspondence R Berman The Christie NHS FoundationTrust Wilmslow Road Manchester M20 4BX UK Tel ¾447710509402Email address RichardBermanchristienhsuk R Bermannow live nearly six times longer after their cancer diagnosisthan was the case years ago [] Half of people diagnosedwith cancer in England and Wales survive their disease for years or more [] Currently in England around million people are living with a diagnosis of cancer and thisnumber is increasing by over a year The total figure is setrise to over million by []Many more cancer patients are being treated closer todeath with novel less toxic high efficacy anticancer therapeutic agents developing with increasing pace within thelast decade The advent of molecular targeted agents forexample has brought new benefits as well as challenges tomodern cancer therapy potentially blurring the distinctionbetween active and palliative interventions []Yet despite this significant progress a large proportion ofpatients with cancer still experience morbidity and symptoms resulting from the cancer andor its treatment []Increases in cancer incidence [] emergency care hospitalisations [] earlier intensive care unit admissions [] andtreatment costs [] have all added to the global burden ofcancer care The disease is becoming a major economicexpenditure for all developed countries [] In the UK andin the USA cancer care costs are substantial and expected to101016jclon20200702009366555“ The Royal College of Radiologists Published by Elsevier Ltd All rights reservedPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxrise significantly in the future due to growth and aging ofthe population and improvements in survival as well astrends in treatment patterns and costs of care followingcancer diagnosis []cancerandcancerManagingtreatmentrelatedmorbidity is therefore a significant public health andeconomic challenge The coronavirus pandemic has deepened this challenge with many cancer outpatient visitsbeing replaced by telephone consultations and deferral ofsome routine therapy tests and procedures This has placedadditional pressures on an already fragile and vulnerablepopulation [] Patients and carers are experiencing moreuncertainty and anxiety associated with COVID19 A recentstudy found that although patients continue to feel wellsupported by their healthcare teams they have concernsabout the longerterm impact of changes to aspects of theirtreatment Patients and carers are no longer able to accessother support services in the way that they had previouslysuch as hospices and peer support groups []There is a growing body of evidence that timely access tosupportive treatments can lead to improvements in qualityof life and survival as well as benefitting the health economy [15e17] The development of a broad multiprofessionalbasis for the study and expansion of supportive carethrough the Multinational Association of Supportive Care inCancer MASCC has been an important step in fostering thegrowth of an evidence base [] MASCC's success has undoubtedly been underpinned by successful integration ofoncological and nononcological specialties []However variations in the definition of supportive careallocation of resources and a lack of clarity on who shouldlead onprovide services means that a clinical model forsupportive care in cancer does not yet exist [] Most specialties whilst they overlap other specialties are based on acore of knowledge or skill that is specific to that specialty[] Supportive care is currently provided by a patchwork ofdifferent medical specialties and is unique because it traverses the entire spectrum of the disease Figure fromdiagnosis through to survivorship or end of life care Theneed for ˜supportive oncology™ to become a specialty in itsown right is borne out not just by the progress in its development in the UK and abroad but by the unmet supportivecare need [] amplified by the rising incidence of cancerworldwide with many patients living longer with incurableillness because of more effective cancer treatments [] Asignificant next step would be to produce an evidencebasednational strategy for supportive care implemented throughappointment of supportive care lead clinicians within eachUK cancer centre This alongside support from the medicalRoyal Colleges and NHS England would be fundamental indeveloping a sustainable clinical modelPerhaps working as a distinct branch of oncology ˜specialists™ in supportive care medicine should have the skillsand resources to manage a broad range of effects associatedwith longterm cancers and cancer survival This paperexplores areas that are showing promise in this development and identifies key next steps needed to recognisesupportive care as an indispensable component of modernoncologyDefinition of Supportive CareThe Inuit may or may not have words for snow butsupportive care seems to have that number of definitions orconnotations [] Supportive care has been used as aeuphemism for palliative care and ˜early palliative care™[] and research suggests that a change in name frompalliative care to supportive care results in more and earlierreferrals to hospitalbased services [] Palliative care is anintegral component of supportive care but supportive careis much more than palliative care or even ˜early palliativecare™MASCC defines supportive care as ˜the prevention andmanagement of the adverse effects of cancer and its treatment This includes management of physical and psychological symptoms and side effects across the continuum ofthe cancer experience from diagnosis through treatment toposttreatment care Enhancing rehabilitation secondarycancer prevention survivorship and endoflife care areintegral to supportive care™ []Strategy for Implementation of SupportiveCare Within Cancer CareThe potential benefits of supportive care includedecreased morbidity improved quality of life and potentially decreased mortality ie secondary to optimal cancertreatment the potential benefits for healthcare servicesinclude decreased utilisation of healthcare resources andimproved treatment outcomes [] Indeed supportivecare offers patients more than many ˜palliative™ oncologicaltreatments and should be considered an essential not justan optional extraCurrently many cancer centres in the UK have supportivecare services either as a result of NHS England's EnhancedSupportive Care ESC Programme discussed below andFig The supportive care umbrellaPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxrelated Commissioning for Quality and Innovation CQUIN[] or as a result of local initiatives However the format ofthese teams is variable as is the patient cohort ie restrictedto specific cancer diagnoses and the interventions offeredie often restricted to symptom controlThus a national strategy is required to standardise supportive care services in relevant settings This needs to beevidencebased and ensure equity of care for all cancer patients irrespective of their cancer diagnosis or stage Thestrategy needs to address the current situation but alsoacknowledge evolving oncology practice ie new treatmentswith new toxicities changing disease patterns ie cancer as˜chronic disease™ and changing patient characteristicsIt needs to address education and training discussedbelow and be supported by benchmarking of servicesincluding inspections of clinical services incorporatingpatient feedback Investment will be required to standardise supportive care services and research fundingshould be allocated to determine the optimal model of careas well as the effectivenesscost effectiveness of the individual components of the servicesImplications for TrainingSupportive care encompasses the entire cancer journeyand so necessitates the involvement of most clinical specialties and many nonclinical services Figure Indeedmodern supportive care cannot be provided by a singleclinical specialty alone However as with other cancermultidisciplinary teams a dedicated ˜core team™ is neededto manage everyday problems with timely input from an˜extended team™ if the need arises Importantly the coreteam needs specificongoing education and training inprinciples of supportive careIt is also important to recognise that although manysupportive care services may have evolved from palliativecare services palliative care healthcare professionalsgenerally have limited formal training in supportive careand it is often not appropriate to extrapolate dataexperience from patients with advanced cancer to patientsreceiving anticancer treatment or cancer survivors Forexample the management of nausea and vomiting inadvanced cancer [] is very different from the management of chemotherapyinduced nausea and vomiting []The development of specialist supportive care servicesmust be supported by the educationtraining of the wideroncology workforce in the principles of supportive careand the management of common symptomsproblemsIndeed specialist supportive care services will only ever beable to see the ˜tip of the iceberg™ and so will need to focuson more complex problems and ones requiring specialistinterventions Moreover for example it is much moreappropriate for the team that gives the oncological intervention to manage the adverse effects of that oncologicalinterventionThus supportive care needs to be incorporated into thecurricula of all healthcare professionals involved in cancercare including primary care physicians AppropriateFig The extended supportive care teamcontinuing professional development opportunities need tobe developed for these groups Patients and their familiesneed access to appropriate educational resources in order tofacilitate rapidsuccessful treatment of the complications ofthe cancer andor the cancer treatmentEnhanced Supportive Care Programme eNHS EnglandNHS England promoted early development of supportivecare within some cancer centres via the ESC CQUIN programme CQUIN is the framework supporting improvements in the quality of services and the creation of newimproved patterns of care [] ESC CQUIN was developedby The Christie NHS Foundation Trust and was based uponsix key principles for the implementation and delivery ofsupportive care Figure [] The programme developedthrough recognition of what specialist palliative care professionals working alongside other cancer care disciplinescould offer across the whole cancer pathway e and throughrecognition of barriers to achieving earlier involvement[] Palliative care and supportive care are often differentlyanised across locations on the basis of resources andtraditions In some centres the two are anised as oneservice whereas in others they are completely separate[] The ESC programme required rebranding a closercollaboration with oncology and referral within weeks ofdiagnosis of incurable cancerNHS England's Specialised Commissioning ImprovingValue Team worked with commissioners and clinical teamsin ESC development Fourteen cancer centres took part inthe ESC CQUIN over a 3year period 2016e2019 Aninterim evaluation of the scheme took place in OctoberPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxx The programme was associated with a variety ofpositive outcomes including timelier referral of patientswith supportive care needs improved symptom controlimproved quality of life reduced 30day mortality fromchemotherapyimproved overall survival and reducedhealthcare costs [] ESC's principles of early referral andintervention may have impacted positively on these outcomes by better supporting patients who decide to proceedwith chemotherapy as well as those who decide not toproceedA limitation of the ESC CQUIN related to variation inservice delivery model across the centres Further robustresearch needs to be undertaken to determine the ˜optimal™approach for delivery of supportive care services withincancer centres and in other settingsDeveloping the Research and Evidence inSupportive CareWhen the American Society for Clinical Oncology ASCOcelebrated its 50th anniversaryit listed the five topachievements in oncology over that period Prominentlylisted was the development of highly effective antiemetictreatment [] What has been the impact of this keyadvancement in cancer supportive care and how did we getthere Does this progress guide us in improving other areasin supportive careThe impact of preventing emesis is broad and largeNausea and vomiting affect all aspects of daily living thequality of life benefits of antiemetic prevention have beendocumented Economically this advance allowed nearly allchemotherapy to be given on an outpatient basis ratherthan requiring hospitalisation This also allows people tohave less disruption and to remain with their families whilepursuing normal activitiesThese improvements are the result of thoughtful andlogical research Principles of this research included thefollowing which can be applied to many supportive caresettings i an understanding of appropriate physiology[] ii establishment of good clinical methodology []and iii evidence that affecting specific neurotransmitterpathways resulted in major clinical benefit [] As aresult of this work 80e90 of patients can be spared emesisin difficult settings as opposed to in the pastAs we enter an era where chemotherapy is progressivelyless used new areas for supportive care emerge Are weprepared to understand in depth unanticipated challengesin supportive care Can we prevent dermatological toxicities with tyrosine kinase inhibitormediated molecularlytargeted approaches through better understanding of themechanisms of these agents and skin physiology Can wepredict who is likely to have autoimmune sideeffects withcheck point inhibitors []Skills in caring for patients with cancer and methods oftreating malignancy continue to improve The advancesmade in preventing chemotherapyinduced nausea andvomiting provide a model that can influence approaches tomany other aspects of supportive care in cancerInterface with Acute Oncology eAmbulatory Supportive CareAdvances in cancer management continue to improvepatient outcomes This has expectedly been associatedwith an increase in emergency presentations with disease or treatmentrelated complications The challengesof emergency oncology presentations have led to an interest in developing optimal care models for meetingpatients' needs [] Cancer patients seeking emergencycare generally have higher admission rateslongerFig NHS England ESC CQUIN programme principles of ESCPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxlengths of stay and higher mortality than noncancerpatients []Ambulatory care is recognised as a key tenet in ensuringthe safety and sustainability of acute care services Thefundamental basis for ambulatory care is that patientspresenting with acute illnesses can be stratified as low riskfor developing complications and therefore do not requiretraditional inpatient care []Individualised management of acute cancer presentations is a key issue for emergency oncology services sothat it can mirror routine cancer care [] There are anincreasing number of acute cancer presentations that can berisk assessed for care in an emergency ambulatory settingThese include lowrisk febrile neutropeniacancerincidental pulmonaryassociated deep vein thrombosisembolism chemotherapyrelated acute kidney injurychemotherapyinduced nausea and vomiting indwellingline infections acute management of pain crises malignanthypercalcaemiaabnormalitiesasymptomatic brain metastases and malignant pleuraleffusion [43e46]and otherelectrolyteAmbulatory models offer the opportunity to integratepalliative care and supportive care with oncology and acuteservices This facilitates improved access for patients toexpertise in cancer care and immediate management of thecomplications of cancer treatment with the goal of preventing downstream complications and future emergencypresentations For example ambulatory enhanced supportive care models have shown utility in the managementof lowrisk febrile neutropenia []Modelling of ambulatory emergency oncology serviceswithin integrated supportive care services is therefore keyin the provision of highquality personalised and sustainable emergency oncology careThe Importance of Supportive Care inExperimental Cancer MedicineExperimentalcancer medicine trialsECMTs arefundamental to the development of novel cancer therapiesThe primary aims of ECMTs are to identify treatmentrelated toxicities and determine the recommended drugdose [] These trials are increasingly complex []intensive with risks of toxicity for patients but there is agrowing recognition that they are a valid therapeutic option []ECMTs have strict eligibility criteria with the need forpatients to have a performance status of or indicatinghigh levels of day to day functioning [] However thesepatients typically have advanced disease multiple previouslines of treatment and therefore a high associated symptom burden [] Hui [] found that patients referredfor ECMTs have a similar symptom burden to those whowere not despite the perception of higher levels of fitness Ahigh symptom burden has also been associated with earlydiscontinuation from trials [] highlighting the potentialrole for supportive care Br 13edart [] suggested thatthis patient group is more likely to accept increased toxicityto facilitate continued access to trial drugs In one studyECMT patients stated that they would still participate in atrial despite the potential risk of serious toxicities and a chance of death []Research suggests that ECMT patients are less inclined toaccept traditional palliative care due to a general andsometimes unrealistic optimism regarding trial participation[] alongside the perception that palliative care is onlyapplicable at the end of life [] However supportive carepractices within the early phase trials setting have the potential to reduce the impact of symptom burden and adverseevents on patients [] potentially increasing trial recruitment and the length of time patients spend on an experimental therapy Evidence in an ongoing study by Ferrell et al[] indicates that additional support can improve thequality of life for this patient group On top of the benefit topatients of access to additional therapies prolonged exposure to trial drugs supports research through increasednumbers of evaluable patients aiding efficient and accurateassessment of novel therapies Thus there is growing evidence for the role of supportive care for ECMT patients withthe need for increased research to assess potential benefitsand identify optimal routes for its deliveryLearning from Other CountriesImplementation of Supportive Care inFranceWith the aim of increasing and improving communityinvestment in supportive care MASCC is promoting severaldifferent approaches to engage countries such as 0f The creation of accreditation for hospitals withdedicated supportive care units 0f Promotion of MASCC and collaboration with localassociations at MASCC meetings 0f Special links with these associations such as jointmembershipsFrance committed to the supportive care approach at theend of the 1990s and as part of its first cancer plan in The French Speaking Association for Supportive Care inCancer AFSOS affiliated to MASCC was created in with the objectives ofaccompaniment 0f Promoting knowledge and execution of supportivecare in oncology 0f Sharing experience with all professionals involved insymptomsthethroughout all phases of the disease 0f Identifying and understanding the impact of thetransferability and interdependency between disciplines facilitating key aspects obstacles interestsand limitations of work 0f Heightening ethical awareness among medical staffand careofAFSOS has set up a research committee with four strategic priority directions healthcare anisation crossPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxdisciplinary meetings and departments supportive careunits dedicated teams management of cancer symptomsand treatments health behaviour and human and socialsciencesIts actions are targeted towards institutions eg TheFrench National Cancer Institute Ministry of Health professionals guidance and symposia on specific topics such asemesis or nutritional disorders as well as patients andtheir specific associations through a patientfacing website a roadshow truck crisscrossing France and an inventoryof supportive care resources AFSOS has developed nationalmeetings devoted to physicians and nurses physiotherapists or other health caregivers Guideline resources with atoolkit app are discussed during a specific 2day event andupdated every years AFSOS is involved in promoting international collaboration with other MASCCaffiliated societies eg Network Italiano Cure di Supporto in Oncologia[NICSO] and the Japanese Association of Supportive Care inCancer [JASCC]This French national mobilisation has led many regionalteams to get involved in cancer safety management projectsfor the benefit of patients and their relatives and can becopied in other countriesof these is poor in the UK [] and there are a number ofreasons why this may be even lower in an oncology setting[] The risk of poor bone health and fracture is increasingly recognised across a number of malignancies forexample a recent large Danish registry study showedincreased risk of fragility fracture in adults with haematological malignancy with the largest risk in the first 2e4years following initiation of treatment [] Given thedevastating nature of fractures there is much supportivecare work to be done to identify and treat at risk patientsand manage fragility fractures effectively across the spectrum of the cancer journeyEndocrinologists have had a traditional role in cancersurvivorship [] For example managing the longtermeffects of brain radiotherapy on the pituitary gland inchildhood brain tumour survivors As the prognosis foradult brain tumour survivors improves similar issues mayarise [] More recently endocrine toxicities such ashypophysitis and insulindeficient diabetes caused byimmunotherapy treatments are also keeping endocrinologists busy [] in collaboration with acute oncology []This will be become an even more complex issue asimmunotherapy moves into the adjuvant arena with expertinput into decision making algorithms crucial []Interface with Other Specialities egEndocrinology and DiabetesDiscussionOptimal supportive care of cancer patients requires inputfrom a range of specialties outside of oncology to assistaccurate diagnosis and management and ultimatelyimprove outcomesUp to of inpatients with cancer have diabetes or areat risk of diabetes from the treatments they receive []The importance of this is increasingly recognised patientswith diabetes and cancer have an increased length of hospital stay [] and mortality [] Although there iscurrently a lack of data demonstrating that improving glycaemic control reduces mortality for cancer patients it iscertainly true that effective and timely management ofhyperglycaemia improves quality of life and reduces inpatient length of stay but this requires specialist input from adiabetes teamSimilarly up to of inpatients with cancer experiencehyponatremia commonly secondary to syndrome of inappropriate antidiuretic hormone secretion although in theera of immunotherapy cortisol deficiency is an importantand increasing cause which can be fatal if missed []Untreated hyponatremia can delay oncology treatmentsand extend the length of hospital stay [] Diagnosis andmanagement of hyponatremia is poorly managed in general and the oncology population are no exception [] Weconsider expert supportive care input into the managementof hyponatremia in oncology patients to be essential inimproving this situationFractures particularly those of the hip and spine aredevastating with up to mortality at year following hipfracture and significant ongoing morbidity Vertebral fractures are highly predictive of further fracture but reportingThe current focus of cancer care is on initial diagnosisand treatment and the last year of life end of life care []However a large proportion of patients with cancer experience debilitating morbidity and complex symptomsresulting from cancer andor its treatment across the entirecancer journey Supportive care has been shown to improvequality of life symptom burden and survival as well asbenefitting the health economy [15e17] Thus supportivecare should be an integral component of modern oncologymanagement and should involve input from a range ofspecialties within and outside of oncology Furthermore itscontinued development perhaps most effectively as a subspecialty of oncology is essential in supporting advances inoncology and the changing demographic of the cancerpopulationConflicts of interestR Berman is a director of Supportive Care UK Ltd Thisis outside the scope of the submitted workReferences[] Macmillan Cancer Support Living after diagnosis mediancancer survival times Available at wwwmacmillanukdocumentsaboutusnewsroomlivingaftercancermediancancersurvivaltimespdf[] Cancer Research UK Cancer survival statistics Available atwwwcancerresearchukhealthprofessionalcancerstatisticssurvivalheadingZeroPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxx[] National Cancer Survivorship Initiative NCSI Living withand beyond cancer taking action to improve outcomesAvailableassetspublishingservicegovukgovernmentuploadssystemuploadsattachment_datafile1810549333TSO2900664NCSI_Report_FINALpdfat[] Clarke G Johnston S Corrie P Kuhn I Barclay S Withdrawal ofanticancer therapy in advanced disease a systematic literature review BMC Cancer [] Klastersky J Supportive care do we need a model Curr OpinOncol [] Global Burden of Disease Cancer Collaboration The GlobalBurden of Cancer JAMA Oncol 20151505e527[] National Audit Office Delivering the cancer reform strategyAvailable at wwwnaoukreportdeliveringthecancerreformstrategy[] Mokart D Pastores SM Darmon M Has survival increased incancer patients admitted to the ICU Yes Intens Care Med2014401570e1572[] National Institutes of Health Cancer trends progress report e update National Institutes of Health [] Sullivan R Peppercorn J Sikora K Zalcberg J Meropol NJAmir E Delivering affordable cancer care in highincomecountries Lancet Oncol 201112933e980[] Yabroff Y Lund J Kepka D Mariotto A Economic burden ofcancer in the US estimates projections and future CancerEpidemiol Biomarkers Prev 201120102006e2014[] Aggarwal A Sullivan R Affordability of cancer care in theUnited Kingdom e is it time to introduce user chargesJ Cancer Policy 2014231e39[] Saini K Heras B Castro J Venkitaraman R Poelman MSrinivasan G Effect of the COVID19 pandemic on cancertreatment and research Lancet Haem 202076e432ee435[] Radcliffe E Khan A Wright D Berman R Demain S RestorickBanks S Understanding the importance of selfmanagement support in people living with cancer reportReport on the impact of COVID19 in press[] Monnery D Benson S Griffiths A Cadwallader C HamptonMatthews J Coackley A Multiprofessionaldeliveredenhanced supportive care improves quality of life for patientswith incurable cancer Int J Palliat Nurs 20182410510e514[] Basch E Deal AM Dueck AC Scher HI Kris MG Hudis C et alOverall survival results of a trial assessing patientreportedoutcomes for symptom monitoring during routine cancertreatment JAMA 20173182197e198[] Cooksley T Campbell G AlSayed T LaMola L Berman RA novel approach to improving ambulatory outpatient management of low risk febrile neutropenia an Enhanced Supportive Care ESC clinic Support Care Cancer 2937e2940[] Klastersky J Christel F Editorial Supportive care in cancerpatients a constantly evolving field Curr Opin Oncol 314257e258[] Cooksley T Rice T Emergency oncology development current position and future direction in the USA and UK SupportCare Cancer 2017253e7[] Guly H Preface A history of accident and emergency medicine1948e2004 London Palgrave Macmillan [] Hui D Hannon BL Zimmerman C Bruera E Improving patientand caregiver outcomes in oncology teambased timely andtargeted palliative care CA Cancer J Clin 201868356e376[] Whelan TJ Mohide EA Willan AR Arnold A Tew M Sellick S The supportive care needs of newly diagnosed cancerpatients attending a regional cancer center Cancer 8081518e1524[] Hui D De La Cruz M Mori M Parsons HA Kwon JH TorresVigil I Concepts and definitions for œsupportive careœbest supportive care œpalliative care and œhospice care inthe published literature dictionaries and textbooks SupportCare Cancer 201321659e685[] Boyd K Moine S Murray SA Bowman D Brun N Shouldpalliative care be rebranded B

Thyroid_Cancer

inhibitor with Temozolomide results in significant apoptosis in glioblastoma via the p65 and actin cytoskeleton regulatory pathwaysNaze G Avci1 Sadaf Ebrahimzadeh‘Pustchi1 Yasemin M Akay1 Yoshua Esquenazi2 Nitin Tandon Jay‘Jiguang Zhu Metin Akay1Glioblastoma GBM is the most malignant brain tumor characterized by intrinsic or acquired resistance to chemotherapy GBM tumors show nuclear factor‘κB activity that has been associated with tumor formation growth and increased resistance to therapy We investigated the effect of inhibitor BAY ‘ with Temozolomide TMZ on the signaling pathways in GBM pathogenesis GBM cells and patient‘derived GBM cells cultured in 3D microwells were co‘treated with BAY ‘ and TMZ or BAY ‘ and TMZ alone and combined experiments of cell proliferation apoptosis wound healing assay as well as reverse‘phase protein arrays western blot and immunofluorescence staining were used to evaluate the effects of drugs on GBM cells The results revealed that the co‘treatment significantly altered cell proliferation by decreasing GBM viability suppressed pathway and enhanced apoptosis Moreover it was found that the co‘treatment of BAY ‘ and TMZ significantly contributed to a decrease in the migration pattern of patient‘derived GBM cells by modulating actin cytoskeleton pathway These findings suggest that in addition to TMZ treatment can be used as a potential target to increase the treatment™s outcomes The drug combination strategy which is significantly improved by inhibitor could be used to better understand the underlying mechanism of GBM pathways in vivo and as a potential therapeutic tool for GBM treatmentGlioblastoma multiforme GBM is the most malignant primary brain tumor in the central nervous system Current standard of care therapy includes surgery followed by radiotherapy and concomitant and adjuvant chemotherapy with the alkylating agent Temozolomide TMZ which provides survival benefits for patients with GBM1 However even with the advances in surgical resection combined with TMZ therapy and irradiation the prognosis for newly diagnosed GBM patients remains poor In fact due to its rapid proliferation increased invasion and migration capacity and chemoresistance to the alkylating agents a0the median survival is only a0months with the ˜Stupp™ regimen radiation with daily TMZ — “ a0weeks followed by cyclic TMZ2 and 5year survival rate is less than which is the lowest longterm survival rate of malignant brain tumors3“ TMZ methylates DNA at the O6 positions of guanine and DNA repair enzyme O6methylguanine methyltransferase MGMT removes alkyl groups from O6 position of guanine in DNA making cells resistant to TMZ6 Therefore new therapies are necessary to prevent cell proliferation and induce apoptosis for GBM patientsNuclear factorkappa B NFκB is a regulatory transcription factor of the Rel gene family including p50 cRel RelB or p65 subunits It is involved in the control of tumor cell proliferation migration immune response and apoptosis7“ Studies have shown that NFκB gene was involved in the regulation pathways of different cancer types such as thyroid cancer head and neck squamous cell carcinoma and colorectal cancer711“ Increased 1Department of Biomedical Engineering University of Houston Cullen Blvd Houston TX USA 2UTHealth Neurosurgery McGovern Medical School Memorial Hermann at Texas Medical Center The University of Texas Health Science Center at Houston Houston TX USA email makayuheduScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cactivation of NFκB has also been identified in GBM tumors where the expression of NFκB was much higher in GBM tissue compared with nonGBM tissue1415 NFκB also promotes chemoresistance to TMZ and regulates MGMT activity in GBM by promoting MGMT gene expression through NFκB binding sites within the MGMT promoter16 NFκB inhibitors such as parthenolide do not completely eradicate tumors therefore they are mostly used in combination with other drugs17 When used in combination with TMZ NFκB inhibitor parthenolide has been shown to activate mitochondrial apoptosis signaling in U87MG and U373 GBM cells which lead to cell death18 and had a combined effect on cell cytotoxicity in LN18 and T98G glioma cells19 NFκB inhibitor CBL0137 has been shown to bind DNA leading the functional inactivation of the Facilitates Chromatin Transcription FACT complex a chromatin remodeling complex regulating transcription replication and DNA repair2021 In a0vitro evaluation of the CBL0137 on FACT p53 and NFκB has been done using U87MG and A1207 GBM cells It was shown that CBL0137 induced loss of chromatinunbound FACT activated p53 and inhibited NFκB dependent transcription21 In a0vivo studies showed that CBL0137 was effective in increasing survival rates in TMZresistant orthotopic mouse models21 Moreover Wang et a0al indicated that NFκB inhibitor BAY suppresses the expression of MGMT and enhances the TMZinduced apoptosis in TMZ resistant U251 cells22 However there is still a lack of characterization of the precise pattern of NFκB activation in combination with TMZ in GBM cell populations that have been a0surgically resected from patientsIn vitro and in a0vivo identifications and validations of molecular targets of GBM are important as they can progress into clinical studies Studies reported that combining multiple gene targets may prevent tumor growth and improve the treatment strategy for GBM23“ Both Bay and TMZ exert antitumoral activities individually in different tumor types28“ Therefore in this study we aimed to analyze functionally the combined effect of Bay and TMZ in different GBM cells For this purpose first we used our 3D PEGDAbased hydrogel microwell platform31“ to provide reliable preclinical models that can recapitulate in a0vivo features of the GBM tumors We cultured GBM cells U87 and LN229 and patientderived GBM cells in 3D microwells for a more precise and personalized treatment approach We then treated GBM cells with Bay and TMZ in combination or alone Our results indicated that the cotreatment of Bay and TMZ significantly reduced cell viability in all three cell lines in correlation with a significant decrease in the spheroid size The levels of NFκB protein and its subunits p65 and p50 were also significantly decreased compared with the control and single drug applications Similar a0decreases in the cell viability and protein levels were observed in all three GBM cells Tumor biopsy samples could give more realistic information about how tumors respond to drugs when they are used for in a0vitro or in a0vivo studies35“ Therefore we decided to continue our experiments with only using our patientderived GBM cells We treated patientderived GBM cells with Bay and TMZ or alone and analyzed specific cellular proteins along with their posttranslational modifications via reversephase protein arrays RPPA to elucidate the mechanism of action of the proteins3839 We observed that several cell signaling pathways including cell metabolism proliferation apoptosis were significantly affected by the combination of the drugs which were consistent with the literature4041 Furthermore our RPPA data revealed that there was a significant change in the modulation of actin cytoskeleton and following experiments including western blot analysis for the expression of FAK protein and wound healing assay for cell migration patterns confirmed the RPPA results We observed a significant decrease in both actin fluorescence intensity and migration pattern in the a0cotreated patientderived GBM cells To the best of our knowledge the effect of cotreatment of Bay and TMZ has never been studied previously on the actin modulation of patientderived GBM cells These results suggested that Bay and TMZ induced alteration in the a0actin filament anization by reducing the level of focal adhesion protein which might implicate in cell apoptosis The effect of Bay with TMZ necessitates further exploration to better understand its mechanism of action in GBM and potential therapeutic tools for GBM treatmentResultsCo‘treatment of Bay ‘ and TMZ reduced viability of GBM cells We used our previously a0published data to select the most effective drug concentrations for this study42 We cultured LN229 U87 and patientderived cells in the microwells for a0days where they formed 3D spheroids and we added a0µM of Bay and a0µM of TMZ in combination or alone Then we cultured the spheroids for more days with or without drug Control group did not receive any treatment The cell viability assay was performed on day after drug administration The results showed that the a0cotreatment significantly reduced cell viability of GBM cells LN229 and U87 and patientderived GBM cells cultured in 3D PEGDA microwells respectively as shown in Fig a01ac When they were used alone TMZ reduced cell viability to and p and Bay reduced cell viability to and in LN229 U87 and patientderived GBM cells respectively compared to control groups Fig a01d However when they were used in combination the viability of the cells significantly decreased to and in LN229 U87 and patientderived GBM cells respectively compared to control groups p Fig a01d Tumor cells are generally less sensitive to drug treatments in 3D cultures than in 2D cultures4344 This could reflect reduced compound access or differences in the response to cell death To confirm that cotreatment was more effective compared to single drug use we quantified the size of the spheroids using ImageJ45 Our data showed that after a0days of drug treatment the spheroids™ sizes were significantly reduced in the cotreatment by and in LN229 Fig a01e U87 Fig a01f and patientderived GBM cells p Fig a01g respectively compared to control group p When we compared the spheroids™ sizes of the cotreatment with TMZ alone there was a reduction of and in LN229 U87 and patientderived GBM cells respectively p Finally the spheroids™ sizes of the cotreatment compared with Bay alone showed a decrease of and in LN229 U87 and patientderived GBM cells respectivelyScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Representative images of the GBM tumor cells cultured in the PEGDA microwells a“c LN229 U87 and patientderived GBM cells were cultured in the microwells for a0days respectively After day Bay and TMZ were applied either alone or in combination onto the cell spheroids Control group did not receive any treatment The cells were cultured with or without drugs additional more days The images were taken on Day Day and Day after the drug application to observe the disruption in the spheroids Dotted black lines represent the edge of the tumor spheroid Scale bars a0µm d Bar graph showing trypan blue staining for cell viability of LN229 U87 and patientderived GBM cells e“g Spheroid size quantification was done using ImageJ for LN229 U87 and Patientderived GBM cells respectively Twotailed ttest followed by Wilcoxon test were done GraphPad Prism v5 Data represent the mean ± SD of three biological replicates p and p Suppression of activity in GBM cells by co‘treatment of Bay ‘ and TMZ As a readout of NFkB activity after drug treatment we first quantitatively assessed the cytoplasmic activation of phosphorylated NFκB p65 subunit in both treated and untreated groups in all GBM cells NFκB pp65 subunit activity was observed in the control groups of all three GBM cells Fig a02a NFκB pp65 subunit activity decreased to and when TMZ applied alone and and when Bay was applied alone in LN229 U87 and patientderived cells respectively However the decrease in NFκB pp65 subunit was reduced to when LN229 U87 and patientderived cells respectively were cotreated p Fig a02a Bay specifically inhibits NFκB activation by blocking phosphorylation of IκBα46 In independent experiments we analyzed the abundance of phosphorylated NFκB p65 NFκB p50 and IκBα in all three GBM cells Qualitative and quantitative western blot analysis revealed that the exposure to Bay with TMZ significantly downregulated the abundance of NFκB p65 NFκB p50 and IκBα compared with control and Bay or TMZ alone Fig a02b Please note that loading controls were used for each experiment but only the representative loading control for p and tP65 and p and tP50 was presented Fig a02b The cell viability assay cells™ size and protein expressions in all three GBM cells revealed similar results without any dramatic change Therefore considering the importance of using patientderived tumor cells to elucidate the mechanism of drugs and respective signaling pathways35“ we further continued our experiments using patientderived GBM cellsApoptosis was promoted by co‘treatment of Bay ‘ and TMZ RPPA technology is designed for multiplexed antibodybased relative quantification where each array is tested with a validated antibody specific to a particular protein along with their particular posttranslational modifications47 In the attempt to elucidate the mechanism of action of Bay with TMZ by which NFκB subunits were modulated and to identify downstream signaling molecules we employed RPPA platform using our drug treated or untreated patientderived GBM cells RPPA results showed that many oncogenic pathways were altered by the drug treatments but more specifically by the cotreatment Fig a03a Decreased expression of NFκB was not only associScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 NF“kB activity in LN229 U87 and patientderived GBM cell lines a NF“kB p65 subunit activity in LN229 U87 and patientderived GBM cell lines respectively The cells cultured with or without drugs for a0days were collected from the microwells and subjected to ELISA Data represent the mean ± SD of three biological replicates p and p b Representative immunoblots LN229 U87 and patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels bottom panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p ated with changes in the a0NFκB pathway but also with apoptosis cell metabolism and proliferation which were confirmed by the analysis of downregulated RPPA proteins in Enrichr KEGG libraries4849 Fig a03c p One of the specific pathways given by RPPA was apoptosis Apoptosis is one of the important mechanisms that regulates cell death and suppress tumorigenesis Studies have demonstrated that Bcl2 family proteins can positively and negatively regulate apoptosis by regulating antiapoptotic protein Bcl2 and proapoptotic protein Bax4050 Our RPPA data using patientderived GBM cells showed that the fold change of Bcl2 relative to control was times higher in cotreated group TMZ alone Bay alone respectively Fig a03b In order to further confirm whether the expression of a0these proteins were downregulated by the cotreatment we performed western blot analysis Our results showed a similar decrease in Bcl2 protein expression in the cotreatment compared with the control and single drug a0treatment Fig a03d In contrast Bax protein fold change relative to control was times higher in cotreated group TMZ alone Bay respectively where we observed a significant increase after the cotreatment of Bay with TMZ compared with the control p Fig a03b Bcl2Bax ratio is a key indicator in susceptibility of the cells to apoptosis Western blot results confirmed the change in Bcl2Bax ratio in the cotreatment compared with the control group and single a0drug treatment Fig a03d Our RPPA data also showed a significant increase in the cleavedcaspase protein Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 The effect of Bay and TMZ on signaling pathways in patientderived GBM cells a Heat map presentation of RPPA analysis showing the changes in the protein expression RPPA was performed on lysates treated with Bay and TMZ alone or in combination All relative protein level data points were normalized to the a0control group Red and green indicate up and down regulations respectively in the heat map The samples were run in duplicate n b Fold change of the a0selected proteins relative to the a0control group via RPPA Data represent the mean ± SD of two biological replicates p p Wilcoxon rank sum test c Analysis of downregulated RPPA proteins shows a a0significant activation in numerous Enrichr KEGG pathways The pathways were a0sorted by p value ranking d Representative immunoblot validation of significantly altered proteins involved in different KEGG pathways Patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p fold change relative to control times higher in the cotreatment compared with times higher in TMZ alone and times higher in Bay alone p Fig a03b To confirm if cotreatment triggered apoptosis correlated with caspase activation we performed western blot analysis with procaspase3 cas3 and cleavedcaspase3 Ccas3 We observed that Bay and TMZ induced apoptosis was associated with cas3 Fig a03d Please note that loading controls were used for each experiment but only the representative loading control for Bax cas3 and Ccas3 was presented Fig a03d Moreover another important mechanism of NFκB activation in GBM regulates through AKT phosphorylation of IκB Our RPPA data showed relative fold changes of in the cotreated group TMZ alone and Bay alone respectively p Fig a03b The western blot results also confirmed a significant decrease in the abundance of AKT pT308 Fig a03dTo further investigate whether cotreatment of Bay with TMZ can lead to glioma cell apoptosis and to confirm our RPPA and western blot results we performed apoptosis assay TUNEL The patientderived GBM cells were cotreated with Bay with TMZ or single drug treated and subjected to TUNEL assay to detect DNA damage Fig a04a The results indicated that TUNEL cells in the cotreatment were increased tenfold compared with control and and 24folds compared with TMZ alone and Bay alone respectively p Fig a04b Additionally in some TUNEL cells we observed a typical ring type chromatin aggregation underneath the nuclear membrane which suggested an early stage apoptosis51 Fig a04a red arrows There were also a few TUNEL cells that lacked the typical apoptotic ringlike nuclear structure indicating that they were either at a different stage of apoptosis or alternatively undergoing necrosis52 that we have not investigated furtherCo‘treatment of Bay ‘ with TMZ changed actin anization by inhibiting FAK phosphorylation and cell migration Actin filaments Factin are one of the main components of the cellular cytoskeleton which regulates actin dynamics and migration process in the cells The disruption of the actin cytoskeleton inhibits cell migration and adhesion53 Depolymerization or cleavage of actin lamins and other cytoskeletal proteins have been also found to be involved in cell apoptosis54“ To confirm the RPPA results showing changes in the actin modulation pathway and to understand the mechanism that regulates cytoskeletal Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Apoptosis assay TUNEL a Fluorescent images of TUNEL cells in patientderived GBM cells TUNEL assay was performed on cells treated with Bay and TMZ in combination or alone in the microwells Cells were collected from the microwells trypsinized and replated into 8well chamber slides TUNEL cells green with ringlike nuclear stain are indicated with red arrows Nuclei were counterstained with DAPI blue b Numbers of TUNEL cells are presented as mean ± SD of three biological replicates p and p X20 objective Scale bars a0µmanization we treated patientderived GBM cells co treated with Bay with TMZ or single drug treated 3D spheroids collected from the microwells were stained with phalloidin green and DAPI blue Staining cells with fluorescently conjugated phalloidin is considered the most reliable method of accurately labeling Factin in fixed cells57 In the control group intact cells formed finemeshed networks with a distinct Factin anization on both day Fig a05a upper panel and day Fig a05a bottom panel In single drug treated cells actin was still found to be polymerized to filaments as it can be seen by its interaction with phalloidin at both days and However the cells which were cotreated with Bay and TMZ lost their Factin anization and their shape compared with the control and the single drug treated groups at day Fig a05a bottom panel Changes in the a0actin distribution within the cells were quantified by measuring the staining intensity using Fiji Macro ImageJ as described previously5859 At day we observed a a0significant decrease in the fluorescence intensity of phalloidin when the cells were cotreated with Bay and TMZ compared with the a0control and single drug treated groups p Fig a05b To investigate the drug related Factin mechanism we examined the levels of FAK protein following cotreatment or single drug treatment As shown in Fig a05c cotreatment significantly decreased the level of phosphorylated FAK compared with both control and single drug applications p Furthermore we investigated cell migration patterns of the patientderived cells that were cotreated with Bay and TMZ or single drug treated We collected 3D spheroids from microwells after drug treatment and replated them in 24well plate to perform scratch wound healing assay We noted a significant increase in cell density in the scratch area in both control and Bay alone after and a0h of scratch formation p Fig a06a Although compared with the a0control cells both cotreatment and TMZ alone groups showed a decrease in the cell migration into the scratch area after a0h we observed that after a0h the migration rate of the cotreated cells was significantly slower than the cells that were treated with TMZ alone p Fig a06b These results indicated that the disanization of actin microfilaments was concomitant with the cell apoptosis after the a0cotreatment of Bay with TMZDiscussionDespite the increase in the median survival of GBM patients from to months4 the clinical efficacy of standard of care therapy including TMZ chemotherapy combined with surgery and radiotherapy is still limited Due to challenges in treating GBM significant attempts have been made to develop single or combined drug treatments60“ However given the cost long time frame and risks of failure associated with developing a new drug repurposing available drugs may be the most effective alternative therapeutic strategy Therefore it is important to evaluate potential drug combinations for GBM treatmentScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Changes in the actin cytoskeleton and migration pattern in patientderived GBM cells cotreated with Bay and TMZ or single drug treated in the microwells a Upper panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin green and DAPI blue Bottom panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin and DAPI Scale bars a0µm b Intensity of staining obtained with phalloidin was measured in each cell using ImageJ and displayed as boxplots with to confidence intervals A twoway ANOVA with Dunnett™s multiple comparisons test was performed to determine statistical relevance Three biological replicates n p p c Representative immunoblots show the levels of FAK pTyr397 and total FAK in patientderived GBM cell lysates cotreated with Bay and TMZ or single drug treated for a0days in the microwells The levels of the proteins were quantified using ImageJ right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p Due to the cell repellent property of PEGDA hydrogel tumor cells can form aggregates at the bottom of the microwells and selfassemble into spheroids in each well within a0days following cell seeding313363 Compared with 2D monolayer cell culture 3D spheroids have an important advantage their larger size Thus often drug effects can easily be monitored over time by measuring the size and shape of spheroids4344 Additionally using 3D in a0vitro tumor models can better recapitulate in a0vivo features of the tumors We used PEGDA hydrogelbased microwell platform313363 in order to culture different types of a0GBM cells commercially available GBM cell lines LN229 U87 and a0patientderived GBM cells However we investigated the effect of the drugs on the patientderived GBM cells more in detail since growing tumors from tumor biopsy samples could give very detailed information about how tumors respond to drugs35“ Considering the precious nature of the patient samples this platform which requires fewer cells compared with 2D monolayer cultures provides us with a robust tool to recapitulate in vivo features of GBM tumors and to test our drug combinationsNFκB is one of the major transcription factors associated with GBM and responsible for activating a series of cellular responses including cell proliferation survival invasion and apoptosis6465 Previous studies have shown that NFκB can activate Akt and promote cell survival and proliferation by downregulating the expression of phosphatase and tensin homolog deleted on chromosome ten1866 NFκB pathway can inhibit cell apoptosis by inhibiting a stressactivated protein kinase and a mitogenactivated protein kinase signaling pathway67 It can also be activated in response to treatment with cytotoxic drugs such as vinca alkaloids and topoisomerase inhibitors Several studies have demonstrated the activation of NFκB in GBM patientderived stemlike cells cultures96869 Moreover alkylating agents TMZ can activate NFκB through DNA damage pathway activation7071 The combination effect of Bay and TMZ have been showed in our previous study where we determined the most effective drug concentrations on GBM cells using our microfluidics platform42 Another study that investigated the combined effect of NFκB inhibitor BAY with TMZ showed that combined drug application induced TMZ resistant in U251 GBM cells22 However the characterization of the precise pattern of NFκB activation in different GBM cell populations from surgically resected tissues still remains elusive Therefore in this study we investigated the interaction of Bay with TMZ and their effects on the LN299 and U87 GBM cell lines as well as patientderived GBM cells in order to recapitulate NFκB activation as in a0vivo features of the GBM and its signaling pathways We applied a0µM of Bay and a0µM of TMZ3442 in combination or alone for all three GBM cell types First we observed a significant decrease in both cell viability and size of the spheroids in the cotreatment compared with control and single drug application Then we showed quantitatively and Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Cell migration of patientderived GBM cells by wound healing assay a Patientderived cells were cotreated with Bay and TMZ or single drug treated in the microwells trypsinized and replated in 24well plates After they reached to their confluency a scratch wound was formed with a 200μl tip and cells were incubated for the next a0h Images were taken 4x at a0h a0hr and a0hr Scale bars a0µm b The wound width was measured with ImageJ and the average wound width was shown Data represent the mean ± SD of three biological replicates p and p oneway ANOVA with Tukey™s post hoc testqualitatively the expression of NFκB in all three GBM cell types a0We noted a significant decrease in the cotreated group compared with control and single drug application Our western blot data also confirmed the decrease in the abundance of pP65 pP50 and pIKBa that Bay has been shown to inhibit its phosphorylation46 However in the cotreated group the decrease was significantly higher compared to both control and single drug application This data showed that cotreatment of Bay and TMZ has more effect on the inhibition of NFκB pathway than Bay or TMZ alone and suggests a a0decreased downstream transcription of oncogenic proteins72 Although there were slight differences in the NFκB expression patterns in three different GBM cell types a0we focused on the patientderived cells in the rest of the study due to their ability to better recapitulate the genomic similarities to primary disease7374Proteins that interact with each other activate multiple pathways which can result in apoptosis according to tissue type and pathological condition Glioblastoma tumors express high levels of antiapoptotic BCL2 family proteins such as Bcl2 and BclxL which may cause glioblastoma cells to resist apoptosis75 The proapoptotic members of Bcl2 family such as Bax and Bak are necessary for their proapoptotic effect Interactions and the ratio between antiapoptotic Bcl2 and proapoptotic Bax are decisive factors in the induction of apoptosis7677 Active NFκB can prevent cells from apoptosis by stimulating the expression of genes and promoting cell proliferation Although patientderived GBM samples have been shown to be highly resistant to apoptosis77 our data revealed changes in the expression of various members of Bcl2 family and NFκB signaling pathway after cotreatment of Bay and TMZ Our RPPA results outlined distinct molecular profiles in which apoptotic P53 signaling and NFκB signaling pathways were significantly affected after the a0cotreatment These results supported that the inhibition of NFκB expression could inhibit the expression of Bcl2 and promote the expression of Bax thus promote apoptosis Our data also suggested the possible interaction between Bcl2 and p53 in Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Proposed schematic of the a0signaling pathways involved in Bay and TMZmediated inhibition in GBM patientderived cells The effect of combined therapy of Bay and TMZ was achieved through the inhibition of SrcFAKVinculin which regulate the cytoskeleton anization through MAPKs JNK and PI3KAKT signaling pathways Exposure to both Bay and TMZ also leads to receptormediated activation of Bax but not Bcl2 in the subsequent inhibition of the downstream NFκB transcription factor Inhibition of NFκB in turn causes cell deathregulating cell survival and death7778 The activation of extrinsic and intrinsic molecular pathways can lead to the proteolytic activation caspases The extrinsic pathway is triggered by proapoptotic ligands that activate cell surface death receptors and procaspase8 which in turn leads to the cleavage of caspase3 and apoptosis79 Our results determined that the a0cotreatment significantly inhibited the expression of caspase3 while the expression of cleaved caspase3 was increased Additionally TUNEL assay which detects DNA strand breaks which could occur as an event in the apoptosis showed a dramatic increase in the TUNEL cells after the cotreatment compared with the a0control and single drug application Altogether these results suggested that the inhibition of cell proliferation Bcl2 and caspase3 by a0the cotreatment of Bay and TMZ may occur through the NFκB mediated apoptosis and they might be tightly coupled8081The literature provides evidence that supports crosstalk between PI3KAktmTOR signaling pathway and NFκB which is downstream of Akt NFκB activation in GBM regulates through AKT phosphorylation of IκB resulting in an activated NFκB that translocates to nucleus8283 Our data showed that when Bay was used with TMZ there was a decrease in the abundance of PI3Kp110 AktpS473 AktpT308 and mTORpS2448 This preliminary data is important to suppo

Thyroid_Cancer

"rapeutic plasma exchange clears circulating soluble PD L1 and PD L1 positive extracellular vesicles Elizabeth Ann L Enninga2 Fabrice Lucien Matteoni3 Jacob J Orme Heather Dale4 Edwin Burgstaler4 Susan M Harrington3 Matthew K Ball4 Aaron S Mansfield1 Sean S Park5 Mathew S Block1 Svetomir N Markovic1 Yiyi Yan1 Haidong Dong3 Roxana S Dronca6 Jeffrey L Winters4To cite Orme a0JJ Enninga a0EAL Lucien Matteoni a0F et a0al Therapeutic plasma exchange clears circulating soluble PD L1 and PD L1 positive extracellular vesicles Journal for ImmunoTherapy of Cancer 20208e001113 101136jitc2020001113 –º Additional material is published online only To view please visit the journal online http dx jitc Accepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Division of Medical Oncology Mayo Clinic Rochester Minnesota USA2Department of Obstetrics and Gynecology Mayo Clinic Rochester Minnesota USA3Department of Urology Mayo Clinic Rochester Minnesota USA4Department of Laboratory Medicine and Pathology Mayo Clinic Rochester Minnesota USA5Department of Radiation Oncology Mayo Clinic Rochester Minnesota USA6Department of Hematology and Oncology Mayo Clinic Florida Jacksonville Florida USACorrespondence toDr Jacob J Orme orme jacob mayo eduBackground Trans acting programmed death ligand PD L1 derives from malignant cells in three known forms High levels of secreted splice variant PD L1 sPD L1 ADAM10ADAM17 shed sPD L1 and PD L1 positive extracellular vesicles evPD L1 each predict poor prognosis and limited response to PD L1 checkpoint inhibitors in cancer To our knowledge no clinical intervention has reduced any of these circulating forms of extracellular PD L1 Here we explore therapeutic plasma exchange TPE as a treatment to reduce circulating extracellular PD L1Results In patients with melanoma sPD L1 levels above ngmL predicted inferior overall survival In patients undergoing TPE for non malignant indications each TPE session removed a mean sPD L1 and evPD L1 detectable in plasma TPE also reduced total and ADAM10 positive extracellular vesiclesConclusion Here we report the first known clinical intervention to remove either sPD L1 or evPD L1 from plasma in vivo TPE reduces plasma sPD L1 and evPD L1 in vivo and may have a role in treatment with immunotherapy TPE may also prove useful in patients with other extracellular vesicle related conditionsINTRODUCTIONOvercoming initial or acquired resistance to programmed death ligand PD L1 immune checkpoint inhibitors is a major area of unmet need for many cancers1 Although the full scope of mechanisms of resistance to these therapies has yet to be determined different forms of tumor derived extracellular PD L1 have been linked to resistance in multiple clinical studies2“in Malignant cells produce trans acting extracellular PD L1 three distinct forms First tumor cells transcribe and secrete soluble PD L1 sPD L1 splice variants4 Second enzymes ADAM10 and ADAM17 shed sPD L1 ectodomain directly from the tumor cell surface6 Both forms of sPD L1 carry known homodimerization domains and can be detected by ELISA sPD L1 can outcompete PD L1 inhibitors kill CD8 T cells and limit the ability of healthy peripheral blood mononuclear cells to kill tumor cells in vitro6 In a third form tumors generate extracellular vesicles EVs bearing surface PD L18 PD L1 positive EVs evPD L1 exhibit similar properties to sPD L1 in systemic circulation9 Each type of trans acting extracellular PD L1 correlates with poor survival in multiple clinical trials online supplementary table While broad spectrum pharmacological inhibitors and genetic manipulation have been shown to reduce release of these forms of PD L1 in culture or animal models none are suitable for clinical use To date we know of no reported clinical intervention that safely and reliably eliminates any of these forms of immunosuppressive systemic extracellular PD L1Therapeutic plasma exchange TPE is a procedure in which blood is passed through an apheresis machine separating plasma from cellular components Removed plasma is discarded and replaced with either colloid eg albumin or crystalloid and colloid solutions Unlike dialysis which removes small ions by diffusion TPE removes plasma restricted substances like antibodies that are too large for rapid diffusion On average each TPE session removes approximately “ of large non cellular plasma restricted intravascular components10It is unknown whether sPD L1 approximately kDa or PD L1 positive EVs “ nm are plasma restricted non diffusing and unbound If so we hypothesized that TPE could efficiently remove these substances from patient blood Such an intervention could if effective improve response to immunotherapyOrme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Figure Soluble programmed death ligand PD L1 suppresses antitumor immunity and predicts overall survival in patients with melanoma A A model of three known tumor derived extracellular PD L1 forms” evPD L1 ADAM10ADAM17 cleaved soluble PD L1 sPD L1 ectodomain and secreted splice variant sPD L1”that downregulate antitumor immunity and prevent response to PDL1 inhibition B A Kaplan Meier plot shows significantly worse overall survival for patients with melanoma exhibiting high ‰¥ ngmL versus low ngmL plasma sPD L1 levels p0005 C Patients with melanoma exhibited a higher mean plasma sPD L1 level ngmL in comparison to healthy controls ngmL p0001RESULTSsPDL1 levels predict overall survival in patients with melanomaEach form of extracellular PD L1 acts in trans as a systemic immunosuppressant through PD1 signaling figure 1A online supplementary table “ To confirm the clinical impact of plasma sPD L1 we measured sPD L1 levels in a retrospective cohort of patients with melanoma Exploratory analysis of overall survival OS determined a working cut off value of sPD L1 ‰¥ ngmL and baseline characteristics at the time of entry into study were similar online supplementary table Patients with high plasma sPD L1 levels experienced inferior median OS compared with patients with low plasma sPD L1 levels figure 1B vs months p0005 In comparison to healthy age matched controls patients with melanoma exhibited higher mean plasma sPD L1 figure 1C ngmL vs ngmL p0001 In a multivariate Cox proportional hazards analysis high sPD L1 prior to treatment predicted worse survival HR CI to p0025 when accounting for advanced age not significant sex not significant late stage p0002 and high serum LDH p001 online supplementary table TPE significantly reduces plasma sPDL1 levelsWe hypothesized that TPE may remove extracellular PD L1 in its various forms figure 2A To address this question we prospectively enrolled patients undergoing planned TPE figure 2B Twenty eight patients met inclusion criteria of which provided informed consent Baseline patient characteristics are in table One patient was excluded for biotin containing supplement use as biotin interferes with the established sPD L1 detection assay The remaining patients underwent plasma exchange and sample collection before and after the procedure as described Discarded plasma samples from the TPE device waste bag for each session were also collected sPD L1 was measured in each sample and most patients undergoing TPE exhibited sPD L1 levels above the clinically relevant ngmL cut off from the retrospective melanoma studyMost patients undergoing TPE did not have an active cancer diagnosis Baseline sPD L1 levels in all patients were compared with matched normal controls and patients with melanoma online supplementary fig and some patients exhibited sPD L1 above the clinically significant cut off level determined in the retrospective melanoma cohort Patients with high baseline sPD L1 levels were significantly more anemic than patients with lower baseline sPD L1 even when controlling for the higher number of female subjects in the high sPD L1 group female only mean Hgb vs p004 male only mean Hgb vs p003 Groups were otherwise similar TPE significantly reduced plasma sPD L1 levels in patients receiving albumin only ie no Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c accessFigure Therapeutic plasma exchange TPE significantly reduces plasma soluble programmed death ligand sPD L1 levels A A model of the TPE procedure in which patient plasma is separated and replaced to extract non cellular substances confined to the plasma B A diagram of the present study in which patients undergo plasma exchange C All plasma levels of sPD L1 immediately prior to pre and after post TPE using albumin replacement fluid are plotted TPE significantly reduced sPD L1 levels in patient plasma by Wilcoxon signed rank test p00001 D In a typical timeline patient sPD L1 levels are reduced by each successive session of TPE gray bars See also table a0 online supplementary figures “FFP replacement fluid figure 2C p00001 Removed sPD L1 was detected in matching plasma samples from the TPE procedure waste bag Each TPE session removed a mean of detectable plasma sPD L1 mean regeneration of sPD L1 between sessions was table TPE sessions were usually separated by “ daysincluding sessions A representative individual patient treatment course showing sPD L1 reduction over four successive TPE sessions is also shown figure 2D All individual patient TPE courses involving donated human blood products eg fresh frozen plasma or FFP are shown in online supplementary fig Pre TPE and post TPE sPD L1 levels for all sessions are also shown online supplementary fig TPE significantly reduced plasma sPD L1 even when sessions requiring donated FFP were included p00001FFP is sometimes given during TPE for patients with increased risk of bleeding We observed that some patients receiving FFP with low baseline sPD L1 experienced rapid increases in sPD L1 levels after TPE presumably passively acquired from donor plasma as this was not observed in patients receiving albumin replacement alone sPD L1 was not detected in the discarded plasma from the procedure for these patients We observed a mild association between post FFP infusion rises in sPD L1 levels and the blood type of the recipient mainly in patients with O type blood Individuals with group Oˆ’ blood are universal recipients of FFP products and universal donors of cellular products due to a lack of ABO group antigens and the presence of preformed anti A and anti B antibodies respectively Recipients of FFP usually receive a mixture of compatible plasma from multiple donors To determine whether blood type in FFP donors is associated with FFP sPD L1 content we measured sPD L1 by ELISA in plasma from multiple FFP donors online supplementary fig O negative plasma donors showed higher sPD L1 levels than donors with most other blood typesTPE efficiently reduces plasma EV levels in vivoWe postulated that TPE may remove PD L1 positive EVs evPD L1 from patient blood To address this question we measured total EV levels and evPD L1 in each sample by flow cytometry We also determined the impact of TPE on platelet derived CD61 positive EVs one of the most abundant EV Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Table Patient baseline characteristicsCharacteristicHigh sPDL1 n17Low sPDL1 n7StatisticStarting sPD L1Age yearsGender FActive cancer YesImmunotherapyNoneAtezolizumabPembrolizumabPlasma exchange indicationCNS demyelination myelitis MS NMO myelopathyImmune encephalitisMyasthenia gravisParaneoplastic syndrome encephalitis neuropathy pemphigusParaproteinemia Waldenström cryoglobulinemia kappa gammopathySusac syndromeTransplant rejection heart kidneyPre TPE white cell countPre TPE hemoglobinPre TPE creatinine to to to to to to to to to to F1223619 p0001F122035 p0558X2070 p0404 X20 p0967 X2288 p0237  X2288 p0315  F122078 p0385F122860 p0008F122382 p0063Patients undergoing therapeutic plasma exchange TPE are compared by starting soluble programmed death ligand sPD L1 level above or below survival cut off established in patients with melanoma ngmL For categorical variables n is given For continuous variables mean quartiles is givenKruskal Wallis PearsonCNS central nervous system MS multiple sclerosis NMO neuromyelitis opticasubpopulations in blood CD61 is a platelet marker and ADAM10 positive low density EVs ADAM10 has been implicated in exosome loading and pathogenesis11“TPE significantly reduced total plasma particle concentration figure 3A average per exchange p00001 TPE sessions requiring FFP or other human blood product were excluded from analysis leaving session pairs PD L1 positive evPD L1 and ADAM10 positive EVs were Table Soluble programmed death ligand sPD L1 reduction and regeneration per exchange Reduction per exchangen44Mean SDMedian min max Regeneration between exchangesMean SDMedian min maxRegeneration per cycle pgmLMean SDMedian min max ˆ’ n44 ˆ’ ˆ’38k 154kFor each exchange not requiring FFP percent sPD L1 reduction and regeneration between each exchange is calculated n44FFP fresh frozen plasmasignificantly reduced by TPE figure 3BC p0028 and p00001 respectively and were detected in waste plasma data not shown Each TPE session using albumin based replacement fluid with pre TPE levels above one million removed a mean of detectable PD L1 positive EVs from patients online supplementary table Platelet derived CD61 positive EVs while abundant were not significantly reduced by plasma exchange figure 3DIndividual patient courses showing total plasma PD L1 positive ADAM10 positive and CD61 positive EV levels before and after each TPE session are shown in online supplementary fig with exemplary nanoflow plots in online supplementary fig Three successive TPE sessions consistently depleted total PD L1 positive and ADAM10 positive but not CD61 positive EVs These trends were less pronounced when sessions in which patients received donor FFP were included online supplementary fig In normal control FFP donors blood type did not correlate with plasma EV concentrations online supplementary fig DISCUSSIONExtracellular PD L1”in the form of splice variant sPD L1 ADAM10ADAM17 cleaved sPD L1 ectodomain or evPD L1 positive EVs evPD L1”mediates resistance to PD L1 inhibitors4“ These forms are resistant to clinically tested Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c accessFigure Plasma exchange efficiently reduces total programmed death ligand PD L1 positive and ADAM10 positive extracellular vesicle EV levels in vivo Plasma levels of total EVs immediately prior to pre and after post therapeutic plasma exchange TPE are plotted TPE significantly reduced A total p00001 B PD L1 positive p0028 and C ADAM10 positive p00001 but not D CD61 positive EVs p094 by Wilcoxon signed rank test See also online supplementary figures “ and online supplementary table combinations of chemotherapy and immunotherapies in vitro and in animal models and are associated with poor prognosis in many cancer types In the present study we found that TPE reliably reduces sPD L1 and evPD L1 This reduction was most pronounced over approximately three consecutive single plasma volume treatment sessions Given the dramatic reduction in sPD L1 and evPD L1 TPE may provide a novel approach to combating these mechanisms of resistanceWhile promising the present study was limited to patients receiving TPE mainly for non oncological indications over a short time horizon One patient in the study had melanoma receiving pembrolizumab and exhibited high pre TPE evPD L1 that was reduced on treatment Another patient had a uterine neuroendocrine tumor receiving atezolizumab and exhibited high pre TPE sPD L1 that was reduced on treatment The purpose of TPE in all cases however was to blunt paraneoplastic autoimmunity or treat some other coexisting autoimmune disorder”not the underlying malignancy Neither of these patients experienced improvement in their autoimmunity after TPE The source of sPD L1 and evPD L1 in these cases is uncertain as no assay currently differentiates tumor derived and non tumor derived PD L1 We observed that these forms of immunosuppressive extracellular PD L1 exist naturally although at lower levels in healthy subjects than in patients with cancer suggesting a potentially beneficial immunoregulatory role While we observed some regeneration for both sPD L1 and evPD L1 between TPE sessions it is unknown to what degree malignant cells may regenerate and maintain extracellular PD L1 homeostasis Relatedly it is uncertain how other plasma substances removed by TPE may affect response to immunotherapy or more broadly cancer immunity overall Nor is it known at what level sPD L1 andor PD L1 positive EV removal would become clinically relevant These facets will be tested in future studiesImmunotherapy resistance is widespread and costly In most instances PD L1 inhibitors such as pembrolizumab nivolumab atezolizumab durvalumab and avelumab are used in situations in which less than half of tumors are expected to respond Of patients that benefit many do not experience a sustained durable response These treatments represent a major investment the cost of PD L1 checkpoint blockade commonly reaches several hundred thousand dollars over the course of therapyTo our knowledge this is the first report of an intervention to achieve consistent rapid reduction in either sPD L1 or PD L1 positive EVs in a clinical setting TPE is safe and commonly prescribed Thus preimmunotherapy TPE may combat immunotherapy resistance In light of the heavy investment that anti PD L1 therapy entails the added cost of TPE in selected patients may be practical14 While the durability of extracellular PD L1 reduction in malignancy will be explored in future studies the present study suggests that this approach warrants further investigationOrme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Beyond evPD L1 this is also to our knowledge the first known intervention to reliably deplete EVs in a clinical setting EVs have been implicated in oncogenesis and metastasis through miRNA carriage and direct protein signaling independent of PD L115 Beyond cancer EVs have also been implicated in autoimmunity17 agingneurodegeneration18 infection19 obesity20 and heart disease21 The selective removal of ADAM10 positive ie likely immune derived versus CD61 positive ie likely platelet derived EVs in this study suggests flexible selective EV depletion may be both possible and expedient in other indications The present study is only a proof of concept and additional exploratory studies in these areas are necessaryIn summary TPE reduces extracellular forms of PD L1 associated with PD L1 checkpoint inhibitor resistance Future studies will explore the potential role of TPE in improving cancer immunotherapyMETHODSRetrospective melanoma outcomes study designIn a retrospective analysis baseline blood samples from patients with melanoma prior to treatment in one of three clinical trials by the North Central Cancer Treatment Group N057e22 N077523 and N087924 between and were tested for sPD L1 of patients were diagnosed with cutaneous melanoma and none received immunotherapy treatments Blood from healthy volunteers undergoing blood donation at Mayo Clinic was also testedProspective TPE study designIn an investigator initiated label single center observational study adults undergoing TPE were approached from December through March In consenting subjects samples of whole blood immediately prior to TPE and on completion of the procedure were collected in ACD vacutainers BD In each case the first mL of blood was discarded to avoid contamination after which an mL sample was obtained in sequence Plasma was isolated by centrifugation A postprocedure blood sample was obtained after completion of the procedure In addition matching samples from discarded plasma from the procedure waste bag were collected Samples were obtained from up to four consecutive procedures for each patient If a patient underwent fewer than four TPE procedures samples were obtained from as many procedures as possiblePatients included were adults able to give consent and undergoing TPE for a variety of hematological neurological and renal diseases as indicated by published guidelines from the American Society for Apheresis ASFA or according to the medical judgment of the referring physicians25 Patients taking biotin supplements were excluded from the study due to biotin interference with the sPD L1 ELISA assay Procedures were performed using centrifugation based cell separators either the Fenwal Amicus Fresenius KABI USA LLC Lake Zurich Illinois USA or the Spectra Optia Terumo BCT Lakewood Colorado USA For each patient a single plasma volume was exchanged using either peripheral intravenous preferred or central lines for vascular access For this study due to the possibility of sPD L1 or PD L1 positive EVs present in donor plasma only TPE sessions using no donor plasma ie fresh frozen plasma FFP in the replacement fluid were included in calculations Anticoagulation consisted of either mL of acid citrate dextrose solution A ACD A or mL of ACD A with units of unfractionated heparin Anticoagulant to blood ratios were when ACD A was used and when ACD Aheparin was used Patients did not receive routine electrolyte replacement but mL of calcium gluconate was administered by slow intravenous push for signs and symptoms of hypocalcemia related to the ACD A anticoagulant in one patientELISAELISA was performed as previously published26 Both secreted splice variant and shed sPD L1 are reliably detected by this ELISA In brief paired mouse IgG2 monoclonal antibody clones H1A and B11 against extracellular human PD L1 were utilized in a capture detection plate assay using biotinylation and HRP streptavidin detection This assay is specific for sPD L1 and does not exhibit cross reactivity to other B7 H homologues nor to evPD L1 Concentrations were determined by optical density measurements along a known standard curve of recombinant human PD L1 ELISAs were performed by team members who were blinded to the identity of the samplesFlow cytometryFlow cytometry for EVs was performed as previously published27 In brief plasma samples were centrifuged twice at 2000g to deplete platelets Resultant platelet free plasma were analyzed using an A60 Micro Plus Nanoscale Flow Cytometer Apogee FlowSystems gating for mid intensity light angle light scatter and markers of interest Anti PD L1 Genentech atezolizumab ADAM10 RD Systems clone and CD61 BioLegend clone VI PL2 antibodies were conjugated to fluorophores Life Technologies Alexa647 PE phycoerythrin and Alexa488 and titrated prior to use Nanoscale flow cytometer calibration was performed using a standard reference bead mix as previously published Flow cytometry was performed by team members blinded to the identity of the samplesStatistical analysisAll statistical analyses were performed using R Statistical Software R Foundation Retrospective progression free survival was analyzed using Kaplan Meier and Cox proportional hazards modeling Optimal cut off values for sPD L1 levels were determined using the greyzoneSurv package for R Wilcoxon signed rank test was used to compare paired pre TPE and post TPE patient sample sPD L1 and EV levels as indicated Baseline clinical characteristics for the study were compared by Kruskal Wallis test for continuous variables and Pearson™s χ2 test for discrete variables as indicated Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0cOtherwise groups were compared by unpaired two sided Student™s t test Figures comprising box plots show quartile values and individual data points Mean values and CI are indicated in corresponding online supplementary figures and tables P was considered statistically significant In figures p values are denoted with with and with Twitter Jacob J Orme JakeOrmeMDPhDAcknowledgements Statistical guidance was provided generously by Nathan Foster of the Mayo Clinic Center for Clinical and Translational Science Some illustrations were created using Servier Medical Art templates which are licensed under a Creative Commons Attribution Unported License https smart servier com Additional illustrations were provided by Mayo Clinic Media Services The authors thank Daniel Summerfield MD MS for use of his likeness in Fig 2AContributors JO originated hypotheses designed the study oversaw experiments performed analyses and wrote the article EALE performed retrospective melanoma cohort analysis FL M performed nanoflow cytometry HD and EB oversaw and performed TPE study enrollment sample collectionprocessing and blinding SMH performed ELISAs MB AM SP MB SNM YY HD RD and JLW helped develop hypotheses provided clinical samples and reagents and contributed support and oversightFunding R21 5R21CA19787802 Role of Bim and soluble B7 H1 in monitoring T cell responses to anti PD1 therapy in melanoma HD and RD L30 CA23154101 Soluble B7H1 as a PD1 Checkpoint œRemote Control in Cancer JJO U10 CA180790 EE K12 CA090628 YY Richard M Schulze Family Foundation HD and RDCompeting interests Intellectual property has been filed addressing discoveries disclosed in this manuscript The authors report no other relevant conflicts of interestPatient consent for publication ObtainedEthics approval All research protocols involving human subjects were approved by Mayo Clinic™s Institutional Review Board and all human subjects gave written informed consentProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available in a public access repository All data will be available for download at the Science Framework at https osf io qtskd access This is an access article distributed in accordance with the Creative Commons Attribution Non Commercial CC BY NC license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial See http creativecommons licenses by nc ORCID iDJacob J a0Orme http orcid REFERENCES O'Donnell JS Long GV Scolyer RA et a0al Resistance to PD1PDL1 checkpoint inhibition Cancer Treat Rev “ Ando K Hamada K Watanabe M et a0al Plasma levels of soluble PD L1 correlate with tumor regression in patients with lung and gastric cancer treated with immune checkpoint inhibitors Anticancer Res “ Fan Y Che X Qu J et a0al Exosomal PD L1 retains immunosuppressive activity and is associated with gastric cancer prognosis Ann Surg Oncol “ Zhou J Mahoney KM Giobbie Hurder A et a0al Soluble PD L1 as a biomarker in malignant melanoma treated with checkpoint blockade Cancer Immunol Res “ access Mahoney KMet a0al œA secreted PD L1 splice variant that covalently dimerizes and mediates immunosuppression Cancer Immunol Immunother “ Orme JJ Jazieh KA Xie T et a0al ADAM10 and ADAM17 cleave PD L1 to mediate PD L1 inhibitor resistance OncoImmunology Romero Y Wise R Zolkiewska A Proteolytic processing of PD L1 by ADAM proteases in breast cancer cells Cancer Immunol Immunother “ Chen G Huang AC Zhang W et a0al Exosomal PD L1 contributes to immunosuppression and is associated with anti PD1 response Nature “ Poggio M Hu T Pai C C et a0al Suppression of exosomal PD L1 induces systemic anti tumor immunity and memory Cell “ Derksen RH Schuurman HJ Meyling FH et a0al The efficacy of plasma exchange in the removal of plasma components J Lab Clin Med “ Berckmans RJ Nieuwland R Böing AN et a0al Cell Derived microparticles circulate in healthy humans and support low grade thrombin generation Thromb Haemost “ Crescitelli R Lässer C Jang SC et a0al Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation J Extracell Vesicles Kowal J Arras G Colombo M et a0al Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes Proc Natl Acad Sci U S A 2016113E968“ Winters JL Brown D Hazard E et a0al Cost minimization analysis of the direct costs of tpe and IVIg in the treatment of Guillain Barré syndrome BMC Health Serv Res “ Lee JC Zhao J T Gundara J et a0al Papillary thyroid cancer derived exosomes contain miRNA 146b and miRNA222 J Surg Res “ Yang J Wei F Schafer C et a0al Detection of tumor cell specific mRNA and protein in exosome like microvesicles from blood and saliva PLoS One 20149e110641 Nakao R Hasegawa H Ochiai K et a0al Outer membrane vesicles of Porphyromonas gingivalis elicit a mucosal immune response PLoS One 20116e26163 Thompson AGet a0al œExtracellular vesicles in neurodegenerative disease pathogenesis to biomarkers Nature Reviews Neurology Nature Publishing Group “ Marcilla A Martin Jaular L Trelis M et a0al Extracellular vesicles in parasitic diseases J Extracell Vesicles Huang Doran I Zhang CY Vidal Puig A œExtracellular Vesicles Novel Mediators of Cell Communication In Metabolic Disease Trends in Endocrinology and Metabolism Elsevier Inc “ Boulanger CM Loyer X Rautou PE et a0al œExtracellular vesicles in coronary artery disease Nature Reviews Cardiology Nature Publishing Group “ Kottschade LA Suman VJ Amatruda T et a0al A phase II trial of nab paclitaxel ABI007 and carboplatin in patients with unresectable stage IV melanoma a North Central Cancer Treatment Group Study N057E1 Cancer “ Kottschade LA Suman VJ Perez DG et a0al A randomized phase study of temozolomide and bevacizumab or nab paclitaxel carboplatin and bevacizumab in patients with unresectable stage IV melanoma a North Central Cancer Treatment Group study N0775 Cancer “ McWilliams RR Allred JB Slostad JA et a0al NCCTG N0879 Alliance A randomized phase cooperative group trial of carboplatin paclitaxel and bevacizumab ± everolimus for metastatic melanoma Cancer “ Padmanabhan A Connelly Smith L Aqui N et a0al Guidelines on the Use of Therapeutic Apheresis in Clinical Practice Evidence Based Approach from the Writing Committee of the American Society for Apheresis The Eighth Special Issue J Clin Apher “ Frigola X Inman BA Lohse CM et a0al Identification of a soluble form of B7 H1 that retains immunosuppressive activity and is associated with aggressive renal cell carcinoma Clin Cancer Res “ Gomes J Lucien F Cooper TT et a0al Analytical considerations in nanoscale flow cytometry of extracellular vesicles to achieve data linearity Thromb Haemost “Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c"

Thyroid_Cancer

Pathway‘specific model estimation for improved pathway annotation by network crosstalkMiguel Castresana‘Aguirre Erik L L SonnhammerPathway enrichment analysis is the most common approach for understanding which biological processes are affected by altered gene activities under specific conditions However it has been challenging to find a method that efficiently avoids false positives while keeping a high sensitivity We here present a new network‘based method ANUBIX based on sampling random gene sets against intact pathway Benchmarking shows that ANUBIX is considerably more accurate than previous network crosstalk based methods which have the drawback of modelling pathways as random gene sets We demonstrate that ANUBIX does not have a bias for finding certain pathways which previous methods do and show that ANUBIX finds biologically relevant pathways that are missed by other methodsImprovements in molecular biology have led to an increase in highthroughput data which typically produces lists of differentially expressed genes or proteins These lists are useful for identifying genes with important roles in certain conditions However more insight about the biological mechanisms is often needed eg which functional gene sets are related to genes in the result list The study of the relation between a query gene set differentially expressed gene list and functional gene sets pathways is called pathway enrichment analysisImprovements in molecular biology have led to an increase in highthroughput data which typically produces lists of differentially expressed genes or proteins These lists are useful for identifying genes with important roles in certain conditions However more insight about the biological mechanisms is often needed eg which functional gene sets are related to genes in the result list The study of the relation between a query gene set differentially expressed gene list and functional gene sets pathways is called pathway enrichment analysisThere are four generations of pathway enrichment analysis approaches Overrepresentation analysis ORA calculates how many genes from a list of genes extracted based on a threshold or criteria eg differentially expressed genes are in a certain pathway1 Statistical significance is assessed repeating this process with a background list of genes eg all the genes in the microarray This is known as Gene Enrichment Analysis GEA and famous tools like DAVID2 use it Similar but taking into account all the genes in the experiment and the gene expression values is the Functional Class Scoring algorithms FCS3 for which known algorithms include Gene Set AnalysisGSA4 and Gene Set Enrichment Analysis GSEA5 However both FCS and ORA have limitations They both consider genes as independent which is often not true only taking into account their overlap and not their associations or interactions6 Another issue with overlapbased methods is their low coverage since they are heavily dependent on pathway knowledge which is still incomplete leading to a high rate of false negatives7 Pathway topologybased methods use the same steps as FCS with additional pathway topology information However the reliance on gene overlap leads to similar limitations as ORA and FCSWe could consider the network crosstalk enrichment tools as the fourth generation They rely on a network such as a functional association network like Funcoup8 or STRING9 These networks integrate different experiments from different data types into a single network providing information about gene to gene functional associations which is translated into links in the network With this limitations such as gene independency and low coverage of overlapbased methods are overcome Association between two sets is measured in terms of links between them in the network known as crosstalk In the past few years different ways to assess enrichment between two gene sets have been published like NEA10 EnrichNet11 CrosstalkZ12 NEAT13 NEArender14 BinoX7 and GeneSetDPGeneSetMC15 EnrichNet defines a network enrichment score based on network distances between two gene sets using random walks with restart but is not able to calculate statistical significance Department of Biochemistry and Biophysics Science for Life Laboratory Stockholm University Box Solna Sweden email eriksonnhammerdbbsuseScientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cof the enrichment The tools NEA and CrosstalkZ assess significance using statistical tests assuming that crosstalk between nonenriched gene sets is normally distributed but this is often not the case Moreover they rely on network randomizations to obtain null model parameters which makes them computationally very slow Computational time is reduced in BinoX which also applies network randomization but uses the binomial distribution to calculate statistical significanceThe methods NEAT NEArender and GeneSetDPGeneSetMC do not use network randomization NEAT calculates the expected number of links between two gene sets based on their degrees and then uses the hypergeometric distribution to assess statistical significance NEArender computes the expected number of links in the same way as NEAT but uses a chisquare test to assess statistical significance GeneSetDP uses dynamic programming to calculate an exact distribution of the expected number of links to a pathway for a certain gene set size GeneSetMC does this approximately using MonteCarlo sampling which is faster These two algorithms are however not implemented to allow large scale pathway enrichment analysisThe null model assumption of NEAT NEArender and BinoX is that compared gene sets are expected to behave like random gene sets For real pathways that are very nonrandom eg highly intraconnected this can lead to underestimating the expected level of crosstalk and produce a high false positive rate FPR To avoid this it is important that the method can cope with the nonrandomness of pathways To this end we have developed a novel networkbased pathway enrichment analysis algorithm called ANUBIX Adaptive NUll distriButIon of Xtalk which is based on scoring random gene sets against real pathways to build its null model We show that ANUBIX clearly outperforms recent network crosstalk methods like BinoX NEArender and NEAT in terms of avoiding False Positives FP showing that it can model expected network crosstalk to pathways more preciselyMaterial and methodsOur networkbased pathway enrichment analysis tool ANUBIX depends on a global functional association network We used the network Funcoup version with a link confidence cutoff of containing genes and links With those genes cid31g1 g2 gnˆ’ gncid30 ˆˆ S and all the pairwise links between them form a symmetric matrix A with dimensions SxS such thataij 1if gi is connected to gj and i � j otherwise aij A gene set Q and a pathway P are a subset of the total number of genes for a certain proteome such that Q P Š† S Notice that S Š† Q we can have some genes from the proteome that are not in the network The crosstalk between Q and P is measured with the degree k cid31iˆˆQcid31jˆˆPaijThe null model is built based on the expected crosstalk between a random gene set of the same size as the original gene set Q and pathway P Since the network connections are binary each link is considered as a Bernoulli trial Y ˆ¼ Bcid31pcid30 where p is the probability of observing a link We also calculate n QP ˆ’ Q ˆ P all the possible links between Q and P We count the links each gene from Q has to the pathway P meaning that if two linked genes are in Q and also in the P we count that link twice boosting the cases where we find overlap Each of these Bernoulli trials are assumed to be independent and the sum of them follows a binomial distributionIn the binomial distribution the mean and variance are defined as µ np and Var npcid311 ˆ’ pcid30 respectively This means that µ ‰¥ Var which may not be true when the random variable is overdispersed leading to an underestimation of its variance16The betabinomial distribution has been extensively used as an alternative to handle overdispersed binomiallike random variables1718 Here the probability of success p is not fixed as it is in the binomial distribution but follows a beta distribution Betaα β with parameters α and β The marginal distribution of the betabinomial is described in Eq a0fcid31kn α βcid30 cid29 nk cid28 Bk α n ˆ’ k βBα βTo estimate the optimal parameters of the betabinomial we use maximum likelihood estimation MLE19 where the loglikelihood is Eq a0lcid31kn α βcid30 logLcid31kn α βcid30 logcid29 n logcid29 nk cid28 logBα k β n ˆ’ k ˆ’ logBα βk cid28 logŴα k logŴβ n ˆ’ kˆ’logŴα β n ˆ’ logŴα ˆ’ logŴβ logŴα βThe negative loglikelihood is optimized with the Nelder and Mead method20 The factorial term in the loglikelihood is removed since it does not depend on the parameters to be optimized Once we have the betabinomial parameters α β of our null distribution we calculate if the crosstalk between Q and P is enriched The null and alternative hypotheses areH0 No more links between Q and P than expected by chanceH1 More links between Q and P than expected by chanceBecause of the discrete nature of the null distributions ordinary pvalues are conservative and therefore mid pvalues were used2122 Mid pvalue is defined as half the probability of the observed statistic plus the probability of observing more extreme values22 The workflow of the ANUBIX algorithm is depicted in Fig a0Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Workflow of ANUBIX The algorithm assesses the significance of the network crosstalk between a query gene set and a pathway A null distribution is generated for each pathway to model the expected crosstalk of random gene sets of the same size as the original gene set This distribution is then fit to a betabinomial distribution to calculate the probability of reaching at least the number of observed links or more between the query gene set and the pathway Software Inkscape version inksc apeIt is important to point out that the networkbased approaches ANUBIX NEAT NEArender and BinoX test three different types of null hypothesis ANUBIX which takes only enrichment into account computes a one tailed test NEAT computes two onetailed tests for enrichment and depletion and takes the minimum pvalue of them multiplied by to emulate a twotailed test BinoX and NEArender compute both enrichment and depletion but only perform one onetailed test since the hypothesis test changes depending on whether the observed number of links is above or below the expected crosstalkPathways To generate the false positive and true positive benchmarks we used KEGG v70123 pathways and REACTOME v6224 pathways for Homo sapiens REACTOME pathways have a deep hierarchical structure including many small pathways on the lower levels that are very specific To reduce Reactome™s specificity we resolved its hierarchy by collapsing lower level pathways below a certain pathway size to their parents until obtaining an average pathway size similar to KEGG pathways genes per pathwayPerformance measures In the FP benchmark we generated random gene sets and tested them against KEGG and REACTOME pathways To make these gene sets representative of real experiments we took the average size of MSigDB25 gene sets which is genesIn the True Positive TP benchmark we bisected the KEGG pathways and REACTOME pathways into two parts Each part gets a similar number of genes and links7 To be able to benchmark GEA we emulated some overlap between the two bisected parts This overlap corresponded to the average overlap between the MSigDB gene sets and the pathway measured individually for each of the pathways in KEGG and REACTOMECorrection for multiple hypothesis testing was done using the Benjamini“Hochberg procedure26Stability Our null distributions are based on random sampling We take random samples of genes from the genome This stochastic procedure makes the null distributions different every time they are generated Since the pvalues are computed from the null distribution their values may change To analyze stability we generated the null distribution times for the crosstalk between the same gene set to the same pathways for increasing numbers of random samples For each sample size we computed the coefficient of variation CV which is the ratio between the standard deviation SD and the mean We required a CV lower than to limit the dispersion of the mean of the null distribution and this was reached at random samples Once the number of random samples were chosen we measured how much the pvalues were varying in each run For that we ran a randomly selected MSigDB gene set times To compute the confidence interval of the pvalues we used the central limit theorem and applied normal distribution statistics to compute themUsed programs ANUBIX bitbu cketsonnh ammer group anubi xBinoX bitbu cketsonnh ammer group binox NEAT cranrproje ctwebpacka gesneatneatpdfNEArender cranrproje ctwebpacka gesNEAre nderNEAre nderpdfGeneSetDP githu bcomstati stica lbiot echno logygenes etdpScientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cFigure a0 Overdispersion of KEGG and REACTOME pathways null distributions when sampling random gene sets of size from the proteome The dispersion for each pathway is calculated as the ratio between the variance and the mean of the crosstalk null distribution For each pathway database we illustrate the dispersion values through a boxplot and also by showing the dispersion distribution Software R version wwwrproje ctFigure a0 Observed crosstalk distribution fit with binomial and betabinomial distributions random gene sets of size were used to generate a null distribution of crosstalk to the A œBetaalanine metabolism B œProstate cancer and C œAlzheimer™s disease pathways Betabinomial shows a much better fit to the observed link distribution than the binomial Software R version wwwrproje ctResultsTo correctly assess the statistical significance of an observed network crosstalk between two gene sets eg one experimental gene set and one known pathway it is paramount that the null distribution appropriately models the crosstalk of random query gene sets Note that it is not necessarily appropriate to assume that the pathway gene set behaves like a random gene set ie the null distributions need to model crosstalk between random query gene sets versus real pathway gene sets It is also paramount to model the expected crosstalk distribution with an appropriate distribution Previous methods such as BinoX or NEAT use binomial and hypergeometric distributions respectively which are not appropriate for overdispersed distributions since they do not allow the variance of the distribution to be greater than the mean To showcase this we generated null distributions for KEGG and REACTOME pathways by sampling gene sets of size from the proteome In Fig a0 we show the dispersion for each pathway as the ratio between the variance and the mean of the crosstalk null distribution We observe that almost all of these distributions suffer from overdispersion meaning that the variance of the distribution is greater than the mean Therefore statistical models that cannot cope with overdispersion are not appropriate to model the null distribution of most pathwaysTo visualize the overdispersion in detail we chose pathways that are in different quartiles of the dispersion distribution We show their null distributions in Fig a0 Figure a03A shows the œBetaalanine metabolism Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Pvalue uniformity test of ANUBIX Binox GEA GeneSetDP NEArender and NEAT random gene sets of genes were tested for crosstalk enrichment against the KEGG pathway œProstate cancer A Reported pvalues are plotted against theoretical quantile rank A perfect method should adhere to the diagonal B Distributions of the FPR for all KEGG and REACTOME pathways tested with ANUBIX BinoX NEArender NEAT and GEA Green distribution for enriched tests and red distribution for depleted The dashed line at FPR denotes the expected FPR level The black triangle and circle represent the mean FPR for enrichment or depletion respectively Software R version wwwrproje ctpathway whose dispersion value is in the first quartile Figure a03B shows the œProstate cancer pathway with a dispersion in the second quartile and Fig a03C shows the œAlzheimer™s disease pathway with a dispersion in the fourth quartile The high variance relative to the mean gives a very poor fit with the binomial distribution yet the betabinomial distribution gives a very good fit This underestimation of variance by the binomial distribution would lead to many false positives With a few pathways there is no overdispersion in the data but these can fit a betabinomial equally well as a binomialBenchmark for false positives Since the null model in ANUBIX is based on random gene sets we expect the pvalue distributions when tested with random query gene sets to behave uniformly for any pathway For almost all pathways we observed a virtually perfectly uniform distribution when plotting ANUBIX pvalues of random gene sets against each KEGG pathway full results at Supplementary Fig a0 A few pathways deviated somewhat from uniform which is the result of the betabinomial fit not being able to model the null distribution with enough precision A second type of deviation from perfect uniform distribution is caused by staggering of observed pvalues This is relatively frequent and arises because the support of the test statistics is limited to a few values and therefore unavoidable We also generated the pvalue distributions for gene sets of size and size against each KEGG pathway Supplementary Fig a0 and respectively which gave similar results However some pathways seem to be affected by the size of the gene set ANUBIX was compared to the top networkbased methods BinoX NEAT and NEArender and a recently published method GeneSetDP For comparison we also tested a popular overlapbased pathway enrichment method GEA Because GeneSetDP and GenesetMC are too computationally heavy for large scale analysis we first tested all the gene sets against one individual pathway We only used GeneSetDP because GeneSetMC produces similar pvalues Pvalues were plotted versus quantiles of a uniform distribution For an unbiased method the pvalues would lie on the diagonal y x Figure a04A shows that for the œProstate cancer pathway Pvalues of ANUBIX adhere to the diagonal much better than for BinoX NEAT NEArender and GEA while performing equally well as GenesetDPFor crosstalk to random gene sets we expect of the pvalues to be lower than However for the œProstate cancer pathway BinoX had of its pvalues lower than NEAT and NEArender GEA whose coverage is small7 had of its pvalues below and highly discrete taking on only four possible values for œProstate cancer due to few overlapping genes ANUBIX and GeneSetDP find a correct fraction of the pvalues with and GeneSetDP under respectivelyWe also ran ANUBIX BinoX NEAT NEArender and GEA for the random gene sets against all pathways in the KEGG database and REACTOME database Full results in Supplementary Data and Data respectively GeneSetDP was not included as it is not implemented to run at a large scale NEAT NEArender and BinoX can also give statistical significance when gene sets have fewer links to a pathway than expected by chance known as depletion To make a more consistent benchmark where all methods can be compared equally we only considered enrichment and depleted pathways were treated as nonsignificant The average FPR for all KEGG pathways was with ANUBIX with BinoX with NEAT with NEArender and with GEA For REACTOME almost the same FPR values were obtained ANUBIX BinoX NEAT NEArender and GEA Roughly the same FPR levels came from significant depletions for BinoX NEAT and NEArender However the averaging of the FPR levels for all pathways does not show the real problem of these methods Some pathways could give very nonconservative pvalues while other pathways could give very conservative pvalues To show how each method performs for each of the pathways we plot the distribution of the FPR fraction of pvalues below for each pathway as violin plots in Fig a04B Since GEA Scientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cand ANUBIX cannot test for depletion they only have the enriched case A perfect method would have all points close to the dashed line at FPR ANUBIX produces FPR values close to this line meaning that the model is robust GEA greatly underestimates FDR and produces almost no false positives but this leads to very poor sensitivity as shown below NEArender NEAT and BinoX produce similar FPR distributions that are very spread out ie the FPR tends to be very different for different pathways For the tests performed per pathway some pathways reach an FPR of for enriched cases and similar for depleted Summing these two can lead to a total FPR above if we take both enriched and depleted cases into account which is very nonconservative The plot also shows that for some pathways these methods are overly conservative giving considerably lower FPR than they should In other words methods like BinoX NEAT and NEArender have a huge variation in the quality of their pvalues depending on the pathway under studyBinoX is implemented in a web server called PathwAX27 where users can submit a query gene set to test for network crosstalk enrichment By analogy we studied false positive rates assuming independence between gene sets where each user submits a single gene set ie multiple testing correction is only performed for number of pathways each query is compared to random gene sets were used against the KEGG database A FDR threshold of was used and enrichment and depletion were grouped separately as shown in Fig a05A The top pathways with highest FPR for BinoX were plotted full results in Supplementary Data all having a highly nonconservative behaviour for BinoX NEAT and NEArender Every time a user submits a random gene set the chance of getting one of these pathways is very high on average if we take both enriched and depleted cases into account In contrast ANUBIX and GEA have less than FPR We observed a very high correlation between perpathway FPR values for BinoX NEAT and NEArender above for each pairwise comparison This indicates that the pathway enrichment analysis results obtained with these methods are highly similar They all had low Pearson correlation to ANUBIX with for BinoX for NEAT and for NEArender The corresponding Spearman correlations were and As for the pathways we noticed that there is a high overlap between some of them For instance the œAlzheimer™s disease and œParkinson™s disease pathways share of their genes The œAlzheimer™s disease the œParkinson™s disease and the œHuntington™s disease pathways have of the genes in common from the union between them Further the œOxidative phosphorylation the œNonalcoholic fatty liver disease the œAlzheimer™s disease the œParkinson™s disease and the œHuntington™s disease have of the genes in common from the union between them Therefore if there is significant crosstalk to one of them crosstalk to the other pathways is very likely The high dependency between some pathways points to opportunities for further improvement of pathway definitions Further exploration was performed in these pathways™ topologies to understand their tendency to generate many FPsWe computed the fraction of intralinks for each pathway as the ratio between the number of internal links and the total number of links We plotted this ratio against the FPR Fig a05B A higher fraction of intralinks means that more links are within the pathway than to the outside suggesting a more isolated pathway The Spearman correlation coefficient between the fraction of intralinks and FPR for BinoX was indicating that the fraction of internal links plays a major role in causing false positives This dependence is also observed with NEAT with a correlation of and with NEArender at However ANUBIX had a correlation of only and GEA This indicates that methods like NEAT NEArender and BinoX cannot deal properly with pathways that are clearly not random and behave more as isolated communitiesAdditionally we calculated the number of maximal cliques each of the KEGG pathways has and we observed a correlation with the FPR for BinoX with a spearman correlation of These maximal cliques were computed using the igraph package in R We considered cliques as all complete subgraphs and a clique is considered maximal if we cannot add more nodes to it This indicates that the higher the number of maximal cliques in a pathway meaning a less random pathway in terms of topology the higher the FPR isBenchmark of true positives Besides a correct FPR it is also important to verify that the power of the method is sufficient for a high true positive rate TPR To this end we devised a benchmark by splitting each KEGG and REACTOME pathway into two parts and then measured each method™s ability to reconnect these parts The splitting into parts included giving an amount of gene overlap between the two parts emulated based on the average overlap between MSigDB gene sets and KEGG and REACTOME pathways We compared the methods by their Receiver Operating Characteristic ROC curves Figure a06A shows only the tests that are statistically significant FDR and only considering enrichment ANUBIX has a TPR of of the enrichment tests as significant without having any FP BinoX has a TPR of with FPR NEArender a TPR of with FPR and NEAT a TPR of with FPR GEA whose coverage is low gives only TPR and no FPs Figure a06B shows the ROC curve for all the enriched tests performed also including insignificant results This shows the coverage of each method ANUBIX recovers of the TP tests without suffering any FPs BinoX NEArender and NEAT have similar curves recovering and of the enriched TP tests respectively GEA can here maximally find of the TP tests since only those tests have some gene overlap This benchmark shows that GEA has very low coverage of what it can potentially find We note that the maximal TPR obtained by GEA corresponds to the amount of significantly enriched crosstalks obtained when running all of MSigDB against KEGG pathways see Pathway annotation of MSigDB gene setsStability and robustness Considering that the null distributions are based on random sampling we studied the number of iterations required to reach a coefficient of variation CV of Figure a07A shows how many pathways pass that threshold depending on different amounts of random samples of the pathways had a CV lower than when using random samples to model the null distribution To verify that this number of random samples is sufficient for every pathway we computed the enrichment of one randomly selected MSigDB Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Analysis of why certain pathways are very prone to produce false positives random gene sets of genes were tested independently for crosstalk enrichment against the KEGG pathways A The top ten pathways that produce the highest false positive rate FPR with BinoX and the FPR obtained with other methods B Fraction of intralinks for each of the KEGG pathways against FPR The size of the point reflects the total number of links in each pathway Software R version wwwrproje ctgene set to all KEGG pathways times The null distributions are thus generated times for each pathway and we would expect some changes in the pvalues between runs Figure a07B shows the standard deviation of the pvalues We observe that the pvalues almost did not vary showing that random samples are enough Moreover because of sampling the pvalue is not an exact pvalue but a point estimate of it we also provide with the confidence interval of each of the pvalues Supplementary Data Compute time Our method relies on random sampling to model the null distribution which makes ANUBIX computationally intensive To benchmark its speed we did runs each time with a randomly chosen biological gene set extracted from MSigDB against KEGG REACTOME and KEGG plus REACTOME We measured the compute time for each of the networkbased methods see Fig a0 With this benchmark we can show that ANUBIX is fast when running single gene sets One should take into account that ANUBIX and BinoX need a precomputation step before running the actual analysis However the ANUBIX precomputation step takes around a0s whereas in BinoX it takes around a0min To compute the randomized network for BinoX Scientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cFigure a0 Receiver Operating Characteristic ROC curve For the TP tests each KEGG and REACTOME pathway is divided into two and a TP is interpreted as the crosstalk between two parts from the same pathway For the FP tests random gene sets of size are tested for enrichment against KEGG and REACTOME pathways A ROC curve for only the significantly enriched tests FDR B ROC curve for all enriched tests Software R version wwwrproje ctFigure a0 Stability analysis of ANUBIX A Fraction of KEGG pathways with Coefficient of variation CV below for different number of iterations B ANUBIX pvalues are stable”their variance is low and proportional to the magnitude of the pvalue A randomly chosen MSigDB gene set DAIRKEE_CANCER_PRONE_RESPONSE_BPA was run times against KEGG pathways Standard deviation of the logpvalue is plotted against the meanlogpvalues for each pathway Software R version wwwrproje ctwe used iterations A drawback for ANUBIX compared to methods like BinoX or NEAT is that the computation time for large scale analyses take more time For instance the time required to compute the large scale pathway annotation study for the MSigDB gene sets against KEGG pathways took a0min for ANUBIX using cores a0min for NEArender a0min for BinoX and a0min for NEAT Compute times were measured on an i77700 CPU a0GHz with a0Gb RAMPathway annotation of MSigDB gene sets We carried out a largescale pathway analysis study by running MSigDB gene sets against KEGG pathways using ANUBIX BinoX NEAT NEArender and GEA Full results are in Supplementary Data In total crosstalk tests were done per method and to get a more fair comparison between different methods we only considered enriched crosstalk considering that ANUBIX and GEA can only test for enrichmentNEArender BinoX and NEAT found the highest number of significantly FDR enriched crosstalks with and of all pairs respectively followed by ANUBIX with and GEA with Many MSigDB gene sets thus appear to have a high occurrence of pathway enrichments Even if we do not know whether those enrichments are TPs or FPs we show above Figs a0 and 5A that BinoX NEArender and NEAT are prone to produce FPsThe Venn diagram in Fig a0 shows that the overlap between BinoX NEAT and NEArender is very high having of their significant pathway annotations in common This was expected since all these methods consider pathways as random The overlap is even higher between NEAT and NEArender because they compute Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Compute time when running a random experimental gene set from MSigDB different gene sets were tested against KEGG REACTOME and KEGG plus REACTOME pathways for each of the networkbased methods Since ANUBIX allows parallelization we also added another run with cores The error bars show the variability in compute time for each of the methods in each of the databases The BinoX precomputation step is not included since it takes a0min Software R version wwwrproje ctFigure a0 KEGG pathway annotation for MSigDB gene sets with five methods The Venn diagram shows the number of shared pathway annotations at FDR Note that ANU

Thyroid_Cancer

"Pressure sores are sometimes refractory to treatment often due to malnutrition Small intestinalbacterial overgrowth SIBO obstructs absorption in the digestive tract and causes malnutrition However little isknown about the association between pressure sore wound healing and SIBO Here we report a case of a patientwith a refractory sacral pressure sore and SIBOCase presentation A 66yearold woman who was spinal cord injured years before visiting our hospitalpresented with the chief complaint of a sacral pressure sore — cm in size which was refractory totreatment Physical examination showed abdominal distension and emaciation with a body mass index of Further examination revealed elevated serum alkaline phosphatase UL bilateral tibial fracture multiple ribfracture and osteoporosis We diagnosed the patient with osteomalacia with vitamin D deficiency Despite oralsupplementation serum levels of calcium phosphorous and vitamin D remained low Also despite concentrativewound therapy for the sacral pressure sore by plastic surgeons no wound healing was achieved Due to asuspicion of disturbances in nutrient absorption we performed bacterial examination of collected gastric andduodenal fluid which showed high numbers of bacteria in gastric content E coli Streptococcus speciesand Neisseria species and duodenal content E coli Candida glabrata Therefore we diagnosed thepatient with SIBO and started selective decontamination of the digestive tract using polymyxin B sulfate andamphotericin B After starting treatment for SIBO the sacral pressure sore began to heal and was nearly healed after days The patient™s serum levels of calcium phosphorous vitamin D and other fatsoluble vitamins alsogradually increased after starting treatment for SIBOContinued on next page Correspondence 2m2hy4gmailcom1Department of Plastic Surgery Chiba University Inohana ChuokuChibacity Chiba JapanFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cKubota BMC Gastroenterology Page of Continued from previous pageConclusion We report a case of a patient with a refractory sacral pressure sore that healed after starting treatmentfor SIBO We conclude that SIBO may be an overlooked cause of malnutrition and poor wound healing in patientswith chronic pressure soresKeywords Pressure wound Small intestinal bacterial overgrowth Spinal cord injury Malnutrition Wound healingCase reportBackgroundPressure sores in patients with spinal cord injury SCIare sometimes refractory to treatment Chronic gastrointestinal symptoms are also frequently seen in patientswith SCI [ ] and malnutrition caused by decreasedgastrointestinal motility in SCI patients is a major causeand exacerbating factor of pressure sores Evaluation ofnutritional status in patients with pressure sores is essential [] as nutritional intervention can be a valuabletreatment option for pressure sores However small intestinal bacterial overgrowth SIBO is rarely consideredin the evaluation of malnutrition in SCI patients withpressure sores SIBO is defined as the presence of morethan — colony forming units CFUmL of bacteriaor any amount of E coli in the proximal small bowelcontent [] Here we report the case of an SCI patientwith a refractory sacral pressure sore that healed afterstarting treatment for SIBO To the best of our knowledge this is the first report of an association between apressure sore and SIBOCase presentationA 66yearold woman visited our hospital for the purpose of treating her sacral pressure sore day whichshe developed months prior due to bed rest duringtreatment of a left humeral fracture in another hospitalShe had paraplegia as well as bladder and rectal disturbance due to SCI at the fourth lumbar level L4 causedby a suicidal jump in response to paranoid delusions at years of age Spinal fusion surgery and cystostomywere performed early after SCI Otherwise she had ahistory of hysterectomy due to uterine cancer at yearsof age lymphaticovenular anastomosis as a treatment forposthysterectomy lymphedema in the bilateral lower extremities at years of age and cholecystectomy at years of ageWhen she visited our hospital she was taking the following oral medicines propiverine hydrochloride vitamin B12 etizolamflunitrazepam sodium bicarbonateanhydrous monobasic sodium phosphate mixture Clostridium butyricum tablets sodium risedronate hydraterebamipide sodium ferrous citrate fursultiamine hydrochloride alfacalcidol and potassium Lasparate She didnot take proton pump inhibitors PPI Her vital signswere as follows body temperature of °C low bloodpressure of mmHg pulse rate of bpm and respiratory rate of per min Physical examinationshowed abdominal distension emaciation with a bodymass index of and a sacral pressure sore — cm in size including a pocket entrance of — cmFig 1a Most of the surface of the pressure sore wascovered by granulation Our evaluation of the pressuresore with DESIGNR [] was D3 e3 s8 i0 g3 N3 P24with a total score of Table Bacterial culture examination ofthe pressure soreshowed Corynebacterium striatum and methicillinresistant Staphylococcus aureus Laboratory data showedan elevated serum alkaline phosphatase level of UL and low serum levels of hemoglobin gdL albumin gdL calcium mgdL and zinc μgdL Onday we observed a sudden decrease of hemoglobin to gdL with a positive fecal occult blood test bilateralpleural effusion on chest xray and serum albumin levelof gdL Upper gastrointestinal endoscopy showed agastric ulcer at H2 stageAs a result of searching for the cause of alkaline phosphatase elevation bilateral tibial fracture multiple ribfracture and osteoporosis were found Fig 1e and fFemoral bone density was of the young adult meanA low serum inanic phosphorous level was foundTable along with a low serum level of 25hydroxyvitamin D3 25OHVitD3 below the detection limit andelevated level of parathyroid hormone Table Levelsof other fatsoluble vitamins were also low vitamin A μIUdL vitamin K1 ngdL and vitamin E mgdL Examination using ultrasound and computedtomography showed normalthyroid and parathyroidglands Basing on these finding we diagnosed osteomalacia with vitamin D deficiencyOn day oral supplementation of calcium phosphorous and vitamin D was started Despite supplementationserum levels of calcium phosphorous and 25OHVitD3on day showed poor improvement calcium mgdLphosphorous mgdL and 25OHVitD3 below the detection limitOn day we performed bacterial examination ofcollected gastric and duodenal fluid with suspicion of adisturbance in absorption which showed elevated num E coli bers of bacteria in gastric contentStreptococcus species and Neisseria species and 0cKubota BMC Gastroenterology Page of Fig See legend on next page 0cKubota BMC Gastroenterology Page of See figure on previous pageFig Patient images a b c and d Sacral pressure sore a Day sore — cm in size with an entrance of — cm DESIGNR score wasD3 e3 s8 i0 g3 N3 P24 with a total score of b Day ie days after starting SDD for treating SIBO reduced size of sore DESIGNR scorewas D3 e3 s3 i0 g1 n0 p0 with a total score of c Day ie days after staring SDD healed sore DESIGNR score was d0 e0 s0 i0 g0 n0p0 with a total score of d Day ie days after staring SDD no recurrence of the sore e and f Osteoporosis and multiple fractures eXray showing left tibial fracture f Tc99 m bone scan showing accumulation in multiple ribs vertebrae and right ulna g h and i Endoscopicexamination and results of bacterial culture of the upper digestive tract All stomach duodenum and proximal jejunum samples were positive forE coli g Stomach Food residue can be seen Acid level was decreased to pH h Duodenum Food residue is evident i Proximal jejunum Flatvilli and a jejunal ulcer are observedTable DESIGNR assessment tool for pressure sore Reprinted with permission from John Wiley and Sons In Matsui et alDevelopment of the DESIGNR with an observational study an absolute evaluation tool for monitoring pressure ulcer woundhealing Wound Repair Regen “Depthd No particular skin lesion and no rednessD Lesion extends into the subcutaneous tissuePersistent rednessLesion extends into dermisExudatee NoneSlight does not require daily dressing change Moderate requires daily dressing changeSizes NoneSmaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2Lesion extends to the muscle tendon and boneLesion extends into the articular or body cavityU It is impossible to measure the depthE Heavy requires dressing change more than twice a dayS cm2 or largerInflammationInfectioniNoneSigns of inflammation fever redness swelling and pain around thewoundIClear signs of local infection eg inflammation pus and foulsmellSystemic impact such as feverGranulation tissueg Granulation cannot be assessed because the wound is healed or tooshallowG Healthy granulation tissue occupies or more but lessthan Healthy granulation tissue occupies or moreHealthy granulation tissue occupies or more but less than Healthy granulation tissue occupies less than No healthy granulation tissue existsNecrotic tissuen NonePocketp NoneN Soft necrotic tissue existsHard and thick necrotic tissue is attached to the woundP Smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger 0cKubota BMC Gastroenterology Page of Table Laboratory data before starting supplementation withvitamin DWBCμL104μLgdL104μLgdLgdLULULULULULULULULmgdLmgdLmLmin173 m2mEqLmEqLmgdLmgdLmgdLμgdLμgdLμgdLμgdLμIUmLmgdLmgdLmgdLmgdLsecondpgmLμgdLμIUmLpgmLngdLof woundirrigationtreatmentdebridementstarting SDD the pressure sore was refractory to multiple methodsincludingdepressurizationointmentbasic fibroblast growth factor and negative pressurewound therapy After starting SDD the pressure sorebegan to heal On day ie days after startingSDD the pressure sore DESIGNR score was D3 e3 s3i0 g1 n0 p0 with a total score of Fig 1b Regardingthe nutritional status the serum albumin level increasedfrom gdL just before starting SDD to gdL at days after starting SDD Also the hemoglobin levelincreased to gdL and the serum zinc level increasedto μgdL Serum levels of calcium phosphorous vitamin D and other fatsoluble vitamins also gradually increased Fig and Table A repeat diagnosticbacterial examination of the upper digestive tract contents for SIBO was not performed because of obviousimprovements in most of the laboratory data Therewere no adverse effects of SDD such as antibioticassociated diarrhea In contrast the patient presentedwith constipation that was noted before starting SDDThe sacral pressure sore was completely healed on day ie days after starting SDD with a DESIGNRscore of d0 e0 s0 i0 g0 n0 p0 and total score of Fig1c In addition the patient showed improved nutritionalstatus and had a serum albumin level of gdL Wesuccessfully reduced the dose of polymyxin B from to million units daily similarly the dose of amphotericin B was reduced from to mg daily on day ie days after starting SDD without any signs ofSIBO recurrence There was no recurrence of the sacralpressure sore with a serum albumin level of gdL onday ie days after staring SDD Fig 1d Onday we successfully ended the use of amphotericinB however the use of polymyxin B at million unitsper day continued On day ie days afterstarting SDD while still using polymyxin B at million per day the serum albumin level was gdL thehemoglobin level was gdL and the serum zinc levelwas μgdL There were no signs of SIBO recurrenceor the sacral pressure soreDiscussion and conclusionsWe report the case of a patient whose sacral pressuresore and osteoporosis were improved by treatment forSIBO Although nutrition status is known to be important for the healing of pressure sores SIBO is rarelychecked as a cause of malnutrition in patients with pressure sores However SIBO is a potential cause of malnutrition in patients with SCI due to decreased intestinalmotility resulting from autonomic disturbances and reduced physical activity [] SCI is also a risk factor forpressure sores [] However to the best of our knowledge there are no previous reports of an associationRBCHbPltTotal ProteinAlbuminASTALTγGTPLDHALPChECKAmyBUNCreatinineeGFRNaKCaiPMgFeZnUIBCFerritinErythropoietinTotal CholesterolTriglycerideHDLCholesterolLDLCholesterolPTINRAPTTACTHCortisolTSHFT3FT4duodenal content E coli Candida glabrataFig 1g h and i Therefore we diagnosed SIBO Onday we started selective decontamination of the digestive tract SDD using oral administration of polymyxin B sulfate million units daily and oraladministration of amphotericin B mg daily Before 0cKubota BMC Gastroenterology Page of Table Vitamins and bone metabolism markers before starting supplementation of vitamin DVitamin AVitamin K1Vitamin K2Vitamin E125OH2 Vitamin D25OH Vitamin D3Retinol binding proteinVitamin B1Vitamin B12Nicotinic acidFolic acidTRACP5bNTxBone type ALPIntact P1NPOsteocalcinintact PTHPTHrPFGF23TRPTmPGFR Below the detection limitIUdLngmLngmLmgdLpgmLpgmLmgdLngmLpgmLμgmLngmLμUmLnmolBCELμgLμgLngmLpgmLpmolLpgmLmgdLnormal range““ “““““““ ““““““ “““between pressure sores and SIBO Thus our case drawsattention to the fact that SIBO can be an overlookedcause of poor wound healing during the treatment ofpressure soresSIBO was first reported by Vantrappen as an increased concentration of 14CO2 in a bile acid breath testfor patients with an absent interdigestive motor complex[] Today consensus diagnostic criteria for SIBO arethe presence of more than — CFUmL of bacteriaor any amount of E coli in the proximal small bowelcontent [] Relatively little is known about commensalsinhabiting the small intestine mainly due to the limitedaccessibility of this environment for microbiological analysis [] In the healthy state the numbers of intestinalbacteria range from to CFUmL and mainly include gramnegative and grampositive aerobes such asStreptococcus Lactobacillus and Bacteroidesspecies[] Regarding the amount of bacteria in proximal jejunal aspiration Khoshini report that normal subexceed — CFUmL and thereforejectsproposed more than — CFUmL coliform bacteriaas the threshold for SIBO [] In our case CFUmLE coli existed in duodenal content and CFUmL Ecoli existed in gastric content which met the traditionaldiagnostic criteria of SIBOrarelyOther diagnostic methods for SIBO are breath testsusing hydrogen or hydrogen methane with lactulose lucose [] Breath tests have clinical utility for diagnosing SIBO because they are less invasive than obtaining proximal small bowel content However there areno standardized criteria for diagnosing SIBO usingbreath tests [] We did not perform a breath test inour case studyDespite no previous reports of an association betweenunhealed pressure sores and SIBO nutritional status isknown to be important for the healing of pressure sores []In our case the sacral pressure sore which was initially refractory began to heal after starting treatment for SIBOAmong intrinsic factors related to the healing of pressuresores blood levels of hemoglobin albumin and zinc are especially important [] Our patient had anemia hypoalbuminemia and a low zinc concentration which graduallyimproved after starting treatment for SIBOSIBO is caused by multiple factors including disturbances in defense mechanisms of the digestive tractanatomical abnormalities surgical interventions and disturbed gastrointestinal motility [ ] Bures described several endogenous defense mechanisms thatprevent bacterial overgrowth [] including secretion ofgastric acidintestinal motility a properly functioning 0cKubota BMC Gastroenterology Page of Fig Bone metabolism markers After starting SDD levels of bone metabolism markers gradually improvedTable Vitamins and trace elements before and after supplementation and selective digestive decontamination SDDVitamin K1 ngmLVitamin K2 ngmLVitamin E mgmL125OH2 Vitamin D pgmL25OH Vitamin D3 pgmLNicotinic acid μgmLMg mgdLFe μgdLBefore supplementationAt the time SDD started days after starting SDD days after starting SDDNANANA 0cKubota BMC Gastroenterology Page of ileocecal valve production of secretory immunoglobulinson the surface of the gastrointestinal mucous membraneand the bacteriostatic properties of pancreatic juice andbile In our case the patient™s history of cholecystectomyand hysterectomy were possible causes or exacerbatingfactors of SIBO Disturbed gastrointestinal motilitycaused by paraplegia below the L4 level due to SCI is another possible cause of SIBO in our case as well as decreased physical activity due to fracture ofthe lefthumerus and bilateral tibiaChronic gastrointestinalinvolvement is seen in “ of patients with SCI [ ] SCI patients lackcentral nervous system control over the gastrointestinalsystem [] Liu report that bowel problems in SCIpatients are related to high levels of cord injury completeness of cord injury and postinjury durations of years or more [] Moderate or severe grade depressivestatus is also associated with neurologic bowel dysfunction in SCI patients Of these risk factors our patienthad complete cord injury that had occurred more than years ago Also many bowel symptoms appear in patients with SCI eg constipation distension incontinence abdominal pain bowel accidents nausea diarrheastrainingautonomichyperreflexia headaches or sweat relieved by a bowelmovement [ “] However our patient showedno appetite loss a sufficient amount of food intake andnonsevere bowel symptoms Thus the presence of malnutrition despite adequate food intake and low levels oflipidsoluble vitamins that were unresponsive to supplementation led us to suspect SIBOrectal bleeding hemorrhoidsAlthough gastrointestinal symptoms are frequently observed in patients with SCI there are few reports ofSIBO in SCI patients Cheng report that of of SCI patients were diagnosed with SIBO basedon the glucose hydrogenmethane breath test [] However the prevalence of SIBO among SCI patients as confirmed by the consensus diagnostic criteria ofthepresence of more than CFUmL bacteria or anyamount of E coli in upper digestive tract content is unknown In patients with SCI absent central nervous system innervation of the digestive tract can change theinhabiting environment of bacteria Gungor reportdifferences in gut microbial patterns between SCI patients and control individuals as measured by bacterialgenome sequencing [] Specifically they found thatbutyrateproducing bacteria were specifically reduced inSCI patients Thus it is possible that SIBO is overlookedin patients with SCI In our caseit is unclear whenSIBO occurred relative to the time of SCI but we suspect that it arose due to gastrointestinal motility disorder caused by autonomic disturbancesDisturbances in fat absorption and deficiency in fatsoluble vitamins ie vitamins A K E and D3 areobserved in patients with SIBO [] Excess bacteria inthe small intestine promotes a change from conjugatedbile acid into deconjugated bile acid which decreasesthe micellar solubilization of dietary fat Bacterial fermented short chain fatty acid causes osmotic watermovement to the intestinal lumen which results in diarrhea and malabsorption [] Intestinal epithelial damagein SIBO also interferes with fat absorption Mucosaldamage is caused by metabolites of aerobic bacteria endotoxins of anaerobic bacteria and lithocholic acidwhich is a bacterial degradation product of unconjugatedbile acid [“] Our patient however showed constipation rather than diarrhea in spite of SIBO Whether ornot diarrhea occurs in patients with SIBO is determinedby multiple factors Constipation frequently occurs inpatients with SCI due to decreased physical activity andautonomic dysfunction De Looze reported that therate of constipation in the patients with SCI is []A certain proportion of the patients with SCI show constipation in spite of the coexisting SIBO Cheng reported that in patients with both SCI and SIBO showed constipation [] We believe that the factorsleading to constipation in our patient were stronger thanthose leading to diarrhea Vitamin D deficiency in SIBOcauses osteomalacia Our patientshowed multiplefractures and osteoporosis with serum vitamin D3 levelsbelow thetosupplementationrefractorydetectionlimitandThere is no consensus on the choice dose or durationof antibiotics for treating SIBO [] In principle antibiotics should be chosen based on the results of an antimicrobial susceptibility test but this approach cannotaddress the great diversity in microbiota of the digestivetract [ ] Metronidazole is a firstline choice forSIBO [] with other choices being rifaximin ciprofloxacin norfloxacin amoxicillinclavulanate trimethoprimsulfamethoxazole cephalexin or their combination []However these antibodies are selected based on customrather than scientific evidence [] In our case we usedoral polymyxin B and amphotericin B in accordancewith SDD which was first reported as a method of preventing ventilationassociated pneumonia and microbialtranslocation of gramnegative rod bacteria and fungi incritically ill patients treated in the intensive care unit[“] Polymyxin B administered to the digestive tractis nonabsorbent into the human body and has strongbactericidal power against gramnegative rod bacteriaexcept for naturally polymyxinresistant bacteria such asProteus Providencia Manella Burkholderia and Serratia [] Amphoteric B is an antifungal drug that isalso nonabsorbent into the human body when administered to the digestive tract In our case after startingSDD fatsoluble vitamins were increased and osteoporosis was improved No obvious adverse effects of SDD 0cKubota BMC Gastroenterology Page of such as antibioticassociated diarrhea were observed inour caseWhen and how to stop antibiotherapy for the treatment of patients with SIBO are difficult problemsFew reports are available for the method and timingfor making a decision to stop antibiotherapy in SIBOLauritano reported that the recurrence rate at months after stopping antibiotherapy in SIBO patientsis [] They also showed that an older age history of appendectomy and chronic use of PPIs areassociated with SIBO recurrence Bures reportedthat cyclical gastrointestinal selective antibiotics areneeded for SIBO treatment [] These reports indicatethat in many patients with SIBO it is actually impossible to stop antibiotherapy because of the underlyingconditions that lead to SIBO Similarly in our case itwas difficult to ameliorate the underlying condition ofdecreased motility of the digestive tract due to SCIWe were compelled to continue SDD for a long duration We did however succeed in gradually reducingthe dose of polymyxin B and end the use of amphotericin B without signs of SIBO recurrence Withcareful consideration it may be possible and feasibleto stop SDD completelyProbiotics are also a treatment approach for SIBO assome species of bacteria are thought to protect againsthigh numbers of E coli and fungi in the digestive tract[] However the role and effects of probiotics are stillunclear The digestive tract microbiome has both pathogenic potential and a protective role in maintaininghealth However metagenomic analysis reveals that “ of microanisms in the digestive tract cannot becultured under laboratory conditions [] The effects ofSDD and probiotics on the digestive tract microbiome inpatients with SIBO should be investigated to furtherunderstand the pathogenesis of the diseaseIn conclusion we treated a patient with a sacral pressure sore who also had SCI multiple fractures withosteoporosis and malabsorption especially of fatsolublevitamins Based on culture of upper digestive tract content we diagnosed the patient with SIBO and startedSDD using polymyxin B and amphotericin B which effectively ameliorated the absorbency disturbance andallowed healing of the pressure sore In light of severalcommon risk factors between pressure sores and SIBOsuch as decreased physical activity our case providesadditional information on the associations among pressure sores malnutrition and SIBOAbbreviationsCFU Colony forming units SCI Spinal cord injury SDD Selectivedecontamination of the digestive tract SIBO Small intestinal bacterialovergrowth 25OHVitD3 25hydroxy vitamin D3AcknowledgementsWe thank the many personnel involved in this interdisciplinary diagnosticworkup as their effective technical assistance enabled a comprehensiveapproach to this difficult diagnosisAuthors™ contributionsYK and TT treated the patient conceived of and wrote the manuscript KISK and TK treated the patient and collected the data SA and NMinterpreted the data HN analyzed the data and created the figures andtables All authors read and approved the final manuscriptFundingNo funding was receivedAvailability of data and materialsData on this case not reported in the manuscript are available from thecorresponding author upon reasonable requestEthics approval and consent to participateEthical approval was not necessary for the reported investigations as theywere performed in a routine clinical setting with therapeutic intentionConsent for publicationThe patient provided written consent for reporting her case in aninternational published medical journal including clinical details and imagesCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Plastic Surgery Chiba University Inohana ChuokuChibacity Chiba Japan 2Department of Molecular DiagnosisChiba University Inohana Chuoku Chibacity Chiba Japan3Department of Plastic Surgery Chiba Emergency Medical Center Isobe Mihamaku Chiba JapanReceived July Accepted August ReferencesStone JM NinoMurcia M Wolfe VA Perkash I Chronic gastrointestinalproblems in spinal cord injury patients a prospective analysis Am JGastroenterol “Liu CW Huang CC Chen CH Yang YH Chen TW Huang MH Prediction ofsevere neurogenic bowel dysfunction in persons with spinal cord injurySpinal Cord “Eglseer D Hodl M Lohrmann C Nutritional management of olderhospitalised patients with pressure injuries Int Wound J “Bures J Cyrany J Kohoutova D Forstl M Rejchrt S Kvetina J Vorisek VKopacova M Small intestinal bacterial overgrowth syndrome World JGastroenterol “ Matsui Y Furue M Sanada H Tachibana T Nakayama T Sugama J Furuta KTachi M Tokunaga K Miyachi Y Development of the DESIGNR with anobservational study an absolute evaluation tool for monitoring pressureulcer wound healing Wound Repair Regen “Sachdev AH Pimentel M Gastrointestinal bacterial overgrowth pathogenesisand clinical significance Ther Adv Chronic Dis “Groah SL Schladen M Pineda CG Hsieh CH Prevention of pressure ulcersamong people with spinal cord injury a systematic review PM R “Vantrappen G Janssens J Hellemans J Ghoos Y The interdigestive motorcomplex of normal subjects and patients with bacterial overgrowth of thesmall intestine J Clin Invest “Zoetendal EG Raes J van den Bogert B Arumugam M Booijink CC TroostFJ Bork P Wels M de Vos WM Kleerebezem M The human small intestinalmicrobiota is driven by rapid uptake and conversion of simplecarbohydrates ISME J “ Miazga A Osinski M Cichy W Zaba R Current views on theetiopathogenesis clinical manifestation diagnostics treatment andcorrelation with other nosological entities of SIBO Adv Med Sci “ 0cKubota BMC Gastroenterology Page of Khoshini R Dai SC Lezcano S Pimentel M A systematic review ofdiagnostic tests for small intestinal bacterial overgrowth Dig Dis Sci “ Heintschel M Heuberger R The potential role of zinc supplementation onpressure injury healing in older adults a review of the literature Wounds“Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations Han TR Kim JH Kwon BS Chronic gastrointestinal problems and boweldysfunction in patients with spinal cord injury Spinal Cord “Ebert E Gastrointestinal involvement in spinal cord injury a clinicalperspective J Gastrointestin Liver Dis “ Gungor B Adiguzel E Gursel I Yilmaz B Gursel M Intestinal microbiota inpatients with spinal cord injury PLoS One 2016111e0145878 Harari D Sarkarati M Gurwitz JH McGlincheyBerroth G Minaker KLConstipationrelated symptoms and bowel program concerning individualswith spinal cord injury Spinal Cord “ Menter R Weitzenkamp D Cooper D Bingley J Charlifue S Whiteneck GBowel management outcomes in individuals with longterm spinal cordinjuries Spinal Cord “ De Looze D Van Laere M De Muynck M Beke R Elewaut A Constipationand other chronic gastrointestinal problems in spinal cord injury patientsSpinal Cord “Lynch AC Wong C Anthony A Dobbs BR Frizelle FA Bowel dysfunctionfollowing spinal cord injury a description of bowel function in a spinalcordinjured population and comparison with age and gender matchedcontrols Spinal Cord “Krogh K Nielsen J Djurhuus JC Mosdal C Sabroe S Laurberg S Colorectalfunction in patients with spinal cord lesions Dis Colon Rectum “ Chen CY Chuang TY Tsai YA Tai HC Lu CL Kang LJ Lu RH Chang FY LeeSD Loss of sympathetic coordination appears to delay gastrointestinaltransit in patients with spinal cord injury Dig Dis Sci “ Cheng X Zhang L Xie NC Xu HL Lian YJ Association between smallintestinal bacterial overgrowth and deep vein thrombosis in patients withspinal cord injuries J Thromb Haemost “Kirsch M Bozdech J Gardner DA Hepatic portal venous gas an unusualpresentation of Crohn's disease Am J Gastroenterol “Jones RM Neish AS Recognition of bacterial pathogens and mucosalimmunity Cell Microbiol “ Hoog CM Lindberg G Sjoqvist U Findings in patients with chronicintestinal dysmotility investigated by capsule endoscopy BMCGastroenterol Singh VV Toskes PP Small bowel bacterial overgrowth presentationdiagnosis and treatment Curr Treat Options Gastroenterol “ Quigley EM AbuShanab A Small intestinal bacterial overgrowth Infect DisClin N Am “ viiiix Melchior C Gourcerol G Bridoux V Ducrotte P Quinton JF Leroi AMEfficacy of antibiotherapy for treating flatus incontinence associated withsmall intestinal bacterial overgrowth a pilot randomized trial PLoS One2017128e0180835 VandenbrouckeGrauls CM Vandenbroucke JP Effect of selectivedecontamination of the digestive tract on respiratory tract infections andmortality in the intensive care unit Lancet “Silvestri L van Saene HK Casarin A Berlot G Gullo A Impact of selectivedecontamination of the digestive tract on carriage and infection due togramnegative and grampositive bacteria a systematic review ofrandomised controlled trials Anaesth Intensive Care “ Camus C Salomon S Bouchigny C Gacouin A Lavoue S Donnio PYJavaudin L Chapplain JM Uhel F Le Tulzo Y Shortterm decline in allcause acquired infections with the routine use of a decontaminationregimen combining topical polymyxin tobramycin and amphotericin Bwith mupirocin and chlorhexidine in the ICU a singlecenter experienceCrit Care Med “ Olaitan AO Morand S Rolain JM Mechanisms of polymyxin resistanceacquired and intrinsic resistance in bacteria Front Microbiol Lauritano EC Gabrielli M Scarpellini E Lupascu A Novi

Thyroid_Cancer

inhibitor with Temozolomide results in significant apoptosis in glioblastoma via the p65 and actin cytoskeleton regulatory pathwaysNaze G Avci1 Sadaf Ebrahimzadeh‘Pustchi1 Yasemin M Akay1 Yoshua Esquenazi2 Nitin Tandon Jay‘Jiguang Zhu Metin Akay1Glioblastoma GBM is the most malignant brain tumor characterized by intrinsic or acquired resistance to chemotherapy GBM tumors show nuclear factor‘κB activity that has been associated with tumor formation growth and increased resistance to therapy We investigated the effect of inhibitor BAY ‘ with Temozolomide TMZ on the signaling pathways in GBM pathogenesis GBM cells and patient‘derived GBM cells cultured in 3D microwells were co‘treated with BAY ‘ and TMZ or BAY ‘ and TMZ alone and combined experiments of cell proliferation apoptosis wound healing assay as well as reverse‘phase protein arrays western blot and immunofluorescence staining were used to evaluate the effects of drugs on GBM cells The results revealed that the co‘treatment significantly altered cell proliferation by decreasing GBM viability suppressed pathway and enhanced apoptosis Moreover it was found that the co‘treatment of BAY ‘ and TMZ significantly contributed to a decrease in the migration pattern of patient‘derived GBM cells by modulating actin cytoskeleton pathway These findings suggest that in addition to TMZ treatment can be used as a potential target to increase the treatment™s outcomes The drug combination strategy which is significantly improved by inhibitor could be used to better understand the underlying mechanism of GBM pathways in vivo and as a potential therapeutic tool for GBM treatmentGlioblastoma multiforme GBM is the most malignant primary brain tumor in the central nervous system Current standard of care therapy includes surgery followed by radiotherapy and concomitant and adjuvant chemotherapy with the alkylating agent Temozolomide TMZ which provides survival benefits for patients with GBM1 However even with the advances in surgical resection combined with TMZ therapy and irradiation the prognosis for newly diagnosed GBM patients remains poor In fact due to its rapid proliferation increased invasion and migration capacity and chemoresistance to the alkylating agents a0the median survival is only a0months with the ˜Stupp™ regimen radiation with daily TMZ — “ a0weeks followed by cyclic TMZ2 and 5year survival rate is less than which is the lowest longterm survival rate of malignant brain tumors3“ TMZ methylates DNA at the O6 positions of guanine and DNA repair enzyme O6methylguanine methyltransferase MGMT removes alkyl groups from O6 position of guanine in DNA making cells resistant to TMZ6 Therefore new therapies are necessary to prevent cell proliferation and induce apoptosis for GBM patientsNuclear factorkappa B NFκB is a regulatory transcription factor of the Rel gene family including p50 cRel RelB or p65 subunits It is involved in the control of tumor cell proliferation migration immune response and apoptosis7“ Studies have shown that NFκB gene was involved in the regulation pathways of different cancer types such as thyroid cancer head and neck squamous cell carcinoma and colorectal cancer711“ Increased 1Department of Biomedical Engineering University of Houston Cullen Blvd Houston TX USA 2UTHealth Neurosurgery McGovern Medical School Memorial Hermann at Texas Medical Center The University of Texas Health Science Center at Houston Houston TX USA email makayuheduScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cactivation of NFκB has also been identified in GBM tumors where the expression of NFκB was much higher in GBM tissue compared with nonGBM tissue1415 NFκB also promotes chemoresistance to TMZ and regulates MGMT activity in GBM by promoting MGMT gene expression through NFκB binding sites within the MGMT promoter16 NFκB inhibitors such as parthenolide do not completely eradicate tumors therefore they are mostly used in combination with other drugs17 When used in combination with TMZ NFκB inhibitor parthenolide has been shown to activate mitochondrial apoptosis signaling in U87MG and U373 GBM cells which lead to cell death18 and had a combined effect on cell cytotoxicity in LN18 and T98G glioma cells19 NFκB inhibitor CBL0137 has been shown to bind DNA leading the functional inactivation of the Facilitates Chromatin Transcription FACT complex a chromatin remodeling complex regulating transcription replication and DNA repair2021 In a0vitro evaluation of the CBL0137 on FACT p53 and NFκB has been done using U87MG and A1207 GBM cells It was shown that CBL0137 induced loss of chromatinunbound FACT activated p53 and inhibited NFκB dependent transcription21 In a0vivo studies showed that CBL0137 was effective in increasing survival rates in TMZresistant orthotopic mouse models21 Moreover Wang et a0al indicated that NFκB inhibitor BAY suppresses the expression of MGMT and enhances the TMZinduced apoptosis in TMZ resistant U251 cells22 However there is still a lack of characterization of the precise pattern of NFκB activation in combination with TMZ in GBM cell populations that have been a0surgically resected from patientsIn vitro and in a0vivo identifications and validations of molecular targets of GBM are important as they can progress into clinical studies Studies reported that combining multiple gene targets may prevent tumor growth and improve the treatment strategy for GBM23“ Both Bay and TMZ exert antitumoral activities individually in different tumor types28“ Therefore in this study we aimed to analyze functionally the combined effect of Bay and TMZ in different GBM cells For this purpose first we used our 3D PEGDAbased hydrogel microwell platform31“ to provide reliable preclinical models that can recapitulate in a0vivo features of the GBM tumors We cultured GBM cells U87 and LN229 and patientderived GBM cells in 3D microwells for a more precise and personalized treatment approach We then treated GBM cells with Bay and TMZ in combination or alone Our results indicated that the cotreatment of Bay and TMZ significantly reduced cell viability in all three cell lines in correlation with a significant decrease in the spheroid size The levels of NFκB protein and its subunits p65 and p50 were also significantly decreased compared with the control and single drug applications Similar a0decreases in the cell viability and protein levels were observed in all three GBM cells Tumor biopsy samples could give more realistic information about how tumors respond to drugs when they are used for in a0vitro or in a0vivo studies35“ Therefore we decided to continue our experiments with only using our patientderived GBM cells We treated patientderived GBM cells with Bay and TMZ or alone and analyzed specific cellular proteins along with their posttranslational modifications via reversephase protein arrays RPPA to elucidate the mechanism of action of the proteins3839 We observed that several cell signaling pathways including cell metabolism proliferation apoptosis were significantly affected by the combination of the drugs which were consistent with the literature4041 Furthermore our RPPA data revealed that there was a significant change in the modulation of actin cytoskeleton and following experiments including western blot analysis for the expression of FAK protein and wound healing assay for cell migration patterns confirmed the RPPA results We observed a significant decrease in both actin fluorescence intensity and migration pattern in the a0cotreated patientderived GBM cells To the best of our knowledge the effect of cotreatment of Bay and TMZ has never been studied previously on the actin modulation of patientderived GBM cells These results suggested that Bay and TMZ induced alteration in the a0actin filament anization by reducing the level of focal adhesion protein which might implicate in cell apoptosis The effect of Bay with TMZ necessitates further exploration to better understand its mechanism of action in GBM and potential therapeutic tools for GBM treatmentResultsCo‘treatment of Bay ‘ and TMZ reduced viability of GBM cells We used our previously a0published data to select the most effective drug concentrations for this study42 We cultured LN229 U87 and patientderived cells in the microwells for a0days where they formed 3D spheroids and we added a0µM of Bay and a0µM of TMZ in combination or alone Then we cultured the spheroids for more days with or without drug Control group did not receive any treatment The cell viability assay was performed on day after drug administration The results showed that the a0cotreatment significantly reduced cell viability of GBM cells LN229 and U87 and patientderived GBM cells cultured in 3D PEGDA microwells respectively as shown in Fig a01ac When they were used alone TMZ reduced cell viability to and p and Bay reduced cell viability to and in LN229 U87 and patientderived GBM cells respectively compared to control groups Fig a01d However when they were used in combination the viability of the cells significantly decreased to and in LN229 U87 and patientderived GBM cells respectively compared to control groups p Fig a01d Tumor cells are generally less sensitive to drug treatments in 3D cultures than in 2D cultures4344 This could reflect reduced compound access or differences in the response to cell death To confirm that cotreatment was more effective compared to single drug use we quantified the size of the spheroids using ImageJ45 Our data showed that after a0days of drug treatment the spheroids™ sizes were significantly reduced in the cotreatment by and in LN229 Fig a01e U87 Fig a01f and patientderived GBM cells p Fig a01g respectively compared to control group p When we compared the spheroids™ sizes of the cotreatment with TMZ alone there was a reduction of and in LN229 U87 and patientderived GBM cells respectively p Finally the spheroids™ sizes of the cotreatment compared with Bay alone showed a decrease of and in LN229 U87 and patientderived GBM cells respectivelyScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Representative images of the GBM tumor cells cultured in the PEGDA microwells a“c LN229 U87 and patientderived GBM cells were cultured in the microwells for a0days respectively After day Bay and TMZ were applied either alone or in combination onto the cell spheroids Control group did not receive any treatment The cells were cultured with or without drugs additional more days The images were taken on Day Day and Day after the drug application to observe the disruption in the spheroids Dotted black lines represent the edge of the tumor spheroid Scale bars a0µm d Bar graph showing trypan blue staining for cell viability of LN229 U87 and patientderived GBM cells e“g Spheroid size quantification was done using ImageJ for LN229 U87 and Patientderived GBM cells respectively Twotailed ttest followed by Wilcoxon test were done GraphPad Prism v5 Data represent the mean ± SD of three biological replicates p and p Suppression of activity in GBM cells by co‘treatment of Bay ‘ and TMZ As a readout of NFkB activity after drug treatment we first quantitatively assessed the cytoplasmic activation of phosphorylated NFκB p65 subunit in both treated and untreated groups in all GBM cells NFκB pp65 subunit activity was observed in the control groups of all three GBM cells Fig a02a NFκB pp65 subunit activity decreased to and when TMZ applied alone and and when Bay was applied alone in LN229 U87 and patientderived cells respectively However the decrease in NFκB pp65 subunit was reduced to when LN229 U87 and patientderived cells respectively were cotreated p Fig a02a Bay specifically inhibits NFκB activation by blocking phosphorylation of IκBα46 In independent experiments we analyzed the abundance of phosphorylated NFκB p65 NFκB p50 and IκBα in all three GBM cells Qualitative and quantitative western blot analysis revealed that the exposure to Bay with TMZ significantly downregulated the abundance of NFκB p65 NFκB p50 and IκBα compared with control and Bay or TMZ alone Fig a02b Please note that loading controls were used for each experiment but only the representative loading control for p and tP65 and p and tP50 was presented Fig a02b The cell viability assay cells™ size and protein expressions in all three GBM cells revealed similar results without any dramatic change Therefore considering the importance of using patientderived tumor cells to elucidate the mechanism of drugs and respective signaling pathways35“ we further continued our experiments using patientderived GBM cellsApoptosis was promoted by co‘treatment of Bay ‘ and TMZ RPPA technology is designed for multiplexed antibodybased relative quantification where each array is tested with a validated antibody specific to a particular protein along with their particular posttranslational modifications47 In the attempt to elucidate the mechanism of action of Bay with TMZ by which NFκB subunits were modulated and to identify downstream signaling molecules we employed RPPA platform using our drug treated or untreated patientderived GBM cells RPPA results showed that many oncogenic pathways were altered by the drug treatments but more specifically by the cotreatment Fig a03a Decreased expression of NFκB was not only associScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 NF“kB activity in LN229 U87 and patientderived GBM cell lines a NF“kB p65 subunit activity in LN229 U87 and patientderived GBM cell lines respectively The cells cultured with or without drugs for a0days were collected from the microwells and subjected to ELISA Data represent the mean ± SD of three biological replicates p and p b Representative immunoblots LN229 U87 and patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels bottom panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p ated with changes in the a0NFκB pathway but also with apoptosis cell metabolism and proliferation which were confirmed by the analysis of downregulated RPPA proteins in Enrichr KEGG libraries4849 Fig a03c p One of the specific pathways given by RPPA was apoptosis Apoptosis is one of the important mechanisms that regulates cell death and suppress tumorigenesis Studies have demonstrated that Bcl2 family proteins can positively and negatively regulate apoptosis by regulating antiapoptotic protein Bcl2 and proapoptotic protein Bax4050 Our RPPA data using patientderived GBM cells showed that the fold change of Bcl2 relative to control was times higher in cotreated group TMZ alone Bay alone respectively Fig a03b In order to further confirm whether the expression of a0these proteins were downregulated by the cotreatment we performed western blot analysis Our results showed a similar decrease in Bcl2 protein expression in the cotreatment compared with the control and single drug a0treatment Fig a03d In contrast Bax protein fold change relative to control was times higher in cotreated group TMZ alone Bay respectively where we observed a significant increase after the cotreatment of Bay with TMZ compared with the control p Fig a03b Bcl2Bax ratio is a key indicator in susceptibility of the cells to apoptosis Western blot results confirmed the change in Bcl2Bax ratio in the cotreatment compared with the control group and single a0drug treatment Fig a03d Our RPPA data also showed a significant increase in the cleavedcaspase protein Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 The effect of Bay and TMZ on signaling pathways in patientderived GBM cells a Heat map presentation of RPPA analysis showing the changes in the protein expression RPPA was performed on lysates treated with Bay and TMZ alone or in combination All relative protein level data points were normalized to the a0control group Red and green indicate up and down regulations respectively in the heat map The samples were run in duplicate n b Fold change of the a0selected proteins relative to the a0control group via RPPA Data represent the mean ± SD of two biological replicates p p Wilcoxon rank sum test c Analysis of downregulated RPPA proteins shows a a0significant activation in numerous Enrichr KEGG pathways The pathways were a0sorted by p value ranking d Representative immunoblot validation of significantly altered proteins involved in different KEGG pathways Patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p fold change relative to control times higher in the cotreatment compared with times higher in TMZ alone and times higher in Bay alone p Fig a03b To confirm if cotreatment triggered apoptosis correlated with caspase activation we performed western blot analysis with procaspase3 cas3 and cleavedcaspase3 Ccas3 We observed that Bay and TMZ induced apoptosis was associated with cas3 Fig a03d Please note that loading controls were used for each experiment but only the representative loading control for Bax cas3 and Ccas3 was presented Fig a03d Moreover another important mechanism of NFκB activation in GBM regulates through AKT phosphorylation of IκB Our RPPA data showed relative fold changes of in the cotreated group TMZ alone and Bay alone respectively p Fig a03b The western blot results also confirmed a significant decrease in the abundance of AKT pT308 Fig a03dTo further investigate whether cotreatment of Bay with TMZ can lead to glioma cell apoptosis and to confirm our RPPA and western blot results we performed apoptosis assay TUNEL The patientderived GBM cells were cotreated with Bay with TMZ or single drug treated and subjected to TUNEL assay to detect DNA damage Fig a04a The results indicated that TUNEL cells in the cotreatment were increased tenfold compared with control and and 24folds compared with TMZ alone and Bay alone respectively p Fig a04b Additionally in some TUNEL cells we observed a typical ring type chromatin aggregation underneath the nuclear membrane which suggested an early stage apoptosis51 Fig a04a red arrows There were also a few TUNEL cells that lacked the typical apoptotic ringlike nuclear structure indicating that they were either at a different stage of apoptosis or alternatively undergoing necrosis52 that we have not investigated furtherCo‘treatment of Bay ‘ with TMZ changed actin anization by inhibiting FAK phosphorylation and cell migration Actin filaments Factin are one of the main components of the cellular cytoskeleton which regulates actin dynamics and migration process in the cells The disruption of the actin cytoskeleton inhibits cell migration and adhesion53 Depolymerization or cleavage of actin lamins and other cytoskeletal proteins have been also found to be involved in cell apoptosis54“ To confirm the RPPA results showing changes in the actin modulation pathway and to understand the mechanism that regulates cytoskeletal Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Apoptosis assay TUNEL a Fluorescent images of TUNEL cells in patientderived GBM cells TUNEL assay was performed on cells treated with Bay and TMZ in combination or alone in the microwells Cells were collected from the microwells trypsinized and replated into 8well chamber slides TUNEL cells green with ringlike nuclear stain are indicated with red arrows Nuclei were counterstained with DAPI blue b Numbers of TUNEL cells are presented as mean ± SD of three biological replicates p and p X20 objective Scale bars a0µmanization we treated patientderived GBM cells co treated with Bay with TMZ or single drug treated 3D spheroids collected from the microwells were stained with phalloidin green and DAPI blue Staining cells with fluorescently conjugated phalloidin is considered the most reliable method of accurately labeling Factin in fixed cells57 In the control group intact cells formed finemeshed networks with a distinct Factin anization on both day Fig a05a upper panel and day Fig a05a bottom panel In single drug treated cells actin was still found to be polymerized to filaments as it can be seen by its interaction with phalloidin at both days and However the cells which were cotreated with Bay and TMZ lost their Factin anization and their shape compared with the control and the single drug treated groups at day Fig a05a bottom panel Changes in the a0actin distribution within the cells were quantified by measuring the staining intensity using Fiji Macro ImageJ as described previously5859 At day we observed a a0significant decrease in the fluorescence intensity of phalloidin when the cells were cotreated with Bay and TMZ compared with the a0control and single drug treated groups p Fig a05b To investigate the drug related Factin mechanism we examined the levels of FAK protein following cotreatment or single drug treatment As shown in Fig a05c cotreatment significantly decreased the level of phosphorylated FAK compared with both control and single drug applications p Furthermore we investigated cell migration patterns of the patientderived cells that were cotreated with Bay and TMZ or single drug treated We collected 3D spheroids from microwells after drug treatment and replated them in 24well plate to perform scratch wound healing assay We noted a significant increase in cell density in the scratch area in both control and Bay alone after and a0h of scratch formation p Fig a06a Although compared with the a0control cells both cotreatment and TMZ alone groups showed a decrease in the cell migration into the scratch area after a0h we observed that after a0h the migration rate of the cotreated cells was significantly slower than the cells that were treated with TMZ alone p Fig a06b These results indicated that the disanization of actin microfilaments was concomitant with the cell apoptosis after the a0cotreatment of Bay with TMZDiscussionDespite the increase in the median survival of GBM patients from to months4 the clinical efficacy of standard of care therapy including TMZ chemotherapy combined with surgery and radiotherapy is still limited Due to challenges in treating GBM significant attempts have been made to develop single or combined drug treatments60“ However given the cost long time frame and risks of failure associated with developing a new drug repurposing available drugs may be the most effective alternative therapeutic strategy Therefore it is important to evaluate potential drug combinations for GBM treatmentScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Changes in the actin cytoskeleton and migration pattern in patientderived GBM cells cotreated with Bay and TMZ or single drug treated in the microwells a Upper panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin green and DAPI blue Bottom panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin and DAPI Scale bars a0µm b Intensity of staining obtained with phalloidin was measured in each cell using ImageJ and displayed as boxplots with to confidence intervals A twoway ANOVA with Dunnett™s multiple comparisons test was performed to determine statistical relevance Three biological replicates n p p c Representative immunoblots show the levels of FAK pTyr397 and total FAK in patientderived GBM cell lysates cotreated with Bay and TMZ or single drug treated for a0days in the microwells The levels of the proteins were quantified using ImageJ right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p Due to the cell repellent property of PEGDA hydrogel tumor cells can form aggregates at the bottom of the microwells and selfassemble into spheroids in each well within a0days following cell seeding313363 Compared with 2D monolayer cell culture 3D spheroids have an important advantage their larger size Thus often drug effects can easily be monitored over time by measuring the size and shape of spheroids4344 Additionally using 3D in a0vitro tumor models can better recapitulate in a0vivo features of the tumors We used PEGDA hydrogelbased microwell platform313363 in order to culture different types of a0GBM cells commercially available GBM cell lines LN229 U87 and a0patientderived GBM cells However we investigated the effect of the drugs on the patientderived GBM cells more in detail since growing tumors from tumor biopsy samples could give very detailed information about how tumors respond to drugs35“ Considering the precious nature of the patient samples this platform which requires fewer cells compared with 2D monolayer cultures provides us with a robust tool to recapitulate in vivo features of GBM tumors and to test our drug combinationsNFκB is one of the major transcription factors associated with GBM and responsible for activating a series of cellular responses including cell proliferation survival invasion and apoptosis6465 Previous studies have shown that NFκB can activate Akt and promote cell survival and proliferation by downregulating the expression of phosphatase and tensin homolog deleted on chromosome ten1866 NFκB pathway can inhibit cell apoptosis by inhibiting a stressactivated protein kinase and a mitogenactivated protein kinase signaling pathway67 It can also be activated in response to treatment with cytotoxic drugs such as vinca alkaloids and topoisomerase inhibitors Several studies have demonstrated the activation of NFκB in GBM patientderived stemlike cells cultures96869 Moreover alkylating agents TMZ can activate NFκB through DNA damage pathway activation7071 The combination effect of Bay and TMZ have been showed in our previous study where we determined the most effective drug concentrations on GBM cells using our microfluidics platform42 Another study that investigated the combined effect of NFκB inhibitor BAY with TMZ showed that combined drug application induced TMZ resistant in U251 GBM cells22 However the characterization of the precise pattern of NFκB activation in different GBM cell populations from surgically resected tissues still remains elusive Therefore in this study we investigated the interaction of Bay with TMZ and their effects on the LN299 and U87 GBM cell lines as well as patientderived GBM cells in order to recapitulate NFκB activation as in a0vivo features of the GBM and its signaling pathways We applied a0µM of Bay and a0µM of TMZ3442 in combination or alone for all three GBM cell types First we observed a significant decrease in both cell viability and size of the spheroids in the cotreatment compared with control and single drug application Then we showed quantitatively and Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Cell migration of patientderived GBM cells by wound healing assay a Patientderived cells were cotreated with Bay and TMZ or single drug treated in the microwells trypsinized and replated in 24well plates After they reached to their confluency a scratch wound was formed with a 200μl tip and cells were incubated for the next a0h Images were taken 4x at a0h a0hr and a0hr Scale bars a0µm b The wound width was measured with ImageJ and the average wound width was shown Data represent the mean ± SD of three biological replicates p and p oneway ANOVA with Tukey™s post hoc testqualitatively the expression of NFκB in all three GBM cell types a0We noted a significant decrease in the cotreated group compared with control and single drug application Our western blot data also confirmed the decrease in the abundance of pP65 pP50 and pIKBa that Bay has been shown to inhibit its phosphorylation46 However in the cotreated group the decrease was significantly higher compared to both control and single drug application This data showed that cotreatment of Bay and TMZ has more effect on the inhibition of NFκB pathway than Bay or TMZ alone and suggests a a0decreased downstream transcription of oncogenic proteins72 Although there were slight differences in the NFκB expression patterns in three different GBM cell types a0we focused on the patientderived cells in the rest of the study due to their ability to better recapitulate the genomic similarities to primary disease7374Proteins that interact with each other activate multiple pathways which can result in apoptosis according to tissue type and pathological condition Glioblastoma tumors express high levels of antiapoptotic BCL2 family proteins such as Bcl2 and BclxL which may cause glioblastoma cells to resist apoptosis75 The proapoptotic members of Bcl2 family such as Bax and Bak are necessary for their proapoptotic effect Interactions and the ratio between antiapoptotic Bcl2 and proapoptotic Bax are decisive factors in the induction of apoptosis7677 Active NFκB can prevent cells from apoptosis by stimulating the expression of genes and promoting cell proliferation Although patientderived GBM samples have been shown to be highly resistant to apoptosis77 our data revealed changes in the expression of various members of Bcl2 family and NFκB signaling pathway after cotreatment of Bay and TMZ Our RPPA results outlined distinct molecular profiles in which apoptotic P53 signaling and NFκB signaling pathways were significantly affected after the a0cotreatment These results supported that the inhibition of NFκB expression could inhibit the expression of Bcl2 and promote the expression of Bax thus promote apoptosis Our data also suggested the possible interaction between Bcl2 and p53 in Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Proposed schematic of the a0signaling pathways involved in Bay and TMZmediated inhibition in GBM patientderived cells The effect of combined therapy of Bay and TMZ was achieved through the inhibition of SrcFAKVinculin which regulate the cytoskeleton anization through MAPKs JNK and PI3KAKT signaling pathways Exposure to both Bay and TMZ also leads to receptormediated activation of Bax but not Bcl2 in the subsequent inhibition of the downstream NFκB transcription factor Inhibition of NFκB in turn causes cell deathregulating cell survival and death7778 The activation of extrinsic and intrinsic molecular pathways can lead to the proteolytic activation caspases The extrinsic pathway is triggered by proapoptotic ligands that activate cell surface death receptors and procaspase8 which in turn leads to the cleavage of caspase3 and apoptosis79 Our results determined that the a0cotreatment significantly inhibited the expression of caspase3 while the expression of cleaved caspase3 was increased Additionally TUNEL assay which detects DNA strand breaks which could occur as an event in the apoptosis showed a dramatic increase in the TUNEL cells after the cotreatment compared with the a0control and single drug application Altogether these results suggested that the inhibition of cell proliferation Bcl2 and caspase3 by a0the cotreatment of Bay and TMZ may occur through the NFκB mediated apoptosis and they might be tightly coupled8081The literature provides evidence that supports crosstalk between PI3KAktmTOR signaling pathway and NFκB which is downstream of Akt NFκB activation in GBM regulates through AKT phosphorylation of IκB resulting in an activated NFκB that translocates to nucleus8283 Our data showed that when Bay was used with TMZ there was a decrease in the abundance of PI3Kp110 AktpS473 AktpT308 and mTORpS2448 This preliminary data is important to suppo

Thyroid_Cancer

CytoskeletonAssociated Role ofPDLIM5Xiaolan Huang Rongmei Qu Jun Ouyang Shizhen Zhong and Jingxing DaiGuangdong Provincial Key Laboratory of Medical Biomechanics Department of Anatomy School of Basic MedicalSciences Southern Medical University Guangzhou ChinaRegenerative medicine represented by stem cell technology has become one of thepillar medical technologies for human disease treatment Cytoskeleton plays importantroles in maintaining cell morphology bearing externalforces and maintaining theeffectiveness of cellinternal structure among which cytoskeleton related proteins areinvolved in and play an indispensable role in the changes of cytoskeleton PDLIM5 isa cytoskeletonrelated protein that like other cytoskeletal proteins acts as a bindingprotein PDZ and LIM domain PDLIM5 also known as ENH Enigma homolog isa cytoplasmic protein with a molecular mass of about KDa that consists of a PDZdomain at the Nterminus and three LIM domains at the Cterminus PDLIM5 bindsto the cytoskeleton and membrane proteins through its PDZ domain and interactswith various signaling molecules including protein kinases and transcription factorsthrough its LIM domain As a cytoskeletonrelated protein PDLIM5 plays an importantrole in regulating cell proliferation differentiation and cellfate decision in multipletissues and cell types In this review we briefly summarize the state of knowledge onthe PDLIM5 gene structural properties and molecular functional mechanisms of thePDLIM5 protein and its role in cells tissues and an systems and describe thepossible underlying molecular signaling pathways In the last part of this review wewill focus on discussing the limitations of existing research and the future prospects ofPDLIM5 research in turnKeywords PDZ and LIM domain microfilament actin cytoskeleton cytoskeletonassociated proteinINTRODUCTIONPDZ and LIM domain also known as ENH ENH1 L9 and LIM is a cytoskeletonrelated proteinthat was first discovered by Kuroda using yeast twohybrid technology with proteinkinase C PKC as the bait protein PDLIM5 which consists of a PDZ domain and one or moreLIM domains is a PDZLIM family member whose sequence is highly conserved across speciesThe proteins of the PDZLIM family EnigmaLMP1 ENH ZASPCipher RIL ALP and CLP36have been suggested to act as linkers to direct LIM binding proteins to the cytoskeleton Vallenius PDZLIM protein can act as a signal modulator to aï¬ect actin dynamics regulate cellstructure and control gene transcription to promote the assembly of protein complexes The PDZLIM protein family which function as protein“protein interaction modules act as scaï¬olds to bindto filamentous actinassociated proteins a range of cytoplasmic signaling molecules and nuclearEdited byMarina PajicGarvan Institute of Medical ResearchAustraliaReviewed byClment ThomasLuxembourg Institute of HealthLuxembourgHongqiang ChengZhejiang University ChinaCorrespondenceJun OuyangjouyangsmueducnShizhen ZhongzhszhsmueducnJingxing Daidaijxsmueducndaijx2013163comSpecialty sectionThis was submitted toIntegrative Physiologya section of the journalFrontiers in PhysiologyReceived May Accepted July Published August CitationHuang X Qu R Ouyang JZhong S and Dai J AnOverview of theCytoskeletonAssociated Roleof PDLIM5 Front Physiol 103389fphys202000975Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5proteins allowing this family to carry out diverse functionsduring development and adulthood Krcmery Several members of the PDZLIM proteins family play aregulatory role in the invasion and migration of cancer cellsDysfunction of the proteins of the PDZLIM family is knownto aï¬ect the maintenance of an function and weaken theinvasion ability and metastatic potential of cancer cells BagheriYarmand TanakaOkamoto Accordingto recent studies PDLIM5 may be involved in the progressionof multiple tumor types Eeckhoute Edlund Heiliger Li The important role ofPDLIM5 in various anizational systems have led to a deeperunderstanding of its physiological function This review aimsto summarize the state of knowledge and progress related toPDLIM5 from multidisciplinary perspectivesTHE PDLIM5 GENE AND ITSEXPRESSION AND THE STRUCTUREAND DISTRIBUTION OF PDLIM5The PDLIM5 GeneThe PDLIM5 gene is located on the human chromosome 4q223between markers W12900 and W13273 and spans exonsUeki Te Velthuis and Bagowski PDLIM5can be categorized as long isoforms with three LIM domainsand short isoforms without LIM domains according to thepresence or lack of three LIM domains and the long and shortisoforms each contain ˆ¼ subtypes Cheng Ito Mouse PDLIM5 isoform I mENH1 encodes afulllength 591aminoacid protein containing a PDZ domainand three LIM domains two smaller transcripts called mousePDLIM5 isoform and mENH2 and mENH3 encode a aminoacid protein and a 239aminoacid protein respectivelyBoth mouse PDLIM5 isoforms and lack three LIM domainsNiederlander Zheng In humans fourPDLIM5 splice isoforms have been identified one long isoformENH1 which contains three LIM domains at its Cterminaland is widely expressed in all tissues and three short isoformsENH24 which are mainly expressed in cardiac and skeletalmuscle Kuroda Ueki Analysis of humanPDLIM5 transcripts showed that three transcripts hENH12 were similar to those of mice while the fourth transcripthENH4 encodes a 215aminoacid protein lacking three LIMdomains Niederlander The Expression Structure andDistribution of PDLIM5is a memberThe PDLIM5 protein also known as ENHofthe Enigma family which consists of an NterminalPDZ domain and three Cterminal LIM domains The mainfunction of PDZ and LIM domains is to participate inproteinprotein interactions Table The PDZ domain oneofischaracterized by a highly conserved amino acid sequenceconsisting of six antiparallel strands and two αhelicesthe most common proteinprotein binding domainsTABLE Binding partners of PDLIM5 and their functionsProteinDomain FunctionsReferencesProtein kinaseA PKAαactininMyotilinLtype calciumchannelYAPProtein kinaseC PKCProtein kinaseD1 PKD1CREBID2PDZPDZPDZPDZPDZLIMLIMLIMLIMNtype Ca2channelsAMP activatedprotein kinaseKAE1Protein kinaseLin Sarcomere Zline proteinSarcomere Zline proteinCalcium channels inmyocytesTranscriptionalcoactivatorProtein kinaseProtein kinaseCAMP relatedtranscription factorsDifferentiation inhibitorNtype calcium channelsin nervous systemProtein kinaseNakagawa Ito Ito Maturana Elbediwy Kuroda Maturana Maturana Ito Lasorella and Iavarone Nakatani et alMaenoHikichi et alYan Liu Kidney anion exchanger Su Fanning and Anderson Sheng and Sala The PDZdomain provides a proteinbinding interface that facilitates theformation of multiprotein complexes with a variety of partnersincluding membraneassociated proteins cytoplasmic signalingproteins and cytoskeleton proteins Jelen Krcmery Zheng Ito The LIM domainis approximately amino acids long and is characterized byhighly conserved and spatially defined cysteine and histidineresidues that coordinate the binding of two zinc ions to formtwo zinc fingerlike structures LIM domains can exist in proteinsalone or in combination with other domains Dawid Bach Kadrmas and Beckerle Te Velthuisand Bagowski The LIM domains can combine highlydiverse partners ranging from signaling molecules and actincytoskeletal components to transcription factors and it alsosupport cellular functions Dawid In particularthe crosslinking with actin cytoskeleton such as LIM domainprotein can maintain the functional structure of cardiomyocytesby a mechanism involving its own binding and actin filamentcrosslinking which plays an important role in the developmentof heart disease Hoï¬mann In addition LIM domainproteins have also been reported to be involved in the invasionand metastasis of cancer as components and targets of thecytoskeleton Hoï¬mann exhibitThe PDLIM5 splicing isoformstissuespecificexpression the long isoforms are widely expressed in varioustissues while the short isoforms are only highly expressedin cardiac and skeletal muscle Yamazaki Thisdiï¬erential expression of PDLIM5 may be related to theirdiï¬erent roles in diï¬erent tissues and an systems Table Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5TABLE List of different disease involved in PDLIM5Disease and developmentReferencesRelated signalingpathwaysRASERKAMPKPKCMicroRNA17ˆ¼Papillary thyroid carcinomaProstate cancerWei Koutros Liu Li Gastric cancernonsmall cell carcinomaEdlund Cancer associated with steroid use Wang Heart developmentCardiomyocyte hypertrophyTGFSmadPulmonary hypertensionDilated CardiomyopathyBipolar disorderDepressive disorderSchizophreniaAlcohol dependence type diabetes and hypertensionNakagawa Yamazaki Bang Chen Cheng Cheng Zhao Liu Numata Owusu THE DIFFERENTIAL ROLES OF PDLIM5IN VARIOUS AN SYSTEMSThe Role of PDLIM5 in the NervousSystemPDZ and LIM domain is widely expressed in diï¬erent regionsof the brain such as the hippocampus thalamus hypothalamuscerebral cortex and amygdala MaenoHikichi Incentral neurons PDLIM5 is mainly localized in the membraneand cytoplasm where it regulates neuronal calcium signaling andcolocalizes with neurotransmitterprotruding vesicles Numata these observations indicate that PDLIM5 playsa role in brain development Some studies have shown thatthe expression of PDLIM5 is associated with multiple mentaldisorders such as bipolar disorder major depression andsusceptibility to schizophrenia Liu Zhao Herrick Wang In the nervous system PDLIM5 plays an important rolein the formation of nerve growth cones and promotes thediï¬erentiation of nerve cells Some studies have shownthat PDLIM5 expression is upregulated during neuraldiï¬erentiation and it has been shown that its ectopic expressionin neuroblastoma cells leads to the translocation of ID2 whichis one of the four members of the ID protein family called adiï¬erentiation inhibitor from the nucleus to the cytoplasmresulting in the inactivation of transcriptional and cellcyclepromoting functions ofthe latter Lasorella and Iavarone Furthermore PDLIM5 can form large complexes withPKC and Ntype Ca2 channels to promote the regulationof Ntype calcium channel activity MaenoHikichi Ren showed that PDLIM5 and PKCεcoexist in the nerve growth cone Through interaction withαactinin PDLIM5 may be involved in regulating microfilamentremodeling in neurons and the formation of the PDLIM5PKCεcomplex in the nerve growth cone which acts as a scaï¬old tomediate PKCε translocation to the membrane during PMAinduced growth cone collapse It is suggested that PDLIM5participates in a variety of functions of the nervous systemas well as in a signaling pathway involving the sequestrationof the transcription factor ID2 in the cytoplasm Howeverthe precise mechanisms by which PDLIM5 regulates thefunctions of the nervous system via ID2 blockade requiresfurther elucidationPDLIM5 in the Heart and Skeletal MusclePhysiological Roles of PDLIM5 in the HeartThe heart undergoes development and begins to function in theearly stages of embryonic development PDLIM5 is expressedat high levels in the skeletal muscle and myocardium and isconsidered to be a heart and skeletalmusclespecific scaï¬oldprotein to regulates mouse heart development Mu PDLIM5 is capable of binding to αactinin throughthe PDZ domain and colocalizing in the zdisk region ofcardiomyocytesindicating that this protein plays a role incardiac development Studies have shown that PDLIM5 mRNAsare mainly expressed in the heart and skeletal muscle of adultrats and that PDLIM5 acts as a scaï¬old protein to mediatethe transmission of PKC signals in cardiomyocytes playing animportant role in development of the muscle cell in an earlydevelopmental stage Nakagawa Zheng The PDZLIM protein family is highly expressed in the striatedmuscle PDLIM5 is similar to other PDZLIM members in thestriated muscle in which the PDZ domain binds to αactininwhile the LIM domain binds to several protein kinases C andprotein kinase D Kuroda Nakagawa For example PDLIM5 traditionally activates the PKC throughthe direct binding of its LIM domain Maturana and interacts to PKA Lin Transcription factorCREB which is one of the first transcription factors activated byneurohumoral factors stimulation is a transcription factor cAMPresponse element binding protein is a known target of the PKCand protein kinase D1 PKD1 pathways Thonberg Ozgen The interaction between PDLIM5 isoform and CREB is necessary for the phosphorylation of CREB at theaminoacid residue Ser133 which promotes the transcriptionalactivation and nuclear localization of CREB the phosphorylatedCREB enters the cardiomyocyte nucleus to play the role oftranscription factor and promote the growth of cardiomyocytesIto Moreover in neonatal rat cardiomyocytesPDLIM5 interacts with PKD1 through its LIM domain and formscomplexes with PKD1 and cardiac Ltype voltagegated calciumchannelα1C subunits to regulate the activity of Ltype voltagegated calcium channels Maturana Although theformation of protein complexes such as PDLIM5PKCPKD1is well understood the downstream molecular events remainto be elucidatedThese results suggest that the localization of PDLIM5 at somesubcellular sites and its ability to interact with multiple functionalproteins play an important role in cellular and physiologicalfunctions Furthermore the role of PDLIM5 in the heart wasFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5studied using a heartspecific PDLIM5knockout mouse modelIt was found that the ablation of PDLIM5 disrupted the stabilityof the PDLIM5CiphersCalsarcin complex formed in the zdiskregion thus interfering with the connection between adjacentsarcomeres and extracellular matrix These eï¬ects were found toresult in the loss of optimal force transmission and a significantdecrease in cardiac shortening fractionleading to dilatedcardiomyopathy Cheng Novel polymorphisms inthe PDLIM5 gene encoding the Zline protein have also beenshown to increase the risk of idiopathic dilated cardiomyopathyWang that underpinsPDZ and LIM domain is additionally involved in skeletalmuscle development Myogenesis is an important biologicalprocessskeletal muscle regeneration andpostnatal growth The silencing of PDLIM5 increases the nuclearaccumulation of diï¬erentiation inhibitor Id2 which inhibits theproliferation and diï¬erentiation of myoblasts Nakatani Qiu In addition the diï¬erentiation of andmorphological changes in skeletal muscle is regulated by a groupof transcription factors known as myogenic regulators PDLIM5isoform overexpression leads to the upregulation of MyoDand myogenin while PDLIM5 isoform knockout significantlydecreases the expression of these two proteins these findingsindicate that the main eï¬ect of PDLIM5 isoform on musclecells is to stimulate the transcription of MyoD andor myogeninencoding genes Ito Although the main role of PDLIM5 is as a specific scaï¬oldprotein which to bridge the connection between cytoskeletonand membrane proteins and promote the formation of proteincomplexes it is capable of generating numerous splicing isoformsENH24 that exert various eï¬ects on the development ofheart and skeletal muscle In vitro PDLIM5 isoform hasbeen shown to promote the expression of myogenic genes andmyotube formation while the short PDLIM5 isoform hasbeen found to abrogate myotubelike morphological changeswithout altering the expression of the myogenic transcriptionfactors MyoD and myogenin Ito Furthermorethe overexpression of PDLIM5 isoform prevented ventricularcardiomyocyte hypertrophy induced by vascular stress hormonesYamazaki Western blotting analysis of muscle tissueusing a nonisoformspecific antiPDLIM5 antibody showed thatPDLIM5 isoform is only expressed in skeletal muscle witha specific distribution of PDLIM5 members between skeletalmuscle and myocardium Niederlander These results suggest that PDLIM5 plays a key role in thedevelopment of the myocardium and skeletal muscle Howeverthe signal transduction mechanisms underlying the role ofPDLIM5 in heart and skeletal muscle remain to be furtherstudied For example the specific molecular mechanisms bywhich PDLIM5 regulates the development of myocardium andskeletal muscle through binding protein kinases are unknownFurthermore the mechanisms by which PDLIM5 sequestersnuclear protein Id2 in the cytoplasm remain to be elucidatedThe Role of PDLIM5 in Cardiovascular DiseasesPDZ and LIM domain is mainly distributed on the zline ofthe sarcomere of cardiomyocytes therefore the eï¬ect of PDLIM5on myocytes may be related to the contractile function of thesecells PDLIM5knockout mice exhibit dilated cardiomyopathywhich is characterized by thinning of the left ventricular wallenlargement of the left ventricular cavityimpaired cardiaccontraction and reduced ejection function Cheng PDLIM5 can regulates vascular remodeling which canas a new proatherosclerotic factor to be a therapeuticallytargeted Huang Cardiac remodeling which isindicative of progression in many cardiovascular diseases ischaracterized by cardiomyocyte hypertrophy and myocardialfibrosis which lead to heartfailure Swynghedauw Barry and Townsend microRNA miR21 derivedfrom cardiac fibroblasts exosomes is a strong paracrine RNAmolecule that induces cardiomyocyte hypertrophy Thum Recentlyit has been reported that PDLIM5 is thedirect target of miR17ˆ¼ Bang Chen By acting on its target gene PDLIM5 miR participates in the interaction between cardiac fibroblastsand cardiomyocytesthus inducing myocardial pathologicalhypertrophy Bang PDLIM5 also plays a role invascular smooth muscle AMPactivated protein kinase is anintracellular energy receptor of AMPK which is activatedunder hypoxia ischemia glucose loss and stress Steinberg andKemp AMPK is generally considered to be an energysensor kinase and requires AMP for its activation Carling Nakano reported that AMPK controls microtubuledynamics by phosphorylating cytoplasmic connexin170 CLIPthus regulating cell migration Nakano In addition AMPK is involved in the regulation of actincytoskeleton dynamics and plasma membrane reanizationBae Kondratowicz Studies haveshown that AMPK activation plays an important role inneovascularization and metastasis HoyerHansen and Jaattela In vascular smooth muscle cells AMPK phosphorylatesPDLIM5 at Ser177inhibiting the downstream Rac1Arp23signaling pathway to mediate cell migration Yan In pulmonary artery smooth muscle cells PASMCsSMCspecific knockout of PDLIM5 enhances hypoxiamediatedvascular remodeling while overexpression of PDLIM5 inhibitsthe TGFSmad signal pathway and prevents hypoxiainducedpulmonary hypertension elevation in vivo Cheng In addition PDLIM5 silencing induces the activity of TGF3 TR1 and TGF and increases the overall expressionlevel of Smad2 The suppression of PDLIM5 has beenfound to enhance the nuclear staining of Samd23 Chen indicating that PDLIM5 participates in thedevelopment of cardiovascular disease by negatively regulatingthe TGF3Smad23 signal pathway Additionally demonstratedthat PDLIM5 plays an important role in the cardiovascularsystem through miRmediated regulation of the phenotype ofpulmonary artery smooth muscle cells miR17ˆ¼ regulatesthe diï¬erentiation of PASMCs through its target PDLIM5indicating that the miR17ˆ¼92PDLIM5TGFSmad pathwayis essential for vascular remodeling during the developmentof pulmonary hypertension PDLIM5 therefore represents apromising therapeutic target for future cardiovascular drugdiscovery eï¬ortsFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5The Role of PDLIM5 in TumorAs an actin adaptor protein PDLIM5 is not only involved incytoskeletal tissue cellular processes and an development butis also considered to play roles in tumorigenesis and developmentEeckhoute Edlund Heiliger Li PDLIM5 is expressed in many cancer celllines In a study of neurologic tumor it was found that thetranscription factor ID2 binds to the PDLIM5 LIM domains andin these cancer cells high levels of PDLIM5 sequester ID2 in thecytoplasm preventing neuronal diï¬erentiation and promotingcell proliferation Lasorella and Iavarone PDZ and LIM domain is additionally upregulated inpapillary thyroid carcinoma PTC tissues elevated PDLIM5expression promotes the migration invasion and proliferation ofPTC cells by activating the RASERK pathway Wei PDLIM5 may therefore serve as a therapeutic target in a varietyof cancers It can promote the invasion and metastasis of cancercells Genotyping chip detection experiments have shown thatPDLIM5 is overexpressed in prostate cancer tissue Koutros and some studies have proved that the utility of serumand urine PDLIM5 levels as indicators for auxiliary diagnosisof prostate cancer with potential value in predicting the risk ofprogression in advanced prostate cancer PCA Ma PDLIM5 plays a key role in regulating the proliferation invasionand migration of malignant tumor cells by binding to AMPKand regulating its activation and degradation Liu PDLIM5 may therefore act as an oncogene in the developmentand progression of PCAIn view of the important role of PDLIM5 in cancer somestudies have indicated that it is involved in the growth of gastriccancer cells and suggested that PDLIM5 silencing through theuse of small interfering RNAs siRNA may be a potentialstrategy for the treatment of gastric cancer Li In addition Edlund found that the increased expressionof PDLIM5 is related to high tumor proliferation rates innonsmall cell carcinoma Edlund Steroids suchas corticosteroid medications play an important role in thedevelopment of cancer it has been reported that singlenucleotide polymorphisms SNPs in PDLIM5 interact withsteroids thus aï¬ecting the occurrence and development of cancerWang The above findings show that PDLIM5 plays an important rolein the occurrence and development of cancer and that PDLIM5represents a candidate oncogene in various cancersThe Role of PDLIM5 in Other DiseasesreportedIn addition to playing a role in the diseasesinvolved in the link between alcoholabove PDLIM5 isdependence and diabetes Owusu found thatPDLIM5 gene polymorphism is associated alcoholdependentAD type diabetes T2D and hypertension and Severalgenetic variants of the PDLIM5 gene can aï¬ect AD T2Dand hypertension indicating that PDLIM5 is a shared geneamong the three diseases Therefore elucidation oftheunderlying molecular mechanisms and identification of hithertoundiscovered molecular functions of PDLIM5 are expectedto enable the development of eï¬ective clinicalfor these diseasestherapiesIn addition PDLIM5 plays a role in the formation of cellextensions Being a scaï¬old protein PDLIM5 is involved inpromoting the activity of microfilamentassociated proteinsMicrofilamentassociated proteins play a central role in theprocess of cell extension Lanier Some studieshave found that PDLIM5 recruitment to cell extensions and isnecessary to form these extensions and that PDLIM5 knockoutreduces the assembly of actin filaments in cell extensionsYuda THE RELATIONSHIP BETWEEN PDLIM5AND INTEGRINS AND ITS POTENTIALROLE IN THE REGULATION OF STEMCELL DIFFERENTIATIONSome studies have shown that the PDZ domain of PDZLIMprotein binds to αactinin protein at the adhesion junctionwhich is the site of integrin localization Xia Pomies Vallenius Tamura The functional interaction with integrin indicates that PDZLIM protein can participate in the adhesion signal cascadewhich transmits extracellular signals via intracellular regulatorypathwaysthereby modifying the actin cytoskeleton Thesefindings show that the PDZLIM protein plays an overall role incell“cell and cell“matrix interaction and cell migration Krcmery The regulation of PDZLIM activity plays animportant role in preventing uncontrolled actin recombinationproliferation and cell migration For example PDLIM5 canalso bind to the integrin protein kinase ILK acting as ascaï¬old bridge between renal ion exchanger KAE1 and ILKproviding a bridge between KAE1 and potential microfilamentsSu It has been reported that PDLIM5 is recruited from thecytoplasm to the integrin adhesion and Factin stress fibersand responds to stress by directly binding to the key stressfiber component αactinin Microfilaments control the nuclearand cytoplasmic localization oftranscriptional coactivatorsYAP and TAZ to regulate gene expression and mediate thediï¬erentiation of MSCs Dupont Halder The eï¬ective domains of some proteins that are recruited intothe actin structure in a forcedependent manner through theLIM domain regulate actin signal transduction Smith The action of mechanical force on integrin results in therecruitment of PDLIM5 to activate the YAP pathway duringmechanical transduction Elbediwy PDLIM5 acomponents of the integrin adhesion complex mediates therelationship between integrin and the cytoskeleton Horton et al2016ab Proteomic analysis of integrinrelated complexes fromMSCs has demonstrated the formation of significant amountsof a vinculinpositive adhesion complex on a hard substratecoated with fibronectin FN in MSCs a subset of whichcolocalized with or closely to clusters of PDLIM1 or PDLIM5Ajeian Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5PDZ and LIM domain undergoes tensiondependentrelocalization in cells Both embryonic and induced pluripotentstem cells can diï¬erentiate into derivatives of the third germlayer as a result human pluripotent cells are important toolsin regenerative medicine Thomson Takahashiand Yamanaka Studies have reported that duringcardiogenesis embryonic stem cells exhibit dramatic changes inthe expression of metabolic enzymes and cytoskeleton proteinsKonze in particular Zdisk related proteins withPDZ and LIM domain proteins including PDLIM5 are upregulated during cardiogenesisFurthermorethe expression of NKX25 an importantmyocardial transcription factor results in the generation ofspecific PDLIM5 splicing variants during the early developmentof cardiomyocytes which in turn aï¬ectsthe myogenicdiï¬erentiation of myocardium Konze At presentthere are few studies on PDLIM5 in stem cells elucidation of itsmechanism in diï¬erent stem cell types is warranted to identify itsfunctions in these cellsPROBLEMS AND PERSPECTIVESIn this we briefly reviewed the known functionsof the PDLIM5 protein the progress in elucidation ofitsroles in various cellular and physiological processes and thesignaling pathways in which it participates As a member ofthe PDZLIM protein family PDLIM5 is involved in actinbinding αactinin binding protein kinase binding acts asa scaï¬old bridge between connective proteins and plays anindispensable role in various cellular processes However so farthe specific molecular mechanisms underlying the functions ofPDLIM5 remain unclearFuture research directions in investigation of PDLIM5 shouldseek to answer the following questionsFirst research on PDLIM5 has mainly focused on its roles intumor the nervous system and the cardiovascular system As amicrofilamentassociated protein does PDLIM5 play the samerole in other physiological systems or cell lines Is the PDLIM5gene expressed in multiple systemsREFERENCESAjeian J N Horton E R Astudillo P Byron A Askari J A and MillonFrmillon A Proteomic analysis of integrinassociated complexes frommesenchymal stem cells Proteomics Clin Appl “ 101002prcaBach I The LIM domain regulation by association Mech Dev “ 101016S0925477399003147Bae H B Zmijewski J W Deshane J S Tadie J M Chaplin D D andTakashima S AMPactivated protein kinase enhances the phagocyticability of macrophages and neutrophils FASEB J “ fj11190587BagheriYarmand R Mazumdar A Sahin A A and Kumar R LIMkinase increases tumor metastasis of human breast cancer cellsvia regulationof the urokinasetype plasminogen activator system Int J Cancer “ 101002ijc21650Bang C Batkai S Dangwal S Gupta S K Foinquinos A and HolzmannA Cardiac fibroblastderived microRNA passenger strandenrichedSecond although the role of PDLIM5 in diseases hasbeen studied eg its expression is upregulated during tumordevelopment the specific mechanisms by which it exerts theseeï¬ects are unknown and further elucidation of the underlyingmechanisms and other functions is warrantedThird PDLIM5 has four splicing isoforms which performdiï¬erent functions what are the mechanisms by which they playdiï¬erent roles Are these diï¬erences in their roles attributable tothe presence or absence of LIM domains Additionally what isthe mechanism by which they play diï¬erent rolesFourth in regard to strategies used for the inhibition ofPDLIM5 expression only viral transfection has been reportedto date no pharmaceutical compounds that inhibit PDLIM5expression have been identified Therefore additional research onPDLIM5 inhibitors is also criticalFinally although PDLIM5 plays a crucial role in binding actinand has attracted much attention as a connecting protein thereare few studies on its eï¬ects on the actin cytoskeleton or othercytoskeleton such as binding to cytoskeletonrelated proteinsbridging the connection with the cytoskeleton Does PDLIM5aï¬ect the shape and location of the cytoskeleton in the processof participating in the biological functions of cellTheeï¬ects of PDLIM5mediated modulation ofthecytoskeleton on cell diï¬erentiation proliferation and othercellular functions remain to be explored in detail in future studiesAUTHOR CONTRIBUTIONSAll authors participated in the conception and writing of themanuscript SZ JO and JD reviewed and suggested modificationsto the content JD designed the structure of the reviewFUNDINGThis work wassupported by the National Key RDProgram of China grant number 2017YFC1105000 andthe Sanming Project of Medicinein Shenzhen grantnumber SZSM201612019exosomes mediate cardiomyocyte hypertrophy J Clin Invest “ 101172JCI70577Barry S P and Townsend P A What causes a broken heart“molecularinsights into heart failure Int Rev Cell Mol Biol “ S1937644810840031Carling D Mayer F V Sanders M J and Gamblin S J AMPactivatedprotein kinase nature™s energy sensor Nat Chem Biol “ nchembio610Chen T HuangJ B DaiJ Zhou Q RajJ U and Zhou Gthe miR17ˆ¼ signaling that PAI1 is a novel component ofregulates pulmonary artery smooth muscle cell phenotypes Am J PhysiolLung Cell Mol Physiol L149“L161 101152ajplung00137Chen T Zhou G Zhou Q Tang H Ibe J C and Cheng H Loss of microRNA17 approximately in smooth muscle cells attenuatesexperimental pulmonary hypertension via induction of PDZ and LIM domain Am J Respir Crit Care Med “ 101164rccm2014050941OCFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5Cheng H Chen T Tor M Park D Zhou Q and Huang J B A highthroughput screening platform targeting PDLIM5 for pulmonary hypertensionJ Biomol Screen “ Cheng H Kimura K Peter A K Cui L Ouyang K and Shen T Lossof enigma homolog protein results in dilated cardiomyopathy Circ Res “ 101161CIRCRESAHA110218735Dawid I B Breen J J and Toyama R LIM domains multiple roles asadapters and functional modifiers in protein interactions Trends Genet “ 101016s0168952598014243Dupont S Morsut L Aragona M Enzo E Giulitti S and Cordenonsi M Role of YAPTAZ in mechanotransduction Nature “ 101038nature10137Edlund K Lindskog C Saito A Berglund A Pontn F and G¶ranssonKultima H CD99 is a novel prognostic stromal marker in nonsmallcell lung cancer Int J Cancer “ 101002ijc27518Eeckhoute J A celltypespecific transcriptional network required forestrogen regulation of cyclin D1 and cell cycle progression in breast cancerGene Dev “ 101101gad1446006Elbediwy A Vanyai H DiazdelaLoza M Frith D Snijders A P andThompson B J Enigma proteins regulate YAP mechanotransductionJ Cell Sci 131jcs221788 101242jcs221788Fanning A S and Anderson J M PDZ domains fundamental buildingblocks in the anization of protein compl

Thyroid_Cancer

"Ovarian cancer is the second most common gynecologic cancer with high mortality rate andgenerally diagnosed in advanced stages The 5year diseasefree survival is below MicroRNAs subset of thenoncoding RNA molecules regulate the translation in post transcriptional level by binding to specific mRNAs topromote or degrade the target oncogenes or tumor suppressor genes Abnormal expression of miRNAs were foundin numerous human cancer including ovarian cancer Investigating the miRNAs derived from the peripheral bloodsamples can be used as a marker in the diagnose treatment and prognosis of ovarian cancer We aimed to findbiological markers for early diagnosis of ovarian cancer by investigating BRCA1 gene mutation carrier monozygoticdiscordant twins and their high risk healthy family individual™s miRNAsMethods The study was conducted on monozygotic twins discordant for ovarian cancer and the liquid biopsyexploration of miRNAs was performed on mononuclear cells that were isolated from the peripheral blood samplesThe miRNA expression profile changes in the study were found by using microarray analysis miRNA isolationprocedure performed from the lymphocyte in accordance with the kit protocol The presence and quality of theisolated miRNAs screened by electrophoresis Raw data logarithmic analysis was studied by identifying thethreshold normalization correlation mean and median values Target proteins were detected for each miRNA byusing different algorithmsResults After the comparison of monozygotic discordant twins for epithelial ovarian carcinoma upregulation of the miRNAs miR6131 miR1305 miR1973p miR3651 and downregulation of miRNAs miR3135b miR4430 miR664b5p miR7663p were found statically significantContinued on next page Correspondence hy2188istanbuledutrDepartment of Cancer Genetics Istanbul Faculty of Medicine OncologyInstitute Istanbul University Istanbul Turkey The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cTuncer Journal of Ovarian Research Page of Continued from previous pageConclusions The detected miRNAs out of miRNAs might be used in the clinic as new biological indicatorsin the diagnosis and follow up of epithelial ovarian cancer with complementary studies The miRNA expressionprofiles were identified to be statistically significant in the evaluation of ovarian cancer etiology BRCA1 mutationstatus and ovarian cancer risk in accordance with the obtained dataThere is a need for validation of the miRNAs which were particularly detected between monozygotic twins and itsassociation with ovarian cancer was emphasized in our study in wider cohorts including ovarian cancer patientsand healthy individualsKeywords Monozygotic twins miRNA expression profiles BRCA1 and BRCA2 BiomarkersBackgroundOvarian cancer is a significant cause of mortality in gynecologic cancers and one ofthe leading cause ofcancerassociated mortality [] In Turkey ovarian cancer is the 7th most common type of cancer in women inaccordance with worldwide Globocan data™s showthat each year more than women are diagnosedwith ovarian cancer OC worldwide and approximately women die from it The data of Globocan for Turkey shows that annually women are diagnosed with ovarian cancer and women die fromthis malignancy The 5year survival rate was given as These data revealed that ovarian cancer is an important reason of gynecologic cancer associated mortality rate [] The epithelial ovarian cancersEOCoriginating from the ovarian surface epithelium constitutes approximately of ovarian malignancy [] Themajority of EOC patients are diagnosed in advanced stages Stage III and IV and year freesurvivalrate is below [] The standard treatment for newlydiagnosed ovarian cancer is the combination of cytoreductive surgery and platinbased chemotherapy Significant advances in radical surgery and chemotherapystrategies have improved clinical outcomes but unfortunately no progress has been made with relapse andtreatment resistance [] Ninety percent of ovarian cancer occurs sporadically in the population whereas hereditary type appears of ovarian cancer patientsBRCA1 and BRCA2 genes are the most common breastovarian cancer syndrome associated genes Both BRCA1and BRCA2 have roles in the control of the genomic stability cell cycle and apoptosis The mutations occurringin these genes result with the inability of DNA repairand therefore results in the accumulation of the mutations in the cell The rate of the breast cancer susceptibility of women with BRCA1 gene mutation until theage of years was and the rate of ovarian cancersusceptibility rate is breast cancer susceptibilitywomen with BRCA2 gene mutation until the age of years is and ovarian cancer development risk is [] Twin studies became important on genetics by theendandcentury Geneticnineteenthofthethe differentiated genesepidemiologic studies with monozygotic twins were accepted as highly useful investigation models in the pastdecades and have been used recently [] When a similarity for a disease or a quantitative feature betweenmonozygotic and dizygotic twins is compared variationsare excluded according to studies conducted in thepopulation and thereforeit is easier to identify andmake etiological differences visible via twin studies Because the affected siblings and dizygotic twins share theapproximately ofthephenotypic differences between twins are known to beassociated with the genetic variation In addition diversity may be revealed with a very limited patient population Therefore the results of the twin studies can beapplied to the population and can make valuable contributions to the genetic studies Monozygotic twins aregenetically similar and generally expected to be compatible for congenital malformations chromosomal abnormalities and Mendelian disorders There are numerousstudies conducted via discordant monozygotic twins revealing the genetic contribution [] Therefore investigating the genetic variability in monozygotic twins ishighly important and the majority of the human geneticsassociated research focus on finding genetic variability indiscordant monozygotic twins Phenotypically discordantmonozygotic twins are used as the model systems inidentification of the variable in understanding the pathogenesis of a disease The most striking study is the oneconducted with monozygotic twins in Canada and evidencing that multiple sclerosis MS was a genetic disease [] MicroRNAs are one of the subset of the noncoding RNAs generally consisting of single strand in “ nucleotide length not transformed to protein havingroles in post transcriptional regulation or suppression oftranslation of the target mRNAs [ ] The regulatoryroles of miRNAs were demonstrated to occur in tumorigenesis cell differentiation proliferation and apoptosis[“] miRNA genes are known to locate in thechromosomal breaks This DNA breaks cause chromosomal abnormalities frequently associated with cancersusceptibility and tumor development [“] The noninvasive biologicalindicators have been used for the 0cTuncer Journal of Ovarian Research Page of treatment resistance of ovarian cancer The most common of this indicators are the cancer antigen125 CA and cancer antigen153 CA153 These biological indicators can be used in the followup of thetreatment response in the diagnosed patients but cannotbe used in the early diagnosis and in differentiation ofthe malignant disease [] Therefore there is a need forspecial therapeutic agents customized for patients thatmay be used target specific therapies and in the earlydiagnosis of the ovarian cancer in identification of theefficacy of therapy and in the follow up period Thusstudies investigating the target molecules and biologicalindicators are required that will enable the early diagnosis and in the development of the better therapy optionsDifferentially synthesize miRNAs such as miR ˆ’ miR141 miR125b miR2223p or let7 family has beenshown in studies with ovarian cancer patients [] However the use of these miRNAs as a biomarker in ovariancancer is not yet available In order to clearly define therole of miRNAs in the pathogenesis of ovarian cancerwe planned to investigate the BRCA mutant monozygotic twins with the same genetic profile but with discordant for ovarian malignant transformation In thisstudy miRNAs which are thought to have the potential biological indicator role were studied from bloodsamples of both discordant monozygotic twins andBRCA wild type healthy siblingsMethodsPatients recruitmentThe peripheral blood lymphocytes of monozygotic twinsdiscordant for ovarian cancer and healthy individuals inthe same family were used in the study The patient diagnosed with ovarian cancer and all family members applied to the Cancer Genetics Clinic of OncologyInstitute of Istanbul University for BRCA breast cancersusceptibility gene testing were examined for BRCAgene mutation All family members in the study consisted of highrisk individuals with Hereditary Breast andOvarian Cancer HBOC syndrome and the people included in the study were given as BR codes according topatient file number The monozygotic ovarian cancer patient healthy monozygotic twin healthy sisters and niece were found to have BRCA1 gene mutation c5266dupC pGln1756Profs74 rs397507247 on exon Thepatient™s brother and daughter were found negative forBRCA1BRCA2 gene mutations In this study lymphocyte cells separated from peripheral blood belonging tototal of cases including younger age ovarian cancer patient and healthy monozygotic twin a patient™s daughter elder sisters a younger sister a nephew and a brotherwere examined by miRNA microarray method Thepedigree of the family included in the study and theirhierarchical cluster analaysis via Euclidean method isshown on Fig The study was approved by the Ethics Committee ofthe Istanbul Faculty of Medicine Following InstitutionalEthics Committee approval informed consents were obtained from all participants before enrollment into thestudy Ethics Committee Approval Number atstoredthey wereLymphocyte and miRNA isolationFicoll SigmaAldrich Darmstadt Germany density gradient was used to separate white blood cells mononuclear cells from other blood components miRNAisolation procedure was performed from the lymphocytein accordance with the kit protocol using the miRNeasyMini Kit Qiagen cat NoID The proceduresteps in accordance with the protocol are as follows μL QIAzol solution was included on the cells storedin nitrogen tank Cell fractionation was enabled by mixturing using the vortex For complete nucleoproteinfractionation °C roomtemperature for min By adding μL chloroformthey were shacked and mixed on hand The tubes wereincubated for “ min at °C room temperature thenwere centrifuged for min at °C and g Thesupernatant formed after centrifugation was transferredto collection tube using a pipette and was mixed usingvortex by inclusion of μL ethanol The supernatant formed after the ethanol centrifuging was transferred to collection tube was removed with a pipettewas mixed with vortex by including μL ethanol Seven hundred microliters was taken from the obtained mixture and was transferred to the RNeasyMiniElute spin colon placed on mL collection tubeThe tubes were centrifuged for s at g at °Croom temperature Seven hundred microliters RWT buffer was added to spin colons and the colons werewashed by centrifuging at g for sThe centrifuging procedure was repeated by including μL RPE buffer twice consecutively to colons The colons placed into clean tubes with mL were dried bycentrifuging min in maximum speed The colonsplaced in mL sterile tubes were included μL distilled water by centrifuging at g in min and themiRNAs were collectThe quality control of the miRNAsThe presence and quality of the isolated miRNAs werescreened by electrophoresis at V on Agarose gelThen the purity and concentrations of the miRNAswere measured on Thermo Scientific NanoDrop spectrophotometer NanoDrop Technologies Wilmington DE USA device The miRNA purity for each persondevicemeasurement result were obtained with the comparisonaccordance withthe NanoDropin 0cTuncer Journal of Ovarian Research Page of Fig The pedigree of the family included in the study and their hierarchical cluster analysis Legend The pedigree of the family and using thecorrelations between samples plotted a dendrogram for sample grouped by Hierarchical clustering Euclidean distance Complete Linkage BR Healthy Brother BRCA1 negative nonBRCA1 mutation carrier BR Healthy Niece BRCA1 positive BRCA1 mutation carrier BR Healthy Daughter BRCA1 negative BR Healthy Monozygotic twin BRCA1 positive BRCA1 mutation carrier BR Monozygotic twindiagnosed with ovarian cancer BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrierof the measurements at spectrophotometrically at nm and nm wave lengths The measurement rates at nm wave lengths is a sign of quality of the purity of the samples therefore the samples in the idealvalue interval of and for RNA measurementswere included in the study The purity of miRNAs wereevaluated using a Bioanalyser device BioanalyserAgilent Technologies Santa Clara CA USA AgilentRNA Nano Kit Agilent Technologies Santa ClaraCA USA for confirming whether the miRNAs were appropriate and in adequate level for microarray analysisThe evaluated sample concentrations and results wereanalyzed The samples with RNA concentrations between ngμL and rRNA rate over and RNA integrity number values between and were evaluated asthe appropriate samples for array study 0cTuncer Journal of Ovarian Research Page of Microarray trial protocolMicroarray protocol was performed by preparing theSpikein solution sample marking hybridization sampledephosphorylation sample denaturation sample ligationhybridization of the samples slide loading preparationof the hybridization unit and elution and scanning ofslides The slide scanning procedure was performedusing the Agilent Microarray Scanner Agilent Microarray Scanner with Surescan High Resolution Technology Agilent Technologies Santa Clara CA USAdevice The scanning procedure of the slides were performed on SurePrint G3 Human miRNA MicroarrayRelease 8x60K Agilent Inc Santa Clara CA platform and using the Agilent Technologies G2600D scanning protocol The analysis of the œTIFF™ Tagged ImageFile Format extensioned files obtained after scanningprocedure was performed using the Agilent Feature Extraction v11011 programThe success levels of stages developed in all experiment process with this analysis program the quality ofthe levels the process were monitored and evaluatedThen Bioinformatic Analysis procedure was performedData analysisRaw data logarithmic analysis was studied by identifyingthe threshold normalization correlation mean and median values Then the miRNAs demonstrating differentexpression profile among the samples were filteredUsing the Fold change rates and independent twosample T test the possible difference between the compared groups were evaluated All evaluations were performed to enable the cutoff values as the foldchangerates FC ‰¥ and pvalue Hierarchical clusteranalysis was performed using the Euclidean method Fig and Complete Linkage cluster method The control ofthe experimental errors and the detection of the erroneous finding rate were identified using the HochbergmethodBioinformatic analysisTarget proteins were detected for each miRNA by usingtwo algorithms Targetscan71 httpswwwtargetscanvert_71 and MirdbV5 httpsmirdbmiRDBThe targeted genes thought for each miRNA wereconfirmed by also both algorithms and the miRNAtarget relations were also experimentally confirmed mirTarbase70httpsmirtarbasembcnctuedutwphpindexphp databaseComparison groupsIn the study miRNA analysis was performed at the genome level withwithout mutation in cases withwithoutovarian cancer The miRNA data was evaluated by comparing different groups in order to investigate the effectof BRCA mutation in ovarian malignancy developmentand determine the miRNAs that can be important in theovarian cancer pathogenesis In Group the monozygotic twins discordant for ovarian cancer were comparedin order to find the effects of miRNAs in the formationof ovarian cancer In Group the family members withBRCA1 mutation were compared with family memberswithout BRCA1 mutation to identify the changes ofmiRNAs expression levels according to BRCA positivityIn Group the monozygotic ovarian cancer patient withBRCA1 mutation carrier and the other healthy familymembers with mutation carrier were compared for investigate the effects of both ovarian cancer developmentand BRCA positivity on miRNAs expression level InGroup all family members were compared with ovarian cancer monozygotic twin in order to find the miRNAs that might be important in the predisposition ofovarian cancer The comparison groups also showed inTable ResultsWe identified differentially expressed comparisonof miRNAs between the groups The raw data obtainedafter experimental studies were filtered before the comparisons between the groups The upregulated or downregulated miRNAs expression levels more than foldFC and smaller than the p value p wereconsidered in evaluation and the comparisons betweenthe groups were performed based on these values Allthese comparisons were evaluated for ovarian cancer etiology BRCA1 mutation carriage and the ovarian cancerrisk Hierarchical cluster analysis of the expression of miRNAs represents sharp separations of upregulatedyellow from downregulated blue in Fig miRNAs total of miRNAs were found statistically different after the comparison of phenotypicallydiscordant monozygotic twin siblings The miRNAsmiR1273 g3p miR1305 miR1973p miR3651 miR and miR92a3p expressions were found to haveupregulated and the other miRNAs let7i ˆ’ 5p miR125a5p miR15b5p miR22 ˆ’ 3p miR3135b miRTable Comparison groups and cases in the groupsCaseGroup BR1639ControlBR1447Group BR1639BR1447BR1547BR2030BR1546BR1861BR1850BR2028Group BR1639Group BR1639BR1447BR1447BR1547BR2030BR1546BR1861BR1547BR2030BR1546BR1861BR1850BR2028 0cTuncer Journal of Ovarian Research Page of Fig Hierarchical cluster analysis of the expression of miRNAs Legend BR Healthy Brother BRCA1 negative nonBRCA1 mutationcarrier BR Healthy Niece BRCA1 positive BRCA1 mutation carrier BR Healthy Daughter BRCA1 negative BR HealthyMonozygotic twin BRCA1 positive BR Monozygotic twin diagnosed with ovarian cancer BRCA1 positive BR Healthy Sister BRCA1positive BR Healthy Sister BRCA1 positive BR Healthy Sister BRCA1 positive The miRNAs that may be effective in the etiology ofovarian cancer were identified after the comparison of monozygotic twins who were phenotypically discordant for ovarian cancer diagnosis ingroup 320d miR3423p miR4430 miR451a miR664b5pand miR7663p expressions were found to have downregulated After the bioinformatic analysis a total of upregulated and downregulated statistically significantmiRNAs and their target molecules are given in Table and Fig Different miRNAs level were compared between group in order to determine the effect of BRCA1 gene mutation Group was consisted after the comparison of theBRCA1 gene mutation carrier family members and individuals not carrying BRCA12 gene mutation accordingto miRNAs expression profiles After the comparisonsdownregulated and upregulated miRNAsrelated toBRCA1 gene mutation carrier were determined The expression of a total of miRNAs including miR4449miR46533p miR4865p miR5739 miR6165 andmiR ˆ’ 8743p associated with the BRCA1 gene mutationcarrying were upregulated and the expression of a totalof miRNAs including miR1263p miR320a miR320b miR320c miR320d miR320e miR3243p miR miR ˆ’ miR4428 miR4516 miR4741 miR miR564 miR6089 miR68695p miR68915pmiR71075p and miR78473p were found downregulated After the bioinformatic analysis a total of upregulated and downregulated statistically significantmiRNAs and their target molecules are shown in Table and Fig AftercomparisonDifferent miRNA levels were compared between group in order to determine the relation with ovarian cancerdevelopment and BRCA positivity Group consists ofcomparison of miRNAs of BRCA1 positive ovarian cancer patient with all other BRCA1 positive healthy individualsanddownregulated miRNAs related to mutation carriage inBRCA1 gene and epithelial ovarian cancer etiology weredetermined The expression of miRNAs includingmiR1260a miR1260b miR165p miR175p miR181b5p miR ˆ’ 26b5p miR4281 miR4286 miR5100miR68403p miR71145p miR7975 and miR7977were found to have upregulated and the expression of miRNAs including miR12255p miR1423p miR ˆ’26a5p miR ˆ’ miR29a3p miR30d5p miR3196upregulated 0cTuncer Journal of Ovarian Research Page of Table Ovarian cancer etiology related upregulated and downregulated miRNAs and target proteins in monozygotic twinsmiRNAsSequence of miRNAmiRNAStatusTarget genesFold change FCvaluesACCACUGCACUCCAGCCUGAGUpregulatedZNF138 TMEM239 BMP3UUUUCAACUCUAAUGGGAGAGAUpregulatedPTPN4 PRKAA1 PAPD7FRAT2 DEPDC1 FBXO41UUCACCACCUUCUCCACCCAGCUpregulatedCD82 PMAIP1 MTHFD1 CHECK1 AGO1 CASP10CAUAGCCCGGUCGCUGGUACAUGA UpregulatedGGCUGGUCAGAUGGGAGUGUpregulatedRACGAP1 OLA1 TEX261PTGS1 NFIC ZNF200PAGR1 IGF2BP1 CACNG8UAUUGCACUUGUCCCGGCCUGUUGAGGUAGUAGUUUGUGCUGUUUCCCUGAGACCCUUUAACCUGUGA Downregulated ERBB3 CDKN1A TP53 ERBB2 EGFR STAT3MYCSTAT3 PTEN ATM NOTCH2 CDH1 NFKB1UpregulatedDownregulated TLR4 BMP4 EIF2C1 NEUROG1 SOCS1 IGF1VEGFAmiR1273 g3pmiR1305miR1973pmiR3651miR6131miR92a3plet7i5pˆ’miR125a5pmiR15b5pUAGCAGCACAUCAUGGUUUACAmiR223pAAGCUGCCAGUUGAAGAACUGUDownregulated BCL2 VEGFA CCND1 CCNE1 CDK1 CDK4 CDK6 E2F3MAPK1Downregulated CDKN1A WNT1 ERBB3 MYCBP HMGB1 E2F2 PTENPOTED SOD2miR3135bmiR320dmiR3423pmiR4430miR451amiR664b5pGGCUGGAGCGAGUGCAGUGGUGAAAAGCUGGGUUGAGAGGAUCUCACACAGAAAUCGCACCCGUAGGCUGGAGUGAGCGGAGAAACCGUUACCAUUACUGAGUUUGGGCUAAGGGAGAUGAUUGGGUADownregulated BIRC5 ABL2 MAPK1 MYCNDownregulated DCTN5 SYNCRIP FBXO28Downregulated GEMIN4 DNMT1 ID4 SREBF1SREBF2 BMP7Downregulated ZNF485ABL2 MAPK1 MSH5 PTENDownregulated CPNE3 RAB5A IL6R AKT1 MMP2Downregulated CD55MSN RHOBTB3 PLAG1miR7663pACUCCAGCCCCACAGCCUCAGCDownregulated COX1 MAPK1 NF2 RAD51 STK4 STK24 VEGFCFig The upregulated and downregulated miRNAs and foldchanges associated with ovarian cancer etiology in monozygotic twins 0cTuncer Journal of Ovarian Research Page of Table BRCA1 gene mutation positivity related upregulated and downregulated miRNAs and target molecules An additional tablefile shows this in more detail [see Additional file ]miRNAsSequence of miRNATarget genesmiRNAStatusFold changeFC valuesmiR4449miR46533pmiR4865pmiR5739miR6165miR8743pmiR1263pmiR320amiR320bmiR320cmiR320dmiR320emiR3243pmiR3656miR4284miR4428miR ˆ’ miR4741miR484miR564miR ˆ’ miR6869 ˆ’ 5pmiR68915pmiR71075pmiR7847 ˆ’ 3pˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’CGUCCCGGGGCUGCGCGAGGCAUpregulatedZFHX3UGGAGUUAAGGGUUGCUUGGAGAUpregulatedATG2A CREBL2 MAT2A FRS2 TMED4 UBN2UCCUGUACUGAGCUGCCCCGAGGCGGAGAGAGAAUGGGGAGCCAGCAGGAGGUGAGGGGAGCUGCCCUGGCCCGAGGGACCGAUpregulatedUpregulatedUpregulatedUpregulatedOLFM4CD40ARHGAP5 IGF1R DOCK3 CADM1DLX6CD207CHIC1PPL2A PLXDC1PER1TFAP2AFADS1 AMER1LUZP1 COX6B1HDAC1 AQP3 STAT3 CDK9UCGUACCGUGAGUAAUAAUGCGDownregulatedTOM1 CRK VEGFA SOX2 TWF1 PITPNC1 IGFBP2 KRASAAAAGCUGGGUUGAGAGGGCGADownregulatedMCL1 BANP ITGB3 BMI1 NRP1 NFATC3 TRPC5AAAAGCUGGGUUGAGAGGGCAADownregulatedCDK6DCTN5SYNCRIPARF1 BCL9L ZNF600AAAAGCUGGGUUGAGAGGGUAAAAGCUGGGUUGAGAGGAAAAGCUGGGUUGAGAAGGACUGCCCCAGGUGCUGCUGGGGCGGGUGCGGGGGUGGDownregulatedDownregulatedSYNCRIPFBXO28SMARCC NPM3DCTN5 SYNCRIP FBXO28DCTN5 NPM3 ZNF275 DDX19A NCAPD2 TXNL1DownregulatedDownregulated WNT9B CREBBP DVL2 WNT2BDownregulatedMRPL12 LSP1 MNT PRDM2ZNF770 CECR1GGGCUCACAUCACCCCAUDownregulatedBCL2L11RBBP5HNRNPA1 ZNF264 TRIB3 CRTAPCAAGGAGACGGGAACAUGGAGCDownregulatedMSL1MAPRE3MYH14CASP2 CCND2 CDK14TP63GGGAGAAGGGUCGGGGCDownregulatedSTAT3M6PRGPR137CCCND2 CCNT1 CDKN1A SCOC TP53CGGGCUGUCCGGAGGGGUCGGCUDownregulatedDDX39BMAPK1 ZBTB39 HMGA1UCAGGCUCAGUCCCCUCCCGAUDownregulatedFIS1 PAGR1 ZEB1 SLC11A2SMAD2 ANAPC7 TBRG1AGGCACGGUGUCAGCAGGCDownregulatedGID4 CNBP E2F3 RCAN3 AKT2 APPL1 SLC1A2 GPR155GGAGGCCGGGGUGGGGCGGGGCGGDownregulatedNKX2 TPT1 KCTD5 BBX SGCD CDH7 CCNB1GUGAGUAGUGGCGCGCGGCGGCDownregulatedTUBB2AMAPK1NRBF2 WEE1HMGA2 MAPK1 STAG2UAAGGAGGGGGAUGAGGGGDownregulatedCHD4 CD207 DDX6 CHRDL1CCND2 TP63UCGGCCUGGGGAGGAGGAAGGGDownregulatedVAV3 CASP16 CCND1 CASP16 MAPK14CGUGGAGGACGAGGAGGAGGCDownregulatedHAVCR1 POTED DNAJC10 SOD2 M6PR CDK19miR3423p miR3665 miR3960 miR4466 miR4530miR46873p miR47875p miR4943p miR50015pmiR50065p miR5787 miR6068 miR6087 miR miR6090 miR6124 miR6125 miR638 miR65105p miR68005p miR7704 miR8063 and miR were found to have downregulated After bioinformatic analysis a total of upregulated and downregulated statistically significant miRNAs and the targetmolecules are given in Table and Fig For identifying the ovarian cancer predisposition allfamily members were compared with ovarian cancermonozygotic twins in group The upregulated ordownregulated miRNAs in association with the epithelialovarian cancer risk were identified after the comparisonThe expression of the miRNAs consisting of let7a5plet7b5p miR181a5p miR1973p miR215pmiR2233p miR23a3p miR27a3p miR36533pmiR4255p miR572 miR5745p miR6127 and miR were detected to be upregulated The expression ofthe miRNAs consisting of let7i5p miR ˆ’ 125a5pmiR15b5p miR1505p miR22 ˆ’ 3p miR3283pmiR4430 miR451a miR46975p miR664b5p andmiR7663p were detected to have downregulated Atotal of upregulated and downregulated statisticallysignificant miRNAs and the target molecules are givenin Table and Fig DiscussionWomen are diagnosed with ovarian cancer at an advanced stage due to limited number of biologicalmarkers for ovarian cancer patients Although existingovarian cancer biomarkers cancer antigen125 and cancer antigen153 CA125 CA15“ are sensitive in thefollowup of diagnosed gynecological cancers they have 0cTuncer Journal of Ovarian Research Page of Fig The upregulated and downregulated miRNAs and foldchanges associated with BRCA1 gene mutation positivityofearlysensitivityin the diagnosislessstagegynecological cancers and separation of malignant tumorformations from benign formations [] Therefore tounderstand the underlying mechanisms of ovarian cancer and to explore targeting drugs and to improve newtreatment protocols for ovarian malignancy revealingsignificant genetic changes is necessary The genetic andepidemiologic studies conducted on monozygotic twinsare known to provide accurate and direct informationabout the gene and environment interaction with thedisease occurrence mechanism [] The changes in genesthat result in the occurrence of tumors such as miRNAexpression level among the monozygotic twins providesinformation on the etiology of disease and may have arole as a biological indicator in identifying the early stagedisease and in the follow up of the prognosis We aimedto identify the noninvasive biological markers that maybe used in the early diagnosis of ovarian cancer throughinvestigating the miRNAs in the peripheral blood ofmonozygotic twin siblings discordant for ovarian cancerwith the miRNA molecules of the other healthy members in the family Thus that may cause less bias thanthe controls to be selected from the population Ninetynine different miRNA molecules presented in the studywere detected after the comparison of monozygotic twinsiblings who were discordant for ovarian cancer andwith the other healthy individuals Seventeen differentmiRNAs were found that could be used for detectingearly diagnosis and prognosis of ovarian cancer betweenthe monozygotic twin siblings who were discordant forovarian cancer in our study The association between out of miRNAs and ovarian carcinoma is beingreported for the first time in this study Due to the highnumber of newly detected miRNAs in our study the discussion and comparison were only made between thecandidate miRNAs Although miR1973p miR1305miR6131 miR3651 miR3135b miR4430 miR664b5p and miR7663p have not been shown to be associated with ovarian cancer in literature but limited number of studies have suggested the association with othercancersWang found the elevated level of miR1973pinthe same way as we do The upregulated miR1973p expression level was shown to promote the cellular invasion and metastasis in bladder cancer in that studyResearchers reported that LINC00312 gene was responsible for invasion and metastasis mechanisms and thisgene inhibited the cellular migration and invasion bysuppressing the miR1973p expression Similar resultswere detected in thyroid cancer in the study of Liu et al[ ] Jin reported that increased expressionlevel of miR1305 caused pluripotent stem cells to accelerate the cell cycle G1S transfer in addition to causingthe cellular differentiation with the increased miR1305expression [] The expression levels ofreducedmiRNA125a5p and let7i5p found in the scope of ourstudy have been shown to parallel with other studies inthe literature Langhe suggested thatlet7i5pmight be described as a diagnostic indicator in ovariancancer [] The miRNA125a5p expression was upregulated to inhibit the cancer proliferation and migrationin the in vitro study of Qin in human cervical carcinomas [] and miR125a5p upregulated expressionlevel was demonstrated to inhibit the cervical cancer 0cTuncer Journal of Ovarian Research Page of Table BRCA1 mutation carriage and epithelial ovarian cancer etiology related upregulated and downregulated miRNAs targetmolecules An additional table file shows this in more detail [see Additional file ]miRNAsSequence of miRNAmiRNAStatusTarget genesAUCCCACCUCUGCCACCAAUCCCACCACUGCCACCAUUAGCAGCACGUAAAUAUUGGCGUpregulatedUpregulatedUpregulatedCAAAGUGCUUACAGUGCAGGUAGUpregulatedPSAT1UNC13A RPS27 BRD7SFRP1 DKK2 SMAD4 PSAT1UNC13A RPS27CCNE1 ARL2 BCL2 HMGA1 CDK6 CCND1VEGFA RECK PRDM4TGFBR2 PTEN CDKN1A BCL2L11E2F1TP53STAT3AACAUUCAUUGCUGUCGGUGGGUUpregulatedTCL1A TIMP3 PLAG1 BCL2RNF2VSNL1 ATMFold changeFC valuesmiR1260amiR1260bmiR16 ˆ’ 5pmiR175pmiR181b5pmiR26b5pmiR4281miR4286miR5100UUCAAGUAAUUCAGGAUAGGUUpregulatedGGGUCCCGGGGAGGGGGGACCCCACUCCUGGUACCUpregulatedUpregulatedUUCAGAUCCCAGCGGUGCCUCUUpregulatedPTGS2 EPHA2 CHORDC1 EZH2CCNE1ABCA1 GATA4NCDN CDKN1A BCL3LDLR ZNF354B NSD1 RABGAP1TAOK1 MKNK2COX10 DEK KCNN3 RAB11FIP1DYNLT1 NOTCH2SLFN12L CTC1 GXYLT2GDE1FADS1PER1 ATG9AmiR68403pGCCCAGGACUUUGUGCGGGGUGUpregulatedmiR71145pUCUGUGGAGUGGGGUGCCUGUUpregulatedM6PR HNRNPUL1 SHMT1 ZNF529ACVR2B PAICS TAF8miR7975miR7977AUCCUAGUCACGGCACCAUUCCCAGCCAACGCACCAUpregulatedUpregulatedmiR12255pGUGGGUACGGCCCAGUGGGGGGDownregulatedKBTBD8GULP1 CASZ1 RAD51HSPA1B ZNF703 TMEM185BSF3B3COX6B1 CCDC9 CDH7ORC4 ODF2L MTRNR2L7PSMG2 MTRNR2L3miR1423pmiR26a5pmiR2861miR29a3pmiR30d5pmiR3196miR ˆ’ 3423pmiR3665miR3960miR4466miR4530miR46873pmiR47875pmiR4943pmiR50015pmiR50065pmiR ˆ’ miR6068miR6087miR6088miR6090miR6124ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’UGUAGUGUUUCCUACUUUAUGGADownregulatedARNTLTGFBR1 RAC1 ROCK2 CCNT2 TAB2 PTPN23UUCAAGUAAUCCAGGAUAGGCUDownregulatedEZH2RB1ADAM17 HMGA2CCND2 CPEB3 DNMT3BGGGGCCUGGCGGUGGGCGGUAGCACCAUCUGAAAUCGGUUAUGUAAACAUCCCCGAC

Thyroid_Cancer

Incidence Differences Between First PrimaryCancers and Second Primary Cancers FollowingSkin Squamous Cell Carcinoma as Etiological CluesThis was published in the following Dove Press journalClinical Epidemiology“ KristinaGuoqiao ZhengSundquist4“ Jan Sundquist AkseliAsta F¶rsti KariHemminkiHemminki12411“1Division of Molecular GeneticEpidemiology German Cancer ResearchCenter DKFZ Heidelberg D69120Germany 2Division of CancerEpidemiology German Cancer ResearchCenter DKFZ Heidelberg D69120Germany 3Faculty of Medicine Universityof Heidelberg Heidelberg Germany4Center for Primary Health Care ResearchLund University Malm¶ Sweden5Department of Family Medicine andCommunity Health Department ofPopulation Health Science and Policy IcahnSchool of Medicine at Mount SinaiNew York NY USA 6Center forCommunityBased Healthcare Researchand Education CoHRE Department ofFunctional Pathology School of MedicineShimane University Izumo Japan 7HoppChildren™s Cancer Center KiTZHeidelberg Germany 8Division ofPediatric Neurooncology German CancerResearch Center DKFZ German CancerConsortium DKTK HeidelbergGermany 9Cancer Gene Therapy GroupTranslational Immunology ResearchProgram University of Helsinki HelsinkiFinland 10Comprehensive Cancer CenterHelsinki University Hospital HelsinkiFinland 11Faculty of Medicine andBiomedical Center in Pilsen CharlesUniversity in Prague Pilsen CzechRepublicCorrespondence Kari HemminkiDivision of Cancer Epidemiology GermanCancer Research Center DKFZ ImNeuenheimer Feld Heidelberg GermanyTel Fax Email karihemminkidkfzdeBackground Most literature on second primary cancers SPCs focuses on possible factorswhich may increase the risk of these cancers and little attention has been paid for the overallincidence differences between first primary cancers FPCs and same SPCs We wanted tocompare the incidence rates for all common cancers when these were diagnosed as FPCs andSPCs after invasive and in situ squamous cell carcinoma SCC of the skin which are usuallytreated by surgery onlyMethods Cancers were identified from the Swedish Cancer Registry from the years through to and they included in addition to skin cancers male cancers totaling patients and female cancers totaling patients Standardized incidencerates and relative risks RRs were calculated for sexspecific common cancers as FPC and asSPC after skin SCC Spearman rank correlations were used in the analysis of incidenceranking of FPC and SPCResults Of total men and women developed invasive SCC and menand women in situ SCC The total number of other male cancers was andof female cancers it was Rank correlations ranged from to P5—ˆ’indicating that overall skin SCC did not interfere with SPC formation The exceptions wereincreased SPC risks for melanoma sharing risk factors with skin SCC and nonHodgkin andHodgkin lymphoma and cancers of the upper aerodigestive tract connective tissue and maleand female genitals suggesting contribution by skin cancer initiated immune dysfunctionConclusion The incidence ranking of SPCs after skin cancers largely follows the incidenceranking of FPCs indicating that overall skin SCC does not greatly interfere with the intrinsiccarcinogenic process The main deviations in incidence between FPC and SPC appeared tobe due to shared risk factors or immunological processes promoting immune responsivecancer typesKeywords skin cancer second cancer first primary cancer immune disturbancePlain Language SummaryIn this study we compared the incidence of first primary cancers and the incidence of thesame cancers as second primary cancer after squamous cell skin cancer Skin cancers aretreated by surgery which is not a risk for second cancer but skin cancers show immunological disturbances that may increase the risk of immune responsive cancers The resultsshowed that the incidence ranking of second cancer followed closely the incidence rankingof these cancers as first cancer The exceptions were cancers such as nonHodgkin lymphoma the incidence of which was increased as second cancer probably due to shared riskfactors such as immunological disturbancessubmit your manuscript wwwdovepresscomDovePresshttp102147CLEPS256662Clinical Epidemiology “ Zheng This work is published and licensed by Dove Medical Press Limited The full terms of this license are available at wwwdovepresscomtermsphp and incorporate the Creative Commons Attribution “ Non Commercial unported v30 License httpcreativecommonslicensesbync30 By accessing thework you hereby accept the Terms Noncommercial uses of the work are permitted without any further permission from Dove Medical Press Limited provided the work is properly attributed Forpermission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cZheng et alDovepressIntroductionMultiple primary cancers are known to be diagnosed incancer patients and Vogt noted that as far back as a study reported that of cancers appeared to beœof multiple growth Multiple primary cancers are considered when two or more independent tumors are diagnosed in an individual but the exact definitions differinternationally and nationally1 Multiple primary cancersinterest23have been of large etiological and clinicalHowever as the frequency of new primary cancers drastically decreases after the second primary cancer SPCmuch of the literature has focused on SPCs As examplesin prostate cancer patients SPCs account for of firstprimary cancers FPCs and third primaries account for of SPCs in melanoma the respective proportionsare and including multiple melanomas45In most studies the incidence of SPC is compared to theincidence of that cancer as FPC and hence the calculatedrelative risks RRs are used as the outcome measure Ingeneral the studies report SPCs with an increased risk forexample due to carcinogenic chemoor radiotherapiesHowever our recent studies on SPC after prostate cancersuggested that SPCs were œautonomous from prostatecancer because the frequencies of SPC correlated withthe frequencies of these cancers as FPC and the risk ofSPC was increased by the familial history of that cancerirrespective of prostate cancer46 Moreover the RRs forSPCs were equal in screening detected and other prostatecancerWe want to address the question of whether the incidence of cancer X differs when it is FPC or SPC aftercancer Y hypothesizing that a possible difference mayreveal something about cancer etiology For cancer Y weselected skin squamous cell carcinoma SCC becauseinvasive and in situ forms are common thus allowinghigh statistical power We thus assessed the incidence ofcancer X as FPC and as SPC after skin SCC In Swedeninvasive SCC ranks second among male and female cancers and in situ SCC has become more common thaninvasive SCC7 Furthermorethese cancers are usuallytreated by surgery and the patients are not subjected topotentially carcinogenic treatments8 Common risk factorsfor SCC include cumulative exposures to ultraviolet UVradiation viral infections immune dysfunctions and sunsensitive skin8“ The role of immune dysfunction is illustrated by the high risk of SCC in immunesuppressedpatients11“ We used data from the Swedish CancerRegistry to systematically compare the incidence of FPCand SPC when SPC was recorded after invasive or in situSCC the most common cancers were analyzed andtheir incidence ranking was tested by rank correlationWhile our primary hypothesis was thatthe rankingremains uniform the secondary hypothesis was to gainetiological clues about cancers that changed their rankinghistologicalidentifiersMethodsData of cancer patients were obtained from the SwedishCancer Registry based on the international classificationof diseases 7th revision ICD7 and later revisions TheRegistry is populationbased and covers practically allcancers diagnosed in Sweden1415 We identified all individuals who were diagnosed with invasive and in situ SCCwithWHOHSCANC241Histology Code œPAD and respectively Inaddition data on most common cancers were retrievedincluding male and female cancers Upper aerodigestive tract UAT included cancers in the mouth lippharynx and larynx We followed newly diagnosedin situ and invasive skin cancer patients for the diagnosisof any invasive SPC the followup for skin cancers werestarted after from the date of diagnosis until diagnosis of SPC emigration death or December whicheveragestandardized world standard population incidence ratefor cancer X as SPC was calculated Similarly a sexspecific and agestandardized incidence rate for cancerX as FPC was calculated For comparison of incidencerates RRs and the corresponding confidence intervals95CIs were calculated for SPC using the populationincidence of the same FPC as a reference and adjusting therates for 5year age group yearcalendar period socioeconomic status groups and place of residence groups in Poisson regression Correlation of ranking forincidence rates between FPC and SPC was tested bySpearman™s rank correlation rho All statistical analyseswere done with SAS version and R version All thetests were twotailed and P value below was regardedas statistically significantearliest A sexspecificoccurredThe study was approved by the Regional EthicalReview Board in Lund February without requirement for informed consent and was conducted in accordance with the tenets of the Declaration of HelsinkiPeople could opt out of the study which was advertisedin major newspapers before the project datasets wereconstructed This opting is common in Swedish publicallysubmit your manuscript wwwdovepresscomDovePressClinical Epidemiology 0cDovepressZheng et alcollected databases but opting outproject datasets are located atHealth Care Research in Malm¶ Swedenis utterly rare Thethe Center for PrimaryResultsAmong million individuals who were followed from to diagnosis of SPCemigration death or December and men and womendeveloped invasive SCC men and womendeveloped in situ SCC The total number of other malecancers was and that of female cancers was Median interquartile age at diagnosis of invasive SCC was “ years for men and “for women and that of in situ SCC was “ for menand “ for women Median interquartile timefrom first invasive SCC to SPC was “ years for menand “ for women and for in situ SCC it was “for men and “ for womenTable shows incidence rates of FPC and SPC diagnosed after invasive SCC in men The case numbers incidence rates for FPC and SPC and the related ranks are listedin columns to followed by adjusted RR for SPCcompared to FPC Among ranking upper aerodigestivecancer UAT climbed from position to position asSPC RR for UAT after skin SCC compared to UAT as anFPC was also the highest followed by melanoma Other cancers with RRs over were connectivetissue and breast cancers and nonHodgkinlymphoma NHL the RRs over were boldedRRs for all other cancers were also significantly increased CIs did not include except for myeloma andHodgkin lymphoma and for endocrine and thyroid cancersThe overall RR was The rates of common cancers in women as FPC andSPC are shown in Table when SPCs were diagnosedafter invasive SCC Among ranking melanoma NHL andUAT climbed from positions and as FPCs torespective positions and as SPCs RRs for thesecancers exceeded and respectivelyThe RR for Hodgkin lymphoma was “RRs for breast colorectallung endometrial ovarianbladder female genital and connective tissue cancers andTable Incidence of Common Cancers as First Primary Cancer and Second Primary Cancer and Respective Relative Risk RR inMenFirst Primary CancerSecond Primary Cancer After Invasive SCCCancerNumber ofCasesStandardized RateRank1 Number ofCasesStandardized RateRank2 RR CIProstateColorectumLungBladderMelanomaLeukemiaNHLNervous systemKidneyStomachUATLiverMyelomaEndocrineConnective tissueHodgkin lymphomaThyroidSmall intestineMale genitalBreastAll““Notes Skin cancer is removed from all cancers some rare cancers not listed in Table are included Bolding shows RRs200Abbreviations SCC squamous cell carcinoma NHL nonHodgkin lymphoma UAT upper aerodigestive tractClinical Epidemiology submit your manuscript wwwdovepresscomDovePress 0cZheng et alDovepressTable Incidence of Common Cancers as First Primary Cancer and Second Primary Cancer and Respective Relative Risk RR inWomenFirst Primary CancerSecond Primary Cancer After Invasive SCCCancerNumber ofCasesStandardized RateRank1 Number ofCasesStandardized RateRank2 RR CIBreastColorectumLungMelanomaEndometriumNervous systemOvaryLeukemiaCervixNHLEndocrineBladderKidneyLiverThyroidStomachUATMyelomaFemale genitalConnective tissueHodgkin lymphomaSmall intestineAll““Notes Skin cancer is removed from all cancers some rare cancers not listed in Table are included Bolding shows RRs200Abbreviations SCC squamous cell carcinoma NHL nonHodgkin lymphoma UAT upper aerodigestive tractleukemia were also significant RRs for six cancers werebelow but none of these were significant The overallRR was The rates after in situ SCC in men are shown inSupplementary Table All RRs that were over in Table were over in Supplementary Table although some RRsafter in situ SCC were somewhat smaller RRs for leukemia and Hodgkin lymphoma were somewhat higherand for male genital cancer the RR was equal comparedto the results in Table The only difference to Table was formale breast cancer the RR of which was much lower yet CIs overlapped The overall RR was Female rates afterin situ SCC are shown inSupplementary Table The results were consistent withdata in Table however the RR of for melanomawas significantly higher than the RR of for melanomain Table The overall RR was In Table we show results from incidencerankinganalysis conducted for SPCs following invasive andTable Spearman Rank Correlation Between Incidences of theFirst Primary Cancer and Second Primary Cancer After Invasiveand in situ SCCGenderSCCSpearman Rank CorrelationCoefficient rPInvasiveIn situMenWomenMenWomenAbbreviation SCC squamous cell carcinoma—ˆ’—ˆ’—ˆ’—ˆ’in situ SCC in men and women summarizing the resultsfrom the above tables Rank correlations were marginallyhigher for men than for women and higher after in situthan after invasive SCC all correlations were highly significant P5—ˆ’The results for male RRs are summarized in Figure illustrating the systematic covariation of RRs for cancerswhen diagnosed after invasive and in situ SCC UAT aftersubmit your manuscript wwwdovepresscomDovePressClinical Epidemiology 0cDovepressZheng et alFigure Relative risks RRs for second primary cancer in men after invasive and in situ SCC of the skin The error bars show confidence intervalsinvasive SCC wass a real deviation with highest of all RRsand the largest difference when diagnosed after invasiveand in situ SCC Similarly female data are shown inFigure confirming the covariation ofinvasive andin situ results and the high risk of UAT especially afterinvasive SCCDiscussionA novel set of findings was revealed by comparing theincidence ranking of SPCs appearing after skin SCC to theranking of same cancers as FPCs The ranking of FPC waslargely maintained among SPCs in men and women withrank correlations at or above and highly significantPvalues SPCs following in situ SCC showed marginallyhigher correlation than SPCs after invasive SCC and malecorrelations were marginally higher than female correlations The high correlations suggest that skin cancer doesnot influence the formation of SPCs and thus SPCs appearto be autonomous from skin cancer which seems to resemble SPCs after prostate cancer46 The higher correlationsafter in situ than invasive SCC may be rationalized byin situ being a precursor stage of shorter lifespan and sizethan invasive lesions8If ranking was identical for FPC and SPC the correlation would be A perfect ranking would be maintained if the incidence of all cancers remained stable orif systematically increased or decreased for all cancersThe overall RRs were men and womenafter invasive SCC and after in situ SCC indicating thatincidence levels were generally increased forSPCs compared to FPCs The deviation from rho100indicates deviations in ranking and thus positive or negative interference of the underlying carcinogenic processthat drives cancerindividual cancersincidence ForClinical Epidemiology submit your manuscript wwwdovepresscomDovePress 0cZheng et alDovepressFigure Relative risks RRs for second primary cancer in women after invasive and in situ SCC of the skin The error bars show confidence intervalsa positive interference would be shown by an RR100and a negative one by an RR100 We found no indication of negative interference as no single RR was significantly below This is also technically reassuringbecause a deficit in reporting of SPCs would also contribute to low RR1617 this concurs with data reportinga generally high coverage of cancers by the SwedishCancer Registry14Possible causes or contributing factors for SPCs aremany but probably the most important ones are intensivemedical surveillance after the diagnosis of FPC therapyfor FPC shared genetic or nongenetic risk factorsbetween FPC and SPC and immune dysfunction elicitedby FPC21819 In the case of skin cancer therapy is not anissue but medical surveillance probably is because SPCswere diagnosed relatively shortly after skin cancers “years which are generally diagnosed in elderly subjectsmedian diagnostic ages were “ years in this study20However as practically all cancers reported to the SwedishCancer Registry are histologically confirmed the effect ofmedical surveillance would be antedating of diagnosesrather than introducing wrong diagnosesThere was ample evidence for nonrandom positiveinterference which marked a set of particular cancersThe RRs between incidence rates showed some systematicchanges replicated between sexes and invasive and in situforms which can be visualized in Figures and Suchconsistent changes should offer some etiological cluessubmit your manuscript wwwdovepresscomDovePressClinical Epidemiology 0cDovepressZheng et alimmuneforsuppressionThe increased RRs for melanoma are likely a consequenceof shared risk factors solar radiation and sensitive skintype Melanoma is an immune responsive tumor asshown by successes in treatment with checkpoint blockingagents and immune mechanisms may also contribute tomelanoma development21 The increased RRs for NHLand Hodgkin lymphoma and cancers of the UAT connective tissue and male and female genitals may be explainedby immune dysfunction caused by skin SCC or a sharedhost risk factor These cancers are known to be related toiatrogenicantransplantation11122224 UAT and genital cancer arerelated to human papilloma virus HPVinfectionswhich are known to be intensified in immunosuppressedpatients2526 The large difference for RR in UAT betweeninvasive and in situ SCC may illustrate the higher level ofimmune dysfunction in invasive SCC probably presentingwith chronic inflammation13 Cervical cancer is anotherHPV related cancer but it showed no increase in RR thelikely reason is its generally earlier onset compared toSCC Finally the intriguingly high RR for male breastcancer after invasive SCC could be if not a fortuitousfinding due to UVinduced chronic inflammation affectingmale breast ductal system which is in intimate contact withskin different from the female breast anatomyThe study has major strengths in being able to usenationwide and histologically confirmed data on skintumors which are not recorded by most cancer registriesSPCs are still rare and for some types of SPCs statisticalpower was not high For any benign conditions such asSCC particularly in situ SCC an undefined proportion ofcases may not be reported to the Cancer Registry however the present results were not sensitive to underreporting of FPCs Nevertheless reporting of SPC would becritical to this study Importantly the present results tendedto reassuringly indicate that the reporting rate is at thesame level as that for FPCsIn summary we found high Spearman rank correlationsbetween incidences of FPC and SPCs The results supportthe notion that overall skin SCC does not greatly interferewith the intrinsic carcinogenic process for other cancersThe main deviations in incidence between FPC and SPCappeared to be due to shared risk factors or immunologicalprocesses promoting immune responsive cancer typesAcknowledgmentsWe thank Patrick Reilly for excellent language editing Thisstudy was supported by the European Union™s Horizon research and innovation programme grant No Janeand Aatos Erkko Foundation HUCH Research FundsEVO Sigrid Juselius Foundation Finnish Canceranizations University of Helsinki The Finnish Societyof Sciences and Letters and from the Swedish ResearchCouncil and Author ContributionsAll authors made substantial contributions to conceptionand design acquisition of data or analysis and interpretation of data took part in drafting the or revising itintellectual content gave finalcritically for importantapproval of the version to be published and agree to beaccountable for all aspects of the workDisclosureAH is a shareholder in Targovax ASA and an employeeand shareholder in TILT Biotherapeutics Ltd The otherauthors declared no conflict of interestReferences Vogt A Schmid S Heinimann K Multiple primary tumourschallenges and approaches a review ESMO e000172 101136esmo 2017000172 Travis LB Demark Wahnefried W Allan JM Wood ME Ng AKAetiology genetics and prevention of secondary neoplasms in adultcancer survivors Nat Rev Clin Oncol “ 101038nrclinonc201341 Travis LB Rabkin CS Brown LM Cancer survivorship“genetic susceptibility and second primary cancers research strategiesand recommendations J Natl Cancer Inst “ 101093jncidjj001 Chattopadhyay S Zheng G Hemminki O Forsti A Sundquist KHemminki K Prostate cancer survivors risk and mortality in secondprimary cancers Cancer Med “ 101002cam41764 Chattopadhyay S Hemminki A F¶rsti A Sundquist K Sundquist JHemmiinki K Familial risks and mortality in second primary cancersin melanoma JNCI Cancer Spectr 20192pky068 101093jncicspky068 Chattopadhyay S Hemminki O Forsti A Sundquist K Sundquist JHemminki K Impact of family history of cancer on risk and mortality of second cancers in patients with prostate cancer ProstateCancer Prostatic Dis Centre for Epidemiology Cancer Incidence in Sweden Stockholm The National Board of Health and Welfare Green AC Olsen CM Cutaneous squamous cell carcinoma anreview Br J Dermatol “epidemiological101111bjd15324 IARC Personal Habits and Indoor Combustions Vol 100E LyonInternational Agency for Research on Cancer Omland SH Ahlstrom MG Gerstoft J Risk of skin cancer inpatients with HIV a Danish nationwide cohort study J Am AcadDermatol “ 101016jjaad201803024 Hortlund M Arroyo Muhr LS Storm H Engholm G Dillner JBzhalava D Cancer risks after solid an transplantation and afterlongterm dialysis Int J Cancer “ 101002ijc30531Clinical Epidemiology submit your manuscript wwwdovepresscomDovePress 0cZheng et alDovepress Harwood CA Toland AE Proby CM The pathogenesis ofcutaneous squamous cell carcinoma in an transplant recipientsBr J Dermatol “ 101111bjd15956 Bottomley MJ Thomson J Harwood C Leigh I The role of theimmune system in cutaneous squamous cell carcinoma Int J Mol Sci 103390ijms20082009 Ji J Sundquist K Sundquist J Hemminki K Comparability of canceridentification among death registry cancer registry and hospital dischargeregistry Int J Cancer “ 101002ijc27462 Pukkala E Engholm G Hojsgaard Schmidt LK Nordic cancerregistries an overview of their procedures and data comparabilityActa Oncol “ 1010800284186X20171407039 Chen T Fallah M Brenner H Risk of second primary cancersin multiple myeloma survivors in german and swedish cancerregistries Sci Rep 101038srep22084 Chen T Fallah M Jansen L Distribution and risk of the seconddiscordant primary cancers combined after a specific first primarycancer in German and Swedish cancer registries Cancer Lett“ 101016jcanlet201508014 Chattopadhyay S Hemminki A Forsti A Sundquist K Sundquist JHemminki K Second primary cancers in patients with invasive andin situ squamous cell skin carcinoma Kaposi sarcoma and Merkel cellcarcinoma role for immune mechanisms J Invest Dermatol Chattopadhyay S Sud A Zheng G Second primary cancers innonHodgkin lymphoma bidirectional analyses suggesting role forimmune dysfunction Int J Cancer “ 101002ijc31801 Hemminki K Hemminki O F¶rsti A Sundquist K Sundquist JLi X Surveillance bias in cancer risk after unrelated medical conditions example urolithiasis Sci Rep 101038s41598017088395 Emens LA Ascierto PA Darcy PK Cancer immunotherapyopportunities and challengesin the rapidly evolving clinicallandscape Eur J Cancer “ 101016jejca2017 Rama I Grinyo JM Malignancy after renal transplantation the roleimmunosuppression Nat Rev Nephrol “of101038nrneph2010102 Harms PW Harms KL Moore PS The biology and treatment ofMerkel cell carcinoma current understanding and research prioritiesNat Rev Clin Oncol “ 101038s41571018 Rangwala S Tsai KY Roles of the immune system in skin cancer BrJ Dermatol “ 101111j13652133201110507x Zur Hausen H The search for infectious causes of human cancerswhere and why Virology “ 101016jvirol20 IARC Biological agents Volume B A review of humancarcinogens IARC Monogr Eval Carcinog Risks Hum 2012100PtB1“Clinical EpidemiologyPublish your work in this journalDovepressClinical Epidemiology is an international peerreviewed accessonline journal focusing on disease and drug epidemiology identification of risk factors and screening procedures to develop optimal preventative initiatives and programs Specific topics include diagnosisprognosis treatment screening prevention risk factor modificationsystematic reviews risk safety of medical interventions epidemiology biostatistical methods and evaluation of guidelines translationalmedicine health policies economic evaluations The manuscriptmanagement system is completely online and includes a very quickand fair peerreview system which is all easy to useSubmit your manuscript here wwwdovepresscomclinicalepidemiologyjournalsubmit your manuscript wwwdovepresscomDovePressClinical Epidemiology 0c'

Thyroid_Cancer

"incidence of thyroid carcinoma is increasing all over the world Some studies have suggestedthat the change of adipokines expression can induce thyroid carcinoma However other studies have come to theopposite Therefore we studied the relationship between adipokines and thyroid carcinomaMethods Databases”PubMed Cochrane Library SinoMed CNKI Wanfang and clinical trial registries weresearched A metaanalysis was then performed through a fixed or randomeffects model to calculate I values forheterogeneity analysisResults Twentynine s were finally included for analysis The level of serum tumor necrosis factoralpha TNFα [standardized mean difference SMD confidence interval CI to I2 P ]and the ratio of TNFα immunoreactivity in tissues [odds ratios OR CI to I2 P ]in thyroid carcinoma are significantly higher than those in control The serum interleukin6 IL6 in patients withthyroid carcinoma is higher than that in control SMD CI to I2 P There is nosignificant difference of the ratio of IL6 immunoreactivity in tissues between carcinoma and control OR CI to I2 P The ratio of leptin immunoreactivity in tissues is significantly associated with therisk of thyroid carcinoma OR CI to I2 P However after analyzing theexpression level of serum adiponectin in three studies no significant difference is found between thyroidcarcinoma and the control P Conclusions Adipokines TNFα IL6 and leptin show a strong relationship between elevated concentrations inserum andor tissue and thyroid carcinoma However the association between adiponectin and thyroid carcinomaneeds further researchKeywords Thyroid carcinoma Adipokines TNFα IL6 Leptin Metaanalysis Correspondence liaolinsdueducn cwc_llsdueducn Junyu Zhao and Jing Wen contributed equally to this work1Department of Endocrinology and Metabology The First Affiliated Hospitalof Shandong First Medical University Shandong Provincial QianfoshanHospital Jinan China5Department of Endocrinology and Metabology Qilu Hospital of ShandongUniversity Cheeloo College of Medicine Shandong University Jinan ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhao BMC Cancer Page of BackgroundThyroid carcinoma is the most common endocrine malignancy but mostly has good prognosis During the pastdecades a rising incidence of thyroid carcinoma worldwide has aroused the widespread attention of researchers[ ] Someone supposed that the growing use of diagnostic imaging and fineneedle aspiration biopsy may bethe main reason [] But this may be only partial andcan not totally explain the increased incidence of microcarcinoma Changes in the incidence of a cancer are notonly associated with increased detection and other unknown risk factors need further explore Recently somescientists found that the incidence of thyroid carcinomahas increased along with a marked rise in obesity rateand accumulating evidence of an association betweenobesity and increased thyroid carcinoma risk has beenproposed [“] Various hypotheses have been supposedto interpret the relaitonship between obesity and thyroidcarcinoma including hyperinsulinemia upregulation ofaromatase activity chronic œlow grade inflammation altered immune response and DNA damage caused byoxidative stress [] Furthermore recent data supportingthe notion that a changed expression of adipokinescaused by obesity can affect the cell proliferation andeven induce a thyroid tumorigenesis [“] Adipose tissue is a specialized connective tissue composed of fatcells which releases a number of biologically active molecules called adipokines or adipocytokinesincludingleptin adiponectin resistin and many cytokines of theimmune system such as tumor necrosis factoralphaTNFα interleukin6 IL6 and complement factor Dalso known as adipsin Adipokines refer to various enzymes hormones cytokines growth factors proteinsand other biological active substances secreted by adipocytes including adiponectin leptin resistin and interleukin The concentration of adipokines such as TNFαIL6 and leptin were significantly higher in obese subjects and the elevated levels was linked to obesity andeven positively correlated with body mass index [“]It is reported that adipokines took part in the biologicalprocesses of insulin sensitivity inflammation and proliferation [ ] which the proliferation have been recognizedthetumorigenesis and development At present many kindsof adipokines have been reported to be associated withthyroid carcinoma Rehem RA [] suggested thatserum leptin levels were higher in welldeffierentiatedthyroid carcinoma patients and a significant drop aftersurgery Another envidence showed that adiponectin related with tumor size [] However the opposite resultswere also found in other studies [] Some researchesreported the expression of adipokines is lower in tumortissue than normal control [“] It is clearly that certain confounders such as age sex ethnicity and alsoimportantfactorleadingtoasanheterogeneity in study size methodology and original ofsample should be considered when trying to analyze theassociation between adipokines and thyroid carcinomaThese confunding factors above may be the cause of inconsistency results from different researches Additionaly the association between adipokines and thyroidcarcinoma are still not well documented Therfore theaim of this metaanalysis was to investigate the association between adipokines and thyroid carcinoma andpropose that adipokine as a risk factor for thyroidcarcinomaMethodsSearching progressWe conducted a search of all studies published until27th July regarding the association between adipokine and thyroid carcinoma Eligible casecontrol studieswere found by searching the database of PubMedCochrane library Sinomed CNKI and Wanfang and restricted to published results Clinical trial register centers httpwwwclinicaltrialsgov were also searchedThe following search terms œAdipokine or œLeptin orœadiponectin or œresistin or œtumor necrosis factoralpha or œInterleukin6 or œComplement factor D orœAdipocytokines or œtumor necrosis factorᝠor œTNFᝠor œIL6 or œadipsin and œthyroid cancer or œthyroid neoplasm or œthyroid tumor or œthyroid carcinoma or œdifferentiated thyroid carcinoma or œDTC orœPapillary thyroid carcinoma or œThyroid carcinomapapillary or œPTC or œThyroid cancer follicular orœFTC or œThyroid Carcinoma Anaplastic or œATC orœThyroid cancer medullary or œMTC Hand searchingwas used to identify appropriate studies including reference lists of eligible s and related previous reviews Eligible studies met the following criteria published in English or Chinese language studyassessed the association between adipokine and thyroidcarcinoma study designed as the casecontrol study study reported the expression of at least one adipokine either in blood or tissue Studies were excluded ifany of the followings were identified insufficient information concerning adipokine or thyroid carcinomaoutcome cannot directly extract or calculate OR and95CI the type of study was not a casecontrol designhave not fulltext animal trialsStudy selection and data extractionTwo reviewers screened the studies and extracted dataindependently Any disagreement was resolved by discussion or consensus with a third senior reviewer Dataincluded the followingfirst author publication yearcountry participant characteristics ie mean age sample size sex ration pathological type of thyroid carcinoma source of controls measured outcomes or the 0cZhao BMC Cancer Page of scores were considered to be of high quality Disagreements were resolved by reevaluating and discussing between two reviewersinSearchingthis metaanalysisResultsSearch results and characteristics of included studies s regarding the association between adipokine and thyroid carcinoma were searched in therelated database and clinicaltrial websites Afterscreening the title and abstracts s were selected for fulltext review Finally studies were eligibleprogressincluded and excluded details are all shown in Fig Eighteen of these studies are published in Chinese[ “] and the rest are published in English[“] Nineteen studies were conducted in Chinatwo in India and two in Turkey Brazil Greece IranItaly Denmark and Serbia each had one study Totally there are patients with thyroid carcinomain the case group and controls including healthysubjects patients with benign thyroid diseases or normal thyroid tissue near carcinoma were included inthe control group The sample size ranges from to in the case group while to in the controlgroup All the thyroid carcinoma patients were confirmed by pathologically Among these studiesfourteen studies reported papillary thyroid carcinomaPTC eight studies reported differentiated thyroidcarcinoma DTCreported differentpathological types in one paper one study reportedmedullary thyroid carcinoma MTC and the restfour studies did not show the pathological detailsThe detailed characteristics ofincluded studies aresummarized in Table three studiespercentage of samples show immunoreactivity for adipokines antibody both in the case and control groups Thecalculation method is shown below take thyroid cancerfor example the number of samples obtained from thyroid carcinoma that show immunoreactivity for adipokines antibody divided by the total number of thyroidcarcinoma samplesStatistical analysisFor metaanalysis dichotomous outcomes were analyzedby using the odds ratios OR computed using the MantelHaenszel method fixed or random models Continuousvariables measured on the same scale expressed as a meanvalue and standard deviation were analyzed by usingweighted mean differences WMD Otherwise standardized mean difference SMD were used for different scaleAll results were reported with confidence interval CI I2 was used to assess heterogeneity between studies and I2 values of and representing no lowmoderate and high heterogeneity respectively Visual inspection of the funnel plot was done to assess publicationbias The analyses were performed by Review Manager Cochrane Collaboration United Kingdom httpwwwcochraneQuality assessment and risk of biasThe methodological quality of casecontrol study wasassessed by the NewcastleOttawa Scale NOS Supplement Table which consists of the three parameterseight questions with nine possible scores Selection Exposure and Comparability A study can be awarded amaximum of one score for each numbered item withinthe Selection an Exposure categories A maximum oftwo scores can be got for Comparability A higher scoremeans better quality in methodology and five or moreFig Flow chart of the systematic search process 0cZhao BMC Cancer Page of Zhao Jianqiang []ChinaPTC FTC ATCand MTCthyroid adenoma andnormal healthUnknownUnknownTable Characteristic of included studiesFirst authorYearCountryPathologicaltype of thyroidcancerSource of controlsL Kayser []Denmark PTC and FTCCao Guangyao []ChinaUnknownMTrovato []ItalyDTC andundifferentiatedcarcinomamultinodular goitersadenomas Hashimoto™sthyroiditis hyperplasticglandsthyroid adenoma andnodular goiternormal thyroid tissues andbenign nodulesMelih Akinci []Wang Jingxia []ZhuangXiaoming []Yu Xiao []Hou Sen []SnezanaZivancevicSimonovic []Xu Xiaocheng []XeniProvatopoulou []TurkeyPTChealthy volunteersChinaPTC and FTCnormal thyroid tissuesChinaPTC FTC andMTCthyroid adenoma andnormal healthChinaPTCthyroid adenoma andnormal thyroid tissue nearcarcinomaChinaPTCthyroid adenomaSerbiaWDTChealthy subjectsChinathyroidcarcinomaGreecePTCthyroid adenomabenign thyroid disease andhealthy controlsSun Qinnuan []ChinaPTCnormal thyroid tissue nearcarcinoma and healthycontrolsChinaPTCthyroid adenomaChinaPTCthyroid adenomaMean age yearFemale Outcome indexNumber ofparticipants ncases control casesUnknowncontrolcontrolcasesUnknownUnknownUnknownUnknownUnknownTNFα tissueTNFα tissueIL6 tissueIL6ãTNFαblood ± ±Unknown leptinblood Unknown TNFα tissue Unknown IL6ãTNFαUnknownUnknownbloodleptintissueUnknown Unknown leptin ± ± tissueTNFαblood ± ± ± ± ± ± ± IL6blood IL6blood TNFαbloodandtissue ±Unknown Unknown leptinUnknownUnknowntissueadiponectintissueUnknown Unknown adiponectintissue IL6ãTNFαblood ± ± Zhang Zijie []Zhong Xiuxiu []Zhang Bo []Hu Jinhua []SnezanaZivancevicSimonovic []YanLan Fan []ChinaDTCChinaDTCnormal thyroid tissue nearcarcinomathyroid adenoma andhealthy controls ±SerbiaPTCcontrol subjectsUnknownUnknownIL6bloodChinathyroidcarcinomanodular goitre Hashimoto™sthyroiditis follicular adenomaand adjacent nonneoplasticthyroid tissue samplesUnknownUnknownleptintissue 0cZhao BMC Cancer Page of Table Characteristic of included studies ContinuedFirst authorSource of controlsYearCountryPathologicaltype of thyroidcancerChinathyroidcarcinomabenign thyroid disease andnormal thyroid tissue nearbenign thyroid diseaseChinaPTCthyroid adenomaTurkeyPTChealthy volunteersIndiaPTCIndiaPTCbenign thyroid diseases andhealthy individualsbenign thyroid diseases andhealthy individualsNumber ofparticipants ncases control cases ±Mean age yearFemale Outcome indexcontrol ±casescontrol TNFαtissue ± ±TNFα tissue IL6bloodUnknown Unknown TNFαbloodUnknown Unknown IL6bloodWangXinzheng []Song Runbo []Kemal Beksac []Toral PKobawala“ []Toral PKobawala“ []RaziyehAbooshahab []Zhang Bo []ZhouXiaodong []Ma Xiaokai []MarianaBonjiornoMartins []IranMTChealthy subjects ± ± leptinãadiponectinbloodChinaDTCnormal thyroid tissue nearcarcinomaUnknown Unknown leptintissueChinaDTChealthy subjects ± ±IL6ãTNFαbloodChinaPTCthyroid adenomaUnknown Unknown leptinBrazilDTCbenign thyroid nodules andhealthy controls ±tissue IL6blood ± ±ChinaIL6 Sun Zhenhua []tissueTNFα tumor necrosis factora DTC differentiated thyroid carcinoma IL6 interleukin6 PTC papillary thyroid carcinoma FTC follicular thyroid carcinoma ATCanaplastic thyroid carcinoma MTC medullary thyroid carcinoma WDTC welldifferentiated thyroid carcinoma FNAC fine needle aspiration cytologynodular goiterPTCQuality of included studiesThe quality assessment of these studies is assessed bythe NOS and the resultis shown in SupplementalTable Five or more scores are determined as highquality Two studies conducted by Cao G in [] and L Kayser in [] only get two scoresshowing a poor quality in methodology The rest studies are assessed as high qualityTNFα and thyroid carcinomaTwelve studies reported the expression of TNFα bothin patients with thyroid carcinoma and control subjects[“ “ ] Among these sevenstudies [ ] had tested the level ofserum TNFα two studies [ ] had tested the expression of TNFα in tissues and the ratio of TNFα immunoreactivity was tested in four studies [ ] Firstly fixedeffect model is used to merge the SMDvalues of serum TNFα level however a large heterogeneity is found by the heterogeneity analysis heterogeneity test Chi2 P I2 and itmay be due to the different units differenttestingmethods in different researches or other unknown factors Then randomeffect model to merge the SMD isused and pooled effect size in favor of control group is CI to P Fig 2a SMDvalues of the expression of TNFα in tissues is mergedby fixedeffected model and the heterogeneity analysisshow a considerable heterogeneity heterogeneity testChi2 P I2 The different unitsand limited numbers of research may be the original ofheterogeneity So the pooled SMD with randomeffectmodel of the expression of TNFα in tissues is CI ˆ’ to P Fig 2b The pooled ORwith fixedeffect model of the ratio of TNFα immunoreactivity in thyroid carcinoma tissues is CI to P However a significant heterogeneity is detected heterogeneity test Chi2 P I2 The published by L Kayser in with a poor quality in methodology may attributeto this high heterogeneity Then randomeffect model ofpooled OR is used and pooled effect size in favor of 0cZhao BMC Cancer Page of Fig Forest plot of the TNFα level and the ratio of TNFα immunoreactivity in tissues in patients with thyroid carcinoma a Level of serum TNFα b Expression of TNFα in tissue c Ratio of TNFα immunoreactivity in tissuecontrol group is CI to P Fig 2c In level of serum TNFα and theratio of TNFα immunoreactivity in tissues of thyroidcarcinoma patients are significantly higher than controlsubjects which are without thyroid carcinomaIL6 and thyroid carcinomaAmong the included studies reported the level ofserum IL6 in patients with thyroid carcinoma and control subjects [ “] Due to thelarge heterogeneity of the merged SMD values of serumIL6 level by the heterogeneity analysis heterogeneitytest Chi2 P I2 randomeffectmodel was used to pooled the SMD values and thepooled effect size in favor of control subjects is CI to P Fig 3a which meansthat patients with thyroid carcinoma have a significantlyhigher level of serum IL6 than control subjects Twostudies reported the ratio of IL6 immunoreactivity bothin thyroid carcinoma tissue and noncarcinoma tissue[ ] The pooled OR of the limited two studies donot show an increased ratio of IL6 immunoreactivity inthyroid carcinoma tissues OR CI to P and a large heterogeneity always existsheterogeneity test Chi2 P I2 Fig3b Thus the level of serum IL6 is higher in patientswith thyroid carcinoma However it needs more clinicaldata to verify the relationship between the expression ofIL6 and thyroid carcinoma tissueLeptin and thyroid carcinomaTwo studies reported the level of serum leptin [ ]and another five studies reported the ratio of leptin immunoreactivity in tissues [ ] Because ofthe considerable heterogeneity of the pooled WMD ofserum leptin level heterogeneity test Chi2 P I2 and pooled OR of the ratio of leptinimmunoreactivity in tissues heterogeneity test Chi2 P I2 by the heterogeneity analysis with fixedeffect model randomeffect model is further used to merge the values and analysis Howeverthere is no association of higher level of serum leptin 0cZhao BMC Cancer Page of Fig Forest plot of the IL6 level and ratio of IL6 immunoreactivity in tissue in patients with thyroid carcinoma a Level of serum IL6 b Ratio ofIL6 immunoreactivity in tissueFig Forest plot of the leptin level and ratio of leptin immunoreactivity in tissuein patients with thyroid carcinoma a Level of serum leptin bRatio of leptin immunoreactivity in tissue 0cZhao BMC Cancer Page of with risk of thyroid carcinoma WMD 95CI ˆ’ to Fig 4a Moreover the pooled OR of theratio ofleptin immunoreactivity in tissues from fivestudies is 95CI to Fig 4b whichmeans a high ratio of leptin immunoreactivity in tissueis significantly related to thyroid carcinomaAdiponectin and thyroid carcinomaThree studies reported the expression of adiponectin inthyroid carcinoma including serum and tissue [ ] and the result is summarized in Table It could befound that the level of serum adiponectin is not staticallydifferent comparing thyroid carcinoma patients withcontrol subjects P Interestinglyit was foundthat the expression of adiponectin in thyroid carcinomatissue is significantly lower than control tissue while theopposite result is found when comparing the ratio ofadiponectin immunoreactivity However there was onlyone study for each result and this may be the reasonwhy the two results are diametrically opposed Thus itneeds more clinical studies to confirm in the futurePublication biasThe funnel plot was applied for assessing publicationbias of studies included in the three results includingTNFα Fig 5a IL6 Fig 5b and leptin Fig 5c InFig 5a and Fig 5b almost all studies lies inside the95CIs with an even distribution around the verticalindicating no evident publication bias was obtainedthrough the visual distribution of funnel plot Howevera potential publication bias was found in Fig 5c whencomparing the ratio of leptin immunoreactivity in tissues and that might influence the result of this metaanalysisDiscussionCurrently obesity affects one third of population amongUS adults [] and China has become a big country ofobesity with the incidence ranking first worldwide in theyear of [] Nowadays increasing clinical and experimental studies and documented the closely relationship between malignancies including colon esophaguskidney liver breast endometrium pancreas and prostate as well as nonHodgkin™s lymphoma and multiplemyeloma and obesityoverweight which affect its occurrence development and prognosis [“] Becauseof the increasing incidence of thyroid carcinoma duringthe past decades lots of scientists focus on studying therisk factors of thyroid carcinoma It was found that theincidence of thyroid carcinoma has increased along witha marked rising rate of obesity [“] Furthermore obesity is an independent risk factor for thyroid carcinoma[] Increased insulin resistance elevated serum cholesterol level and upregulated COX2 expression may be thetarget of the correlation between obesity and thyroidcarcinoma [] It is reported that people with higherbody mass index have a higher concentration of adipokines [“] Adipokines take part in the followingpathological and physiological processes such as insulinsensitivity inflammation and proliferation [ ] andthese are important in the process of tumorigenesis anddeveloping So adipokines may be one of the targetslinking obesity with thyroid cancer The metaanalysiswas based on previous published studies In previousstudies the analysis of adiponectin and thyroid cancermostly focused on TNF IL6 Leptin and AdiponectinWhile few studies focused on other molecules includingIL1 and IL8 and we failed to combine statisticsTherefore in this metaanalysis only TNF IL6 Leptinand Adiponectin which are the most published adiponectin were analyzedTNFα produced by adipose tissue and inflammatorycells can lead to inflammatory response necrocytosisand assist other cytokines to kill tumor cells and improve the antitumor ability Meanwhile TNFα plays animportant role in the process of inflammation insulinresistance diabetes and obesity A moderate amount ofTNFα has a protective effect while an excessive amountwill cause damage which may lead to a resistant oftumor cells to TNFassociated apoptosisinduced ligandswhen the microenvironment of apoptosis is maladjustedTNFα has the ability to promote the production ofgranulocytecolony stimulating factor by thyroid fibroblasts [] which may play an important role in thyroidcancer Moreover TNFα can stimulate the vasoactivemediators such as interleukin and prostaglandin []and these mediators can promote the proliferation oftumor cells and significantly reduce the immune function TNFα can also induce an increased expression ofvascular endothelial growth factor VEGF [] the laterof that can promote the proliferation of tumor cells andprovide conditions for tumors metastasisTable Summary of adiponectin expression in thyroid carcinomaserum adiponectin []ratio of adiponectin immunoreactivity []Effect sizeWMD OR adiponectin in tissue [] CI confidence interval WMD weighted mean differences OR odds ratiosWMD 95CIˆ’ ˆ’ ˆ’PI2Not applicable 0cZhao BMC Cancer Page of Fig Funnel plots of a TNFα b IL6 and c leptin revealed no significant publication bias SE SMD standard error of standardizedmean differenceIn surprisingly the results of clinical studies provide evidence for basic research Simonovic SZ [] evaluated cytokine profiles determined in supernatants obtained from whole blood cultures in patients with DTC before and days after radioactiveiodine 131Itherapy and control subjects andfound that the expression of TNFα in DTC patients ishigher than control subjects and it showed a decreasedlevel after 131I therapy than those before therapy However no statistical difference found for the limited sample size Another study conducted by Kobawala TP et al[] with more patients patients with benign thyroiddisease PTC patients and healthy individuals determined the circulating levels of TNFα and it wasfound that the serum level of TNFα was significantlyhigher in PTC patients than benign thyroid disease patients and the later was also significantly higher thanhealthy individuals Furthermore serum TNFα was reported to be a significant prognosticator for overall survival in PTC patients It is a pity thatopposite result wasreported in a casecontrol study that included DTCcases and matched cancerfree cohort participantswhich found that TNFa was not associated with thyroidrisk in either gender []Based on current evidence our metaanalysis suggeststhat TNFα exhibit a strong association with thyroid carcinoma It may because that elevated TNFα may involved in the tumorigenesis and development of thyroidcancer Another possible reason is that the TNFα decreased with tumor cells less resulted the activation ofthe immune system by thyroid carcinomaThereforemore clincal studies and basic reseaches should be conducted in the futureIL6 a multifunctional cytokine plays important rolesin different types of cells including tumor cells It is reported that elevated serum IL6 level is closely related tothe tumorigenesis and development of a variety of tumors [] A strong positive association between theserum IL6 and the progression and poor prognosis oftumors in patients with several types of tumor wasalready found [“] Serum IL6 level in thyroid cancer has been evaluated in numerous studies including 0cZhao BMC Cancer Page of in vivo and in vitro studies Provatopoulou X []found that serum IL6 were significantly higher in malignant and benign thyroid diseases compared to healthycontrols However other studies show a different resultthat no significance different of IL6 was found betweenthyroid cancer and nonthyroid cancer [ ] A limited sample size different inclusion criteriadifferent population characteristics or different pathological type of thyroid cancer may explain such a difference For in vitro research IL6 was also found to beexpressed in thyroid cancer cell lines and a potential roleof IL6 in PTC was confirmed indirectly []The underlying mechanism may be the followingsbelow Tumor cells including esophageal cancerlungcancer colorectal cancer and melanoma were foundhave the function of autocrine IL6 which can affect thegrowth and proliferation of tumor cells and participatein the tumor growth and metastasis by acting on themembrane receptors [] Also IL6R was found associated with the characterization of thyroid nodules™ malignancy and tumor aggressivenessIn additionIliopoulos D [] found that Src nonsomatic tyrosine kinase family oncogene can induce the normal epithelial cell transformation by activating NFκB and thistransformation contributes to tumorigenesis IL6 is considered as an important regulatory factor in this processAnother possibility is that the activation of the immunesystem of patients with thyroid cancer leads to an increase in adikopines level[]In general the data above support that IL6 is important for thyroid cancer but the detail mechanism remainto be further studyLeptin a circulating hormone secreted by adipocytesexerts its biological effect by combing with its receptorwhich is mainly presented in the hypothalamus Meanwhile gene of leptin receptor is also expressed in manyother tissues such as lung liver and kidney It is reported that obesity and overweight can lead to a highlevel of serum leptin which may because that obesity always accompanies with insulin resistance and hyperinsulinemia and insulin further enhance the expression ofleptin Moreover leptin acts as a growth factor in a variety of human cellsincluding both normal cells andtumor cells which regulates the process of differentiation proliferation and apoptosis thus stimulate thetumorigenesis and development of tumors through mediatingpathway RhoALIMK1Cofilinpathway and MAPKERK pathway [] Kim WG et al[] evaluated the effect of dietinduced obesity on thyroid carcinogenesis in a mouse model that spontaneously develops thyroid cancer Thrb PVPV Ptenˆ’ mice and found that obesity increases the frequency of anaplasia of thyroid cancer and exacerbatesthyroid cancer progression that were mediated byJAKSTAT3increased activation of the JAK2 signaling transducerand activator of STAT3 signaling pathway and inductionof STAT3 target gene expression Leptin is always reported a high expression on solid tumors [] and it isconfirmed that serum leptin levelis significantly increased in thyroid cancer mainly PTC while otherstudies showed a same results in cancer tissues [ ] Yu Xiao [] conducted a clinical studycomparing the level of serum leptin in PTC patientsincluding patients with lymph node metastasis and thyroid adenoma patients in Dalian China and foundthat patients with lymph node metastasis have a higherlevel of leptin than those without lymph node metastasisLeptin can induce the expression of vascular endothelialgrowth factor and promote neovascularization in tumortissue [] In addition it can also inhibit the apoptosisthrough Bcl2 dependent mechanism Meanwhile leptinreceptor exists in all thyroid cancer cells It is overexpressed in PTC and is involved in tumor invasion andlymph node metastasis [ ] Thus leptin may be involved in the tumorigenesis and metastasis of thyroidcancer through a complex pathway and a monitoringmay have some significance Due to the absence of directevidence elevated leptin levels can also be caused bythyroid carcinoma The cause and effect relationship between leptin and thyroid carcinoma are unclear now andneed further studiesCompared to lean women overweightobese womenhad lower serum adiponectin levels and this differencehas statistical significance [] In addition adiponectinis negatively associated with a variety of benign and malignant tumors especially those associated with obesityand insulin resistance such as leukemia [] renal carcinoma [] gastric carcinoma [] and colon cancer[] Moreover the association of adiponectin with potential tumorlimiting functions has been widely proposed []Otvos L Jr [] tried in vitro experiments andproved that adiponectin can inhibit the metastasis ofcancer cells Mitsiades N [] measured circulatingadiponectin levels in ptaients with PTC and found thatit is independently and inversely associated with the riskof thyroid cancer As the receptor that binds to adiponectin for biological effects adiponectin receptor hadbeen reported closely correlated with the developmentof PTC Adiponectin receptor1 and are higher expression in PTC tissues than that in the surrounding normaltissues and this is thought to be associated with a betterprognosis []However other studies have shown different results[ ] and more studies should be done furtherly tosupport the antitumor effect of adiponectin and thepositive correlation between the increased level of adiponectin in circulating blood and the prognosis of thyroid 0cZhao BMC Cancer Page of neoplasms and provide new ideas for the prevention andtreatment of thyroid neoplasmsFrom the above a strong relationship between elevatedconcentrations of adipokines in serum andor tissueand thyroid cancer can be concluded And this may explain why increased incidence of obesity and thyroidcancer are consistent Thus targeted drugs for adipokinemay be useful for the treatment of thyroid cancer in thefutureHowever some limitations in our metaana

Thyroid_Cancer

THYROID development of thyroid hormone TH analogues was prompted by the attempt to exploit the effects of THon lipid metabolism avoiding cardiac thyrotoxicosis Analysis of the relative distribution of the a and bsubtypes of nuclear TH receptors TRa and TRb showed that TRa and TRb are responsible for cardiac andmetabolic responses respectively Therefore analogues with TRb selectivity were developed and four differentcompounds have been used in clinical trials GC1 sobetirome KB2115 eprotirome MB07344VK2809and MGL3196 resmetirom Each of these compounds was able to reduce lowdensity lipoprotein cholesterolbut a phase trial with eprotirome was interrupted because of a significant increase in liver enzymes and thecontemporary report of cartilage side effects in animals As a consequence the other projects were terminatedas well However in recent years TRb agonists have raised new interest for the treatment of nonalcoholic fattyliver disease NAFLD After obtaining excellent results in experimental models clinical trials have beenstarted with MGL3196 and VK2809 and the initial reports are encouraging Sobetirome turned out to beeffective also in experimental models of demyelinating disease Aside TRb agonists TH analogues includesome TH metabolites that are biologically active on their own and their synthetic analogues ¢triiodothyroacetic acid has already found clinical use in the treatment of some cases of TH resistance due toTRb mutations and interesting results have recently been reported in patients with the Allan“Herndon“Dudleysyndrome a rare disease caused by mutations in the TH transporter MCT8 35diiodothyronine T2 has beenused with success in rat models of dyslipidemia and NAFLD but the outcome of a clinical trial with a syntheticT2 analogue was disappointing 3iodothyronamine T1AM is the last entry in the group of active TH metabolites Promising results have been obtained in animal models of neurological injury induced by bamyloidor by convulsive agents but no clinical data are available so farKeywords TH analogues sobetirome eprotirome resmetirom triac 35diiodothyronine 3iodothyronamineIntroductionT he term thyroid hormone TH analogue is used withregard to compounds that have a similar molecularstructure as TH and can therefore interact with at least someof its molecular targetsIt should be kept in mind that TH signaling is particularlycomplex Canonical TH signaling is based on the interactionwith nuclear TH receptors TRs leading to either activation orrepression of the transcription of a large number of genes Twogene subtypes exist TRa and TRb and different isoformswhich are designated by numerical subscripts can be producedby alternative splicing TRa1 is the major TR isoform in theheart and it is also expressed in other tissues including skeletal muscle brain and bone TRa2 lacks part of the THbinding domain so it is unable to bind TH and is thought tomediate constitutive repression of transcription TRb1 iswidely expressed in most tissues while TRb2 expression ismore circumscribed and it is particularly relevant in the brainpituitary retina and inner ear TRb3 has been identified in ratadipose tissue but it does not appear to be present in humansWith regard to the topic of this review the main functionalconsequence of TR subtype distribution is that the metaboliceffects of TH are largely mediated by TRb1 while cardiacTH actions depend on TRa1In addition to canonical signaling TH may activate anumber of noncanonical signaling pathways The latterinvolves TRs that do not bind DNA directly extranuclearDepartment of Pathology University of Pisa Pisa Italyª Riccardo Zucchi Published by Mary Ann Liebert Inc This Access is distributed under the terms of the CreativeCommons Attribution Noncommercial License httpcreativecommonslicensesbync40 which permits any noncommercial usedistribution and reproduction in any medium provided the original authors and the source are cited 0cZUCCHITRs that activate the phosphatidylinositol 3kinase pathwayand membrane receptors belonging to the integrin family In addition some TH metabolites have been suggested tobe potentially active although their physiological role is stillunclear By definition these compounds should be regardedas TH analogues although they are endogenous moleculesThey have also been used as templates to develop novelclasses of synthetic analogues Active TH metabolites include35diiodothyronine T2 3iodothyronamine T1AM andseveral thyroacetic acids ¢¢tetraiodothyroacetic acid orTetrac ¢triiodothyroacetic acid or Triac 3iodothroaceticacid or TA1 They have been the object of several recentreviews “ and their properties are briefly recalled in thesubsequent paragraphs only in as much as this is relevant to theobject of the present succinct reviewTH analogues have been developed for clinical and therapeutic purposes namely to exploit some aspects of THsignaling to produce beneficial effects in the diseaseTherefore in the following paragraphs these analogues arediscussed with the perspective of their potential therapeuticuse Special emphasis is placed on clinical investigationsalthough some results obtained in experimental models ofdisease are mentioned when appropriateTH Analogues with Selective TRb Activityin DyslipidemiaThe development of selective TRb agonists was promptedby the aim of treating dyslipidemia particularly hypercholesterolemia avoiding cardiac thyrotoxicosis The ligandbinding pocket is more flexible in TRb than in TRa and one ofthe amino acids involved in triiodothyronine T3 binding isdifferent since TRa serine is replaced by asparagine in TRb These differences have been exploited to synthesizeselective TRb agonists The analogues that have reached the clinical field areshown in Figure In GC1 now called sobetirome the iodine atoms and the oxygen linking the two aromatic rings arereplaced by alkyl groups and the amino acid side chain isreplaced by an oxoacetic chain The ratio of TRb to TRaaffinity is 10fold higher for GC1 than for T3 and an additional useful property is represented by selective liver uptakeIn KB2115 now referred to as eprotirome iodine is replaced by bromine in the tyrosyl ring or isopropyl in thephenolic ring and an amidoacetic side chain is presentyielding nearly 20fold TRb selectivityA different strategy was followed by researchers at MetabasisTheir compound known as MB07811 and now renamedVK2809 is a prodrug selectively taken up by the liver where it isconverted into the active principle MB07344 which differsfrom GC1 only for the presence of a phosphoryl group in the sidechainAnother compound that has been used in human is MGL also called resmetirom whose more complex chemicalstructure a substituted pyridazinone ring replaces the phenolicring and a heterocyclic cyanoazauracil group is included in theside chain allows nearly 30fold TRb selectivityAll these compounds have been used in animal models ofhypercholesterolemia where they reduced total and lowdensity lipoprotein LDL cholesterol without significantchanges in heart rate Based on these preclinical findingsclinical trials were started In both sobetirome andeprotirome were reported to produce a significant reduction in total and LDL cholesterol after “ weeks oftreatment in small groups of patients affected by hypercholesterolemia Similar results were obtained with MGL3196which reduced serum triglycerides and LDL cholesterol after weeks of treatment in hypercholesterolemic patients In patients treated with statins the addition of eprotirome caused further reduction in serum LDL cholesterol andtriglycerides No significant side effects were reported in any of theseinvestigations and therefore a phase trial was undertakento compare eprotirome at the daily doses of and lgversus placebo in patients with familial hypercholesterolemia After weeks total cholesterol LDL cholesterolFIG Chemical structureof the synthetic TRb analogues that have been used inclinical trials In the leftlower panel please note thatMB07811 now known asVK2809 is a prodrug in theliver it is converted into theactive principle formerlyknown as MB07344 by thehydroxylase CYP3A in a reaction requiring glutathioneGSH See text for furtherdetails 0cTHYROID HORMONE ANALOGUESand triglycerides were significantly reduced in both treatmentgroups without any change in HDL cholesterol However the trial was interrupted it was originally planned to last“ weeks because parallel experimental investigationsfound that eprotirome caused cartilage damage in dogs Notably during the study period a significant increase in liverenzymes was detected which determined treatment interruption in four patientsResulting in a partial domino effect termination of theeprotirome project caused parallel projects to be terminatedas well In particular the sobetirome project was interruptedand Metabasis announced that a phase investigation withMB07344 was aborted after observing increased liver enzymes in some patients Additional reasons probably contributed to these decisions While selective TRb agonistswere devised and tested excellent clinical results were obtained with statins in the primary and secondary preventionsof major cardiac events At the same time investigationsperformed with other experimental drugs showed that LDLcholesterol reduction per se was not necessarily associatedwith reduced cardiovascular risk In aggregate selective TRbstimulation was no longer regarded as a promising strategy totreat hypercholesterolemiaTH Analogues with Selective TRb Activityin Liver DiseaseDespite the disappointing results obtained with TRb agonists in hypercholesterolemia in recent years new potentialuses have been proposed for these agents The most attractivefield is probably represented by nonalcoholic fatty liver disease NAFLDThis is a highly prevalent condition since it is estimated toaffect “ of the adult population in the United Statesand Europe Its clinical presentation is quite variableMost NAFLD patients show a simple increase in liver enzymes with a histological pattern of increased hepatocytetriglyceride steatosis Symptoms may be minimal or absentand the clinical picture may be stable over time However asignificant fraction of NAFLD patients up to in someseries develop histological evidence of lobular inflammationand hepatocyte ballooning defining a variant of the diseaseknown as nonalcoholic steatohepatitis NASH NASH is aserious condition since it is associated with a high risk about“ of evolution into cirrhosis that is derangement inliver architecture leading to hepatic insufficiency Patientswith NASH andor cirrhosis are also prone to develop hepatocellular carcinoma HCCNAFLD is frequently associated with insulin resistancehypercholesterolemia or other components of the socalledmetabolic syndrome and patients are usually treated forthese underlying conditions but no specific treatment toprevent liver damage has been approved so far In principleTH should be beneficial in NAFLD due to reduced fatty acidsynthesis increased triglyceride and fatty acid breakdownand enhanced hepatocyte regeneration The liver effects of TH are mediated by TRb so selective TRb agonistshave been tested in experimental models and several compounds including sobetirome eprotirome and MB07811were able to reduce liver steatosis “ Sobetirome wasalso reported to prevent the development of HCC induced byactivation of the catenin pathway The study of TRb agonists in NAFLD has just entered theclinical field and the results of two clinical trials have recently been reported In patients with biopsyconfirmedNASH treated with MGL3196 resmetirom mg dailyfor weeks liver fat as assessed by magnetic resonanceimaging was significantly reduced versus with placebo if expressed as percentage of absolute livermass versus if expressed as percentage ofbaseline liver fat The effect was retained after weeksand in a subgroup of patients biopsy revealed a reduction ofhistological markers of inflammationSimilarresults were reported with VK2809 aliasMB07811 in patients with NAFLD treated for weeksat the dosages of mg every other day or mg daily In this trial liver fat content assessed by magnetic resonanceimaging decreased by “ versus with placebo“ vs if expressed as percentage ofbaseline liver fat While these results have raised a greatinterest and discussions have already started aboutthecomparison of these two drugs it should be stressed that thefinal results of the latter trial have not been published yetTH Analogues with Selective TRb Activity in CentralNervous System DiseaseIn recent years theoretical arguments have been developed suggesting the potential usefulness of TH analogues indemyelinating disease since TH favors both oligodendrocytedifferentiation and myelin sheet synthesis AlthoughTRa1 is widely expressed in the central nervous systemefforts have been focused on TRb agonists because of thenecessity to avoid cardiac side effects To increase bioavailability an ethanolamine ester of sobetirome has beensynthesized which acts as a prodrug since it crosses thebloodbrain barrier and is converted into sobetirome withinthe central nervous system This compound has beenrecently tested in different experimental models of demyelination with good results biochemical evidence of remyelination was confirmed by morphological findingsobtained by nuclear magnetic imaging and it was associatedwith improved functional recovery Clinical tests may be imminent for a specific diseaseknown as Xlinked adrenoleukodystrophy ALD This is arare congenital disease due to mutations in the ALD protein atransporter for verylongchain fatty acids ie with ¡carbon atoms physiologically located in the peroxisomalmembrane and encoded by the ABCD1 gene The consequence is the accumulation of verylongchain fatty acids andtheir derivatives which are eventually incorporated in cellular membranes whose structure and function are derangedAffected males develop adrenal insufficiency in childhoodand progressive myelopathy occurs in adulthood Demyelinating lesions in cerebral white matter also appear since theage of years ALD patients may benefit from hematopoietic stem celltransplantation but this treatment is effective only if performed in the early stages of the disease and it carries asignificant risk of mortality “ No specific pharmacological therapy is available for ALD Notably TH inducesthe expression of ABCD2 coding for an additional peroxisomal transporter and in a transgenic mouse model of ALDsobetirome administration reduced the brain and adrenal 0cZUCCHIcontent of verylongchain fatty acids Based on theseobservations a clinical trial with sobetirome in XlinkedALD has been posted in the NIH database NCT01787578The trial is presently labeled as withdrawn and the allegedreason is the need for revisions to the original protocolTriac in Syndromes of Reduced Sensitivity to THReduced sensitivity to TH is diagnosed when symptoms ofhypothyroidism occur despite normal or increased serum THThis finding may be the consequence of mutations in TRstransporters or metabolizing enzymes The expression˜˜resistance to TH™™ is usually reserved for syndromes causedby TR mutations Most cases are associated with TRb mutations Since TRb is involved in the inhibition of thyrotropin TSH secretion by T3 and thyroxine T4 circulatinglevels of T4 and T3 are usually high TSH is normal or highand goiter may be present The clinical picture is quitevariable and includes signs and symptoms of both hypothyroidism in TRbdependent ans and thyrotoxicosisin TRbdependent ans The most common findings aredelayed growth delayed bone maturation cognitive impairment and tachycardia Treating TH resistance is not easy Exogenous T4 or T3administration may improve some symptoms but it mayworsen others and symptomatic therapy is often prescribedfor example betablockers to reduce heart rate The idealtreatment would be represented by a T3 analogue able toactivate the mutated receptorIn some patients this can occur with Triac Fig Thelack of the amine group does not prevent Triac from activating TRs and it has similar affinity as T3 for the wildtypereceptor Apparently the different shape and charge of theTriac molecule allow several classes of mutated TRb to beactivated as well In these patients chronic Triac administration at dosages on the order of “ lg per kg of bodyweight daily represents the best therapeutic regimen Ingeneral Triacsensitive cases of TH resistance are caused bymutations in the carboxyterminal region of the T3 bindingFIG Chemical structure of some active thyroid hormone metabolites that have been used in patients or in animal models of human disease See text for further detailsdomain while mutations located close to the hinge region donot respond to Triac No positive response to Triac has beenreported in TRa mutations so farA different cause of reduced sensitivity to TH is represented by mutations in MCT8 a T3T4 transporter that isthe major pathway mediating T4 and T3 uptake in the centralnervous system The clinical syndrome associated withMCT8 mutations is known as the Allan“Herndon“Dudleysyndrome The MCT8 gene is located on the X chromosome and therefore virtually all patients are male Diagnosis is suspected in the presence of high T3 with low tonormal T4 and low to normal TSH associated with congenital brain hypothyroidism The phenotype is variable depending on the location and type of the mutationSymptoms usually include cognitive impairment associated with congenital hypotonia and weakness which mayprogress to spasticity Paroxysmal dyskinesias and seizuresmay also occur Peripheral hyperthyroidism often causestachycardia muscle wasting and progressive body weightreduction “Since it has been observed that cellular Triac uptake doesnot depend on MCT8 a clinical trial has been undertakenwith this endogenous TH analogue in patients with amedian age of years The results obtained after monthsof treatment at the dosage of “ lg daily have recently been published The treatment was highly effective in reducing serum T3 and Triac dosage was actuallytitrated on T3 reduction Signs of peripheral hypothyroidismnotably tachycardia and body weight reduction were significantly attenuated On the contrary the effects on theneurological symptoms were limited and improvement inthe indices of motor function was limited to patients youngerthan years It seems therefore that once the neurologicalphenotype is fully developed it is hardly reversibleOn this basis another phase trialis underwayNCT02396459 in which Triac treatment will be started asearly as possible in postnatal lifeSynthetic TH analogues might also be useful Notably in asmall clinical study diiodothyropropionic acid DITPA administration “ mgkg per day was able to normalizeT3 in four children affected by MCT8 deficiency aged “months Heart rate decreased in three patients and weightgain occurred in two Although DITPA was introduced over years ago as a relatively weak and poorly selective TRagonist its mode of interaction with TRs has not been specifically investigated so farPotential Uses of T2 and T1AMWhile Triac is basically a thyromimetic other active THmetabolites Fig can interact with different moleculartargets T2 has direct mitochondrial actions allegedly onrespiratory chain complex IV subunit Va and it stimulatesmitochondrial respiration and fatty acid oxidation These metabolic responses are further supported by genomiceffects whose molecular basis is unclear since they appear tobe different from those elicited by T3 In any case in ratstreated with highfat diet exogenous T2 decreased serumtriglycerides and LDL cholesterol as well as liver fat andbiochemicalindices of liver injury suggesting potentialtherapeutic value for either dyslipidemia or NAFLD However it is still controversial whether these positive 0cTHYROID HORMONE ANALOGUESeffects may be achieved without inducing tachycardia orcardiac hypertrophy since cardiac thyrotoxicosis has beenreported in mice A single pilot investigation was performed in two humanvolunteers taking T2 lgkg for weeks A slightbut significant decrease in body weight was reported without any change in serum T3 T4 TSH and cardiac function More recently a synthetic T2 analogue TRC150094was used in patients with metabolic syndrome but theresults were rather disappointing since no significant effect on insulin sensitivity and plasma lipid profile was observed The last entry into the group of active TH metabolites isrepresented by T1AM It does not interact with canonical ornoncanonical TH targets and it was discovered as a highaffinity agonist of TAAR1 a membrane G proteincoupledreceptor GPCR that is expressed in the brain and manyother tissues T1AM is a biogenic amine and sharesseveral properties of this class of compounds includingthe ability to interact with multiple targets namely otherGPCRs eg a2A adrenergic receptor membrane ionicchannels eg TRPM8 and monoamine transporters In experimental animals the administration of exogenousT1AM induced many different functional effects Neurological and metabolic effects are particularly interesting sincethey appear to be elicited at relative low doses and mighthave physiological relevance The former includesmodulation of feeding behavior and sleepwake cycle reduction of pain threshold prolearning and antiamnestic responses The major metabolic effects consistin thestimulation of triglyceride and fatty acid catabolism but antiinsulin effects on glucose metabolism are also elicited atslightly higher dosesAlthough the administration of exogenous T1AM has notbeen tested in humans some experimental results obtainedin murine models of disease have raised interest aboutpotential therapeutic applications T1AM reduced serumcholesterol in spontaneously obese mice as well asliver triglycerides in a mouse model of polycystic ovarysyndrome Neuroprotective effects appear even more promising Intracerebral T1AM rescued longterm potentiation and behavioral evidence of cognitive dysfunction in an in vivomodel of bamyloid toxicity namely transgenic mice overexpressing humanmutated amyloid precursor protein In addition the intracerebral injection of T1AM metabolite iodothyroacetic acid protected from the convulsive effect ofpentylenetetrazole and from kainate toxicity while exogenous T1AM reduced apoptosis and functional injury in amouse model of spinal cord clamp Because of the multitude of T1AM effects an active research line is focused on the development of synthetic derivatives with more favorable biodistribution andor receptorselectivity In conclusion the development TH analogues was initially prompted by the attempt to exploit the effects of THon lipid metabolism while avoiding unwanted cardiac effects TRb agonists have provided good results in a fewsmall clinical trials performed in hypercholesterolemic patients but these projects have been terminated after the report of potential side effects In recent years TRb agonistshave raised new interest for the treatment of NAFLD and acouple of clinical trials have provided encouraging initialresults Triac has already found clinical use in the treatmentof selected cases of TH resistance due to TRb mutationsand interesting results have recently been reported in theAllan“Herndon“Dudley syndromeOther TH analogues are under consideration for neurological diseases although human results are not yetavailable In particular sobetirome derivatives have beensuccessfulin animal models of multiple sclerosis andT1AM has been beneficial in an animal model of bamyloidtoxicityOverall research on TH analogues is experiencing a shiftin its focus but it still appears to be an active and promisingfieldAuthor Disclosure StatementNo competing financial interests existFunding InformationThis work was supported by a grant from Pisa UniversityPRA to RZReferences OrtigaCarvalho TM Sidhaye AR Wondisford FE Thyroid hormone receptors and resistance to thyroid hormone disorders Nat Rev Endocrinol “ Flamant F Cheng SY Hollenberg AN Moeller LC Samarut J Wondisford FE Yen PM Refetoff S Thyroidhormone signaling pathways time for a more precise nomenclature Endocrinology “ Davis PJ Goglia F Leonard JL Nongenomic actionsof thyrid hormone Nat Rev Endocrinol “ Senese R de Lange P Petito G Moreno M Goglia FLanni A 35diiodothyronine a novel thyroid hormone metabolite and a potent modulator of energy metabolism Front Endocrinol Lausanne Groeneweg S Peeters RP Visser TJ Visser WE Triiodothyroacetic acid in health and disease J Endocrinol234R99“R121 Hoefig CS Zucchi R Ko¨ hrle J Thyronaminesand derivatives physiological relevance pharmacological actions and future research directions Thyroid “ Ko¨hrle J Biebermann H 3iodohyronamine”a thyroid hormone metabolite with distinct target profiles andmodes of action Endocrine Rev “ Zucchi R Rutigliano G Saponaro F Novel thyroidhormones Endocrine “ Joharapurak AA Dhote VV Jain MR Selectivethyromimetics using receptor and tissue selectivity approaches prospects for dyslipidemia J Med Chem “ Mondal S Mugesh G Novel thyroid hormone analogues enzyme inhibitors and mimetics and their actionMol Cell Endocrinol “ Tancevski I Rudling M Eller P Thyromimetics ajourney from bench to bedside Pharmacol Ther “ Lin VW Klepp HM Hanley RM Sobetirome is aTRb and liverselective thyromimetic that can affectsubstantial LDLC lowering without significant changes inheart rate or the thyroid axis in euthyroid men [abstract] 0cZUCCHISan FranciscoENDO OR3633the Endocrine Society Annual Meeting Berkenstam A Kristensen J Mellstro¨m K Carlsson BMalmJ Rehnmark S Garg N Andersson CM Rudling MSjo¨berg F Angelin B Baxter JD The thyroid mimeticcompound KB2115 lowers plasma LDL cholesterol andstimulates bile acid synthesis without cardiac effects inhumans Proc Natl Acad Sci U S A “ Taub R Chiang E ChabotBlanchet M Kelly MJ ReevesRA Guertin MC Tardif JC Lipid lowering in healthyvolunteers treated with multiple doses of MGL3196 alivertargeted thyroid hormone receptorb agonist Atherosclerosis “ Ladenson PW Kristensen JD Ridgway EC Olsson AGCarlsson B Klein I Baxter JD Angelin B Use of thethyroid hormone analogue eprotirome in statintreateddyslipidemia N Engl J Med “ Sjouke B Langslet G Ceska R Nicholls SJ Nissen SEOhlander M Ladenson PW Olsson AG Hovingh GKKastelein JJ Eprotirome in patients with familial hypercholesterolaemia the AKKA trial a randomizeddoubleblind placebocontrolled phase study LancetDiabetes Endocrinol “ European Association for the Study of the Liver EASLEuropean Association for the Study of Diabetes EASDEuropean Association for the Study of Obesity EASO ESALEASDEASO clinical practice guidelines forthe management of nonalcoholic fatty liver diseaseJ Hepatol “ Coppola M Glinni D Moreno M Cioffi F Silvestri EGoglia F Thyroid hormone analogues and derivativesactions in fatty liver World J Hepatol “ Sinha RA Singh BK Yen PM Direct effects of thyroid hormones on hepatic lipid metabolism Nat Rev Endocrinol “ Martagon AJ Lin JZ Cimini SL Webb P Phillips KJ The amelioration of hepatic steatosis by thyroid hormonereceptor agonists is insufficient to restore insulin sensitivityin obob mice PLoS One 10e0122987 Puliga E Min Q Tao J Zhang R PradhanSundd TPoddar M Singh S Columbano A Yu J Momga SP Thyroid hormone receptorb agonist GC1 inhibits metbcatenindriven hepatocellular cancer Am J Pathol “ Harrison SA Bashir MR Guy CD Zhou L Moylan CAFrias JP Alkhouri N Bansal MB Baum S NeuschwanderTetri BA Taub R Moussa SE Resmetirom MGL for the treatment of nonalcoholic steatohepatitis amulticenter randomized doubleblind placebocontrolledphase trial Lancet “ Loomba R Neutel J Bernard D Severance R Mohseni RDao M Saini S Margaritescu C Homer K Tran B Mancini M Masamune H Lian B VK2809 a novel liverdirected thyroid receptor beta agonist significantly reducesliver fat in patients with nonalcoholic fatty liver disease aphase randomized placebocontrolled trial [abstract]Hepatology 681447A Zhang M Ma Z Qin H Yao Z Thyroid hormonepotentially benefits multiple sclerosis via facilitating remyelination Mol Neurobiol “ Placzek AT Ferrara SJ Hartley MD SanfordCrane HSMeining M Scanlan TS Sobetirome prodrug esterswith enhanced bloodbrain barrier permeability BioMed Chem “ Hartley MD Banerji T Tagge IJ Kirkemo LL ChaudharyP Calkins E Galipeau D Shokat MD DeBell MJ VanLeuven S Miller H Myelin repair stimulated by CNSselective thyroid hormone action JCI Insights 4e126329 Kemp S Huffnagel IC Linthorst GE Wanders RJ EngelenM Adrenoleukodystrophy”neuroendocrine pathogenesis and redefinition of natural history Nat Rev Endocrinol “ Hartley MD Kirkemo LL Banerji T Scanlan TS Athyroid hormonebased strategy for correcting the biochemical abnormality in Xlinked adrenoleukodystrophyEndocrinology “ Refetoff S Dumitrescu AM Syndromes of reducedsensitivity to thyroid hormone genetic defects in hormonereceptors cell transporters and deiodination Best PractClin Endocrinol Metabolism “ Groeneweg S Peeters RP Visser TJ Visser WE Therapeutic applications of thyroid hormone analogues inresistance to thyroid hormone RTH syndromes Mol CellEndocrinol “ Friesema ECH Visser WE Visser TJ Genetics andphenomics of thyroid hormone transport by MCT8 MolCell Endocrinol “ Schwartz CE Stevenson RE The MCT8 thyroidhormone transporter and AllanHerndonDudley Syndrome Best Pract Clin Endocrinol Metab “ Groeneweg S Peeters RP Moran C Stoupa A Aurial FTonduti D Dica A Paone L Rozenkova K Malikova Jvan der Walt A de Coo IFM McGowan A Lyons GAarsen FK Barca D van Beynum IM van der Knoop MMJansen J Manshande M Lunsing RJ Nowak S den UilCA Zillikens MC Visser FE Vrijmoeth P de Wit MCYWolf NI Zandstra A Ambegaonkar G Singh Y de RijkeYB Medici M Bertini ES Depoorter S Lebl J Cappa MDe Meirleir L Krude H Craiu D Zibordi F Oliver Petit IPolak M Chatterjee K Visser TJ Visser WE Effectiveness and safety of the triiodothyronine analogue Triacin children and adults with MCT8 deficiency an international single arm label phase trial Lancet Diabetes Endocrinol “ Verge CF Konrad D Cohen M Di Cosmo C DumitrescuAM Marcinkowski T Hameed S Hamilton J Weiss RERefetoff S Diiodothyropionic acid DITPA in thetreatment of MCT8 deficiency J Clin Endocrinol Metab“ Goglia F The effects of 35diiodothyronine on energybalance Front Physiol Jonas W Lietzow J Wohlgemuth F Hoefig CS WiedmerP Schweizer U Ko¨hrle J Schurmann A 35DiiodoLthyronine 35T2 exertsthyromimetic effects onhypothalamuspituitarythyroid axis body compositionand energy metabolism in male dietinduced obese miceEndocrinology “ Antonelli A Fallahi P Ferrari SM Di Domenicantonio AMoreno M Lanni A Goglia F 35diiodoLthyronineincreases resting metabolic rate and reduces body weightwithout undesirable side effects J Biol Regul HomeostAgents “ Van der Val F Hassing C Visser M Thakkar P MohananA Pathak K Dutt C Chauthaiwale V Ackermans MNederveen A Serlie M Nieuwdorp M Stroes E Theeffect of a diodothyronine mimetic on insulin sensitivity inmale cardiometabolic patients a doubleblind randomizedcontrolled trial PLoS One 9e86890 0cTHYROID HORMONE ANALOGUES Zucchi R Chiellini S Scanlan TS Grandy DK Traceamineassociated receptors and their ligands Br J Pharmacol “ Rutigliano G Accorroni A Zucchi R The case forTAAR1 as a modulator of central nervous system functionFront Pharmacol Zucchi R Accorroni A Chiellini G Update on iodothyronamine and its neurological and metabolic actions Front Physiol AssadiPorter FM Reiland H Sabatini M Lorenzini LCarnicelli V Rogowski M Selen Alpergin ES Tonelli MGhelardoni S Saba A Zucchi R Chiellini G Metabolic reprogramming by 3iodothyronamine T1AM anew perspective to reverse obesity through regulation ofsirtuin and expression Int J Mol Sci 19e1535 Selen Alpergin ES Bolandnazar Z Sabatini M RogowskiM Chiellini G Zucchi R AssadiPorter FM Metabolic profiling reveals reprogramming of lipid metabolicpathways in treatment of polycystic ovary syndrome with3iodothyronamine Physiol Rep 5e13097 Accorroni A Rutigliano G Sabatini M Frascarelli S BorsoM Novelli E Bandini L Ghelardoni L Saba A Zucchi ROriglia N Exogenous 3iodothyronamine rescues theentorhinal cortex from bamyloid toxicity Thyroid “ Laurino A Landucci E Resta F De Siena F PellegriniGiampietro DE Masi A Mannaioni G Raimondi L Anticonvulsant and neuroprotective effects of the thyroidhormone metabolite 3Iodothyroacetic Acid Thyroid “ Lv J Liao J Tan W Yang L Shi X Zhang H ChenL Wang S Li Q 3Iodothyronamine acting throughan antiapoptotic mechanism is neuroprotective againstspinal cord injury in rats Ann Clin Lab Sci “

Thyroid_Cancer

breast cancer patients especially those with triple‘negative breast cancer is still grave More effective therapeutic targets are needed to optimize the clinical management of breast cancer Although collagen type VIII alpha chain COL8A1 has been shown to be downregulated in BRIP1‘knockdown breast cancer cells its clinical role in breast cancer remains unknownMethods Gene microarrays and mRNA sequencing data were downloaded and integrated into larger matrices based on various platforms Therefore this is a multi‘centered study which contains breast cancer patients and controls COL8A1 mRNA expression in breast cancer was compared between molecular subtypes In‘house immunohistochemistry staining was used to evaluate the protein expression of COL8A1 in breast cancer A diagnostic test was performed to assess its clinical value Furthermore based on differentially expressed genes DEGs and co‘expressed genes CEGs positively related to COL8A1 functional enrichment analyses were performed to explore the biological function and potential molecular mechanisms of COL8A1 underlying breast cancerResults COL8A1 expression was higher in breast cancer patients than in control samples standardized mean differ‘ence confidence interval [CI] “ Elevated expression was detected in various molecular subtypes of breast cancer An area under a summary receiver operating characteristic curve of CI “ with sensitivity of CI “ and specificity of CI “ showed moderate capacity of COL8A1 in distinguishing breast cancer patients from control samples Worse overall survival was found in the higher than in the lower COL8A1 expression groups Intersected DEGs and CEGs positively related to COL8A1 were significantly clustered in the proteoglycans in cancer and ECM‘receptor interaction pathwaysConclusions Elevated COL8A1 may promote the migration of breast cancer by mediating the ECM‘receptor interac‘tion and synergistically interplaying with DEGs and its positively related CEGs independently of molecular subtypes Several genes clustered in the proteoglycans in cancer pathway are potential targets for developing effective agents for triple‘negative breast cancerKeywords COL8A1 Breast cancer Immunohistochemistry staining MechanismCorrespondence fengzhenbo_gxmu163com chenganggxmueducn Wei Peng and Jian‘Di Li contributed equally as first authors Department of Pathology The First Affiliated Hospital of Guangxi Medical University NO6 Shuangyong Road Nanning Guangxi People™s Republic of ChinaFull list of author information is available at the end of the BackgroundBreast cancer poses a grave threat to female health According to the latest American cancer statistics breast cancer is estimated to be the most common cancer and the second most common cause of cancerassociated The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cPeng a0et a0al Cancer Cell Int Page of deaths in women [] Owing to higher distal metastatic and recurrent rates triplenegative breast cancer TNBC patients exhibit worse overall and diseasefree survival than any other type of breast cancer [“] Etiology investigations suggest that hereditary factors account for nearly onetenth of breast cancer cases Other risk factors such as early or delayed menstruation nulliparity hormone replacement therapy and alcohol consumption also contribute to the prevalence of breast cancer [] Clinical practice guidelines recommend that females aged “ or “ who are at higher risk screen for breast cancer [] Imaging examinations such as bilateral breast Xray imaging positron emission tomography“computed tomography and ultrasound histological findings and especially molecular pathology are the predominant methods of breast cancer diagnosis and assessment [ ] Based on the tumor burden optimal treatments namely breastconserving surgery radiotherapy chemotherapy and endocrine therapy are individually designed for breast cancer patients [] For TNBC patients anthracyclines and taxanes are preferred in the initial treatment and neoadjuvant therapy has been recognized as a standard strategy [“] Unfortunately neither endocrine therapy nor trastuzumab treatment is effective for TNBC Targeted drugs for example vascular endothelial growth factor [VEGF] antibodies epidermal growth factor receptor [EGFR] inhibitors and mammalian target of rapamycin [mTOR] inhibitors are gradually being employed in TNBC treatment even though their therapeutic effects are unsatisfactory [“] Therefore the situation faced by breast cancer”especially TNBC”patients is still grave More effective therapeutic targets are needed to optimize the clinical management of breast cancerMolecular events occurring in breast cancer help us better understand the onset and progression of breast cancer It is generally agreed that chromosome 1q amplification chromosome 16q deletion and PIK3CA mutations are the most common pathways leading to luminal breast cancer [ ] Moreover breast cancer gene BRCA1 and P53 mutations EGFR upregulation and cytokeratin downregulation have been associated with TNBC [ ] It has also been reported that circSEPT9 promotes tumor formation and TNBC progression [] Recently ˆ†Np63 has been found to participate in breast cancer metastasis and dissemination [] On the other hand several genes protect patients from breast cancer progression For example ZNF750 miR5745p and circKDM4C can inhibit breast cancer progression by mediating the epigenetic regulation of prometastatic genes indirectly suppressing SKILTAZCTGF and miR548pPBLD axis regulation [“] Based on these discovered molecular mechanisms some progress has been made in breast cancer treatment Delivering dual microRNA using CD44targeted mesoporous silica nanops proved to be effective in TNBC treatment [] Although many studies provided in a0vitro and in a0vivo a detailed molecular picture of TNBC cancers [“] the underlying cause of breast cancer has not been fully understood Further research is required to elucidate the breast cancer mechanisms and discover effective therapeutic targets for TNBCCollagen type VIII alpha chain COL8A1 also named C3orf7 is located at chromosome and encodes alpha chain in collagen type VIII which is an essential component of extracellular matrix ECM [] Previous studies mainly addressed the relevance between COL8A1 and agerelated macular degeneration ADM as well as cell proliferation [“] Recently limited studies demonstrate the deregulation of COL8A1 in various cancers Elevated COL8A1 expression was found in gastric cancer patients and higher COL8A1 correlated with advanced tumor stages and worse overall survival condition and COL8A1 was selected as a candidate diagnostic biomarker in gastric cancer [“] Additionally upregulation of COL8A1 was also reported in adamantinomatous craniopharyngioma [] Furthermore COL8A1 proved to participate in the progression of colon adenocarcinoma possibly by mediating focal adhesionrelated pathways [] COL8A1 upregulation induced by TGFβ1 was found in renal cell carcinoma carcinogenesis and also correlated with poor prognosis [] Moreover elevated COL8A1 in hepatocellular carcinoma promoted tumor cells proliferation invasion and in a0vivo tumorigenicity [] Thus far only few studies mentioned COL8A1 in breast cancer COL8A1 was one of the key genes restored by epigallocatechin3gallate in a murine breast cancer model [] COL8A1 proved downregulated in both BRIP1knockdown breast cancer cells and MCF10A a0CDH1 noncancer breast cells [ ] However the role of COL8A1 in breast cancer remains unknownConsidering this knowledge gap our study aimed to explore the role of COL8A1 in breast cancer We were focused on investigating the expression of COL8A1 messenger RNA mRNA in various molecular subtypes of breast cancer by analyzing gene microarray and RNA sequencing data sets The COL8A1 protein expression level was validated by immunohistochemistry staining We also aimed to determine prognostic value of COL8A1 in breast cancer to pave the way for future clinical applications Moreover we explored the molecular mechanisms of COL8A1 underlying breast cancer to improve our knowledge of breast cancer carcinogenesis and progression 0cPeng a0et a0al Cancer Cell Int Page of MethodsExpression of a0COL8A1 mRNA in a0breast cancerWe integrated gene microarrays and mRNA sequencing data downloaded from Gene Expression Omnibus The Cancer Genome Atlas TCGA the GenotypeTissue Expression the Sequence Read Archive ArrayExpress and Oncomine The search formula based on MESH terms was as follows Breast OR mammary AND neoplasm OR cancer OR adenoma OR carcinoma OR tumor OR BRCA OR neoplasia OR malignant OR malignancy Studies were screened according to the following criteria i the studied species should be Homo sapiens ii the studied specimens should be tissue dissected from patients or healthy individuals rather than cell lines In the case of duplicated studies or samples the most recent version was retained The exclusion criteria were as follows i expression profiles not including COL8A1 ii breast cancer patients receiving hormone therapy or chemotherapy iii stromal rather than epithelial tumors and iv metastatic rather than primary tumors The included data sets were carefully checked and a log2 transformation was performed if any matrices had not been normalized Additionally the data sets were integrated into larger matrices according to various platforms and batch effects between studies were removed using the limmavoom package in R v361 Subsequently COL8A1 expression values were extracted and grouped according to specimen types Standardized mean difference SMD were calculated to compare the expression of COL8A1 mRNA between breast cancer patients and control samples using STATA v120 Heterogeneity between the included studies was assessed with the I2 statistic Statistical significance was set to an I2 value greater than with a Pvalue less than A random effects model was used in the case of significant heterogeneity Sensitivity analysis was performed to probe the potential source of heterogeneity and a publication bias test was used to evaluate the stability of the SMD results Subgroup analysis was performed to compare the COL8A1 expression levels between molecular subtypes luminal A luminal B human epidermal growth factor receptor 2positive [HER2 ] and TNBCDiagnostic value of a0COL8A1 in a0breast cancerA diagnostic test was performed to assess the clinical significance of COL8A1 in breast cancer Based on the expression value of COL8A1 a receiver operating characteristic ROC curve was plotted to compute the area under the curve AUC using IBM SPSS Statistics v190 AUC values of less than between and and greater than represented weak moderate and strong discriminatory capability of COL8A1 respectively between breast cancer patients and control samples The true positives false positives true negatives and false negatives rates were calculated and the cutoff values were identified A summary receiver operating characteristic sROC curve was drawn using STATA v120 to assess the general discriminatory capability of COL8A1 between breast cancer patients and control samples The significance of the area under the sROC curve was consistent with that of the ROC curve The diagnostic odds ratio DOR sensitivity specificity positive diagnostic likelihood ratio DLR P and negative diagnostic likelihood ratio DLR N were also calculated to precisely determine the accuracy and validity of COL8A1 in distinguishing breast cancer patients from control samplesPrognostic value of a0COL8A1 in a0breast cancerTo explore the relation between COL8A1 mRNA expression and prognosis of breast cancer patients information on clinicopathological parameters was collected The independent samples ttest or oneway analysis of variance was used to identify statistically significant differences in COL8A1 expression between two or more groups A Pvalue of was considered statistically significant Kaplan“Meier curves were used to compare high and low COL8A1 expression groups in terms of survival The logrank test was used to determine whether the prognostic difference was statistically significantInvestigation of a0COL8A1 protein expression in a0breast cancer by a0immunohistochemistry stainingA total of nonspecific invasive breast carcinoma and normal breast tissue specimens were obtained from the First Affiliated Hospital of Guangxi Medical University PRCHINA All patients had previously signed informed consent forms and our research was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University The breast cancer and normal breast tissue specimens were fixed with formalin The two steps immunohistochemistry method was used to determine the protein expression of COL8A1 The primary antibody was polyclonal Antibody to COL8A1 concentrated dilution purchased from Wuhan Pujian COLTD Supervision TM MouseRabbitHRP Broad Spectrum Detection System Product No D300415 was purchased from Shanghai Long Island The experimental procedure conformed to the manufacturer™s instructions The patients™ clinicopathological information was analyzed to determine the relationship between COL8A1 protein expression and prognosisEvaluation of a0genetic alteration and a0mutation landscapesThe cBioPortal for Cancer Genomics httpcbiop ortal proved to be a powerful tool and facilitated our online search and cancer genomics data analysis Using 0cPeng a0et a0al Cancer Cell Int Page of cBioPortal we gained insight into the genetic alterations of COL8A1 in breast cancer patients and obtained information on the association between COL8A1 alterations and breast cancer patient survival We selected the Breast Invasive Carcinoma TCGA Firehose Legacy cohort which contains patients and used a method of mRNA expression zscores relative to diploid samples RNASeqV2 RSEM We also considered the mutation types of COL8A1 in breast cancer patients using the Catalogue of Somatic Mutations in Cancer COSMIC which has been recognized as the most detailed resource for somatic mutations in cancerIdentification of a0differentially expressed genes and a0COL8A1 co‘expressed genes in a0breast cancerTo gain insight into the role of COL8A1 in breast cancer we identified DEGs and COL8A1 CEGs using the limmavoom package The DEG criteria were as follows ilog2FoldChange and ii adjusted Pvalue The CEG criteria were as follows irelation coefficient and ii Pvalue Upregulated DEGs and CEGs positively related to COL8A1 were intersected Similarly downregulated DEGs and CEGs negatively related to COL8A1 were intersectedMolecular mechanisms of a0COL8A1 underlying breast cancerOverlapping genes were used to perform function enrichment to shed light on the potential mechanisms of COL8A1 underlying breast cancer The R clusterProfiler package was used to conduct Gene Ontology GO Kyoto Encyclopedia of Genes and Genomes KEGG Disease Ontology DO and Reactome pathway analyses Proteintoprotein interaction PPI network was constructed using STRING https strin gdb to investigate protein interactions Hub genes and functional modules were identified using Cytoscape v361 Based on breast cancer patients the mutation landscapes of genes clustered in essential pathways were visualized using the TCGAmutations package in R v361ResultsUpregulation of a0COL8A1 mRNA in a0breast cancerAdditional file a0 Figure S1 shows the flow diagram of the study inclusion process A total of studies were included and integrated into larger platform matrices covering breast cancer patients and controls Table a0 COL8A1 was generally upregulated in breast cancer compared to normal breast tissue Thirteen of the twenty platforms showed much higher COL8A1 expression in breast cancer patients than in control samples Additional file a0 Figure S2 Because of significant heterogeneity I2 P a random effects model was used An SMD value of confidence interval [CI] “ showed that COL8A1 expression was significantly higher in breast cancer than in nonbreast cancer tissue Fig a0 Sensitivity analysis indicated that the included studies could not explain the source of heterogeneity Additional file a0 Figure S3a No publication bias existed Additional file a0 Figure S3b Subsequently we compared COL8A1 expression levels between different subtypes of breast cancer COL8A1 expression was universally higher in luminal A luminal B HER2 and TNBC patients than in control samples Additional file a0 Figure S4 Additional file a0 Figure S5 and Additional file a0 Figure S6a Furthermore three platforms showed significantly higher COL8A1 expression in nonTNBC than TNBC while only one showed lower expression in nonTNBC than TNBC Additional file a0 Figure S6b However an SMD of ˆ’ CI ˆ’“ showed no difference in COL8A1 expression between TNBC and nonTNBC patients Additional file a0 Figure S7 Subgroup analysis of four molecular subtypes of breast cancer showed no significant differences in COL8A1 expression levels between them Fig a0Diagnostic and a0prognostic value of a0COL8A1 mRNA in a0breast cancerThe clinical value of COL8A1 in breast cancer was found to be promising Among the thirteen platforms showing high COL8A1 expression twelve platforms indicated the ability of COL8A1 in differentiating breast cancer patients and control samples where four platforms showed strong discriminatory capability of COL8A1 between breast cancer patients and control samples Additional file a0 Figure S8 An area under the sROC curve of CI “ with sensitivity of CI “ and specificity of CI “ displayed moderate capacity in distinguishing breast cancer patients from control samples Fig a03a A DOR of CI “ also highlighted the discriminatory ability of COL8A1 in breast cancer Fig a03b The DLR P and DLR N were CI “ and CI “ respectively Additional file a0 Figure S9 As shown in Additional file a0 Figure S10 and Additional file a0 Table a0S1 elevated COL8A1 expression correlated with race white black molecular subtypes of breast cancer luminal B luminal A TNBC ER PR and HER2 status Moreover Kaplan“Meier curves indicated worse overall survival in high compared to low COL8A1 expression groups Fig a0Expression levels and a0clinical significance of a0COL8A1 protein in a0breast cancerClinical data of breast cancer samples used to perform in immunohistochemistry was summarized 0cPeng a0et a0al Cancer Cell Int Page of ldnaoPESGi abarA iduaSESG ecnarFESG ASUESG inapSESG ecnarFESG ecnarFESG adanaCESGkramneDESGi eropagnSESGl yatIESG ASUESG ynamreGESG inapSESG ASUESGASUESG cil bupeRhcezCESG ldnaerIESGi eropagnSESGASUESG ailartsuAESG iocxeMESGi abarA iduaSESGASUESG ASULPG‘ESGadanaCESG ASUESG adanaCESGASULPG‘ESGASULPG‘ESGadanaCESGianhCESG anhCi ESG ASUESG ASUESGianhCESGl aisyaaMESGi anhCESGadanaCESGi anhCESGASUESGi anhCESGASUESGi eropagnSESGl aisyaaMESGASUESG ESG ESG ESG ESG ESGASUESG ESGASU ESGASU ESGanhCi ESGynamreG ESGASU ESGˆ’‘ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’LPGLPGLPGLPGLPGLPGLPGˆ’LPGLPGLPGLPGLPGLPGLPGxETG‘AGCTESGESGESGESGESGsrebmun latoTseirtnuoC seireSOEG NTNFPFPTleuav‘PfdtlortnoC ACRBnoisseccADSMNDSMN stesataddell a0 orneeht a0fonoitamrofni cisaB elbaT 0cPeng a0et a0al Cancer Cell Int Page of Fig General expression status of COL8A1 in breast cancer BRCA compared to non‘BRCA tissues A standardized mean difference SMD value and 95CI with no overlap of zero indicated COL8A1 was significantly upregulated in BRCA compared to non‘BRCA tissuesAdditional file a0 Table a0 S2 Protein expression confirmed the upregulation of COL8A1 in breast cancer The breast cancer patients whose specimens were analyzed in this study were aged between and mean and their followup durations ranged from to a0 days Immunohistochemistry staining showed varying coloration intensity of COL8A1 in breast cancer and normal breast tissue COL8A1 was negatively weakly moderately or strongly stained in normal breast epithelium Fig a05a“d and breast cancer tissue Fig a0 5e“h According to the staining intensity and color range percentages of breast cancer tissue specimens exhibited low and exhibited high COL8A1 expression whereas of normal breast tissue specimens exhibited low and exhibited high COL8A1 expression A Chi square test confirmed the significantly higher expression of COL8A1 in breast cancer than normal breast tissue χ2 P Moreover elevated COL8A1 expression correlated with estrogennegative ER breast cancer P Genetic alterations and a0mutation kinds of a0COL8A1 in a0breast cancerAlterations and mutations of COL8A1 in breast cancer were relatively frequent Based on cBioPortal COL8A1 was altered in of breast cancer patients Additional file a0 Figure S11 Amplification and high and low mRNA were the main alterations No statistically significant difference in overall and diseasefree survival was found between high and low COL8A1 expression breast cancer groups P Furthermore according to COSMIC substitution missense mutations were the most frequent types Additional file a0 Table a0S3DEGs and a0COL8A1 CEGs in a0breast cancerA total of platform matrices were collected to determine the DEGs The approach has been aforementioned Initially upregulated DEGs downregulated DEGs CEGs positively related to COL8A1 and CEGs negatively related to COL8A1 were identified Additional file a0 Figure S12 shows partial DEGs and CEGs After being intersected 0cPeng a0et a0al Cancer Cell Int Page of Fig Subgroup analysis based on the subtypes of breast cancer The result indicated that the elevated COL8A1 expression shared no significant difference among Luminal A Luminal B HER‘ and Three Negative Breast Cancer TNBC subgroupsthe DEGs and CEGs were divided into two gene sets overlapping upregulated DEGs and CEGs positively related to COL8A1 all genes appeared in no fewer than three data sets and overlapping downregulated DEGs and CEGs negatively related to COL8A1Potential mechanisms of a0COL8A1 underlying breast cancerThe GO KEGG DO and Reactome pathway analyses based on the intersected genes are shown in Additional file a0 Table a0S4 Regarding the overlapping upregulated DEGs and CEGs positively related to COL8A1 the following KEGG pathways were significantly aggregated 0cPeng a0et a0al Cancer Cell Int Page of Fig Diagnostic value of COL8A1 in breast cancer BRCA a Summary receiver operating characteristic sROC curve b Forest plot of diagnostic odd ratio DOR An AUC value and a DOR signified COL8A1 possessed moderate capability in distinguishing BRCA from non‘BRCA patients AUC area under the curve 0cPeng a0et a0al Cancer Cell Int Page of Fig The prognostic value of COL8A1 mRNA in breast cancer tissues a GSE25307 b GSE35629‘GPL1390 c TCGA In the GSE25307 cohort high COL8A1 group possessed poor overall survival condition compared to low COL8A1 group in breast cancer patients 0cPeng a0et a0al Cancer Cell Int Page of See figure on next pageFig Protein expression levels of COL8A1 in breast cancer BRCA and normal breast tissues a‘d normal breast tissues e“h BRCA tissues Magnification × COL8A1 was negatively stained in normal breast epithelium a and BRCA e COL8A1 was weakly stained in normal breast epithelium b and BRCA f COL8A1 was moderately stained in normal breast epithelium c and BRCA g COL8A1 was strongly stained in normal breast epithelium d and BRCA h According to staining intensity and percentage of color range BRCA tissues exhibited low COL8A1 expression and BRCA tissues exhibited high COL8A1 expression While normal breast tissues exhibited low COL8A1 expression and normal breast tissues exhibited high COL8A1 expressionFig a06a proteoglycans in cancer Additional file a0 Figure S13 ECMreceptor interaction Additional file a0 Figure S14 and several cancer pathways such as thyroid cancer colorectal cancer and hepatocellular carcinoma Interestingly genes WNT2 GADD45B FZD2 CDKN1A KRAS LEF1 WNT7B BAK1 BRCA2 CDK4 GRB2 HRAS PIK3R3 and POLK were associated with the breast cancer pathway ID hsa05224 even though is not in the top KEGG pathways Moreover DO analysis indicated that these genes are closely associated with myeloma and bone marrow cancer Fig a06b as well as renal cell carcinoma ovarian cancer renal carcinoma and other types of cancer Furthermore Reactome pathway analysis revealed extracellular matrix anization ECM proteoglycans integrin cell surface interactions and degradation of the extracellular matrix as the top four metabolic pathways Fig a0 6c Regarding GO enrichment extracellular matrix anization extracellular matrix and extracellular matrix structural constituent were the most clustered Biological Process BP Cellular Component CC and Molecular Function MF terms respectively Fig a07a The proteoglycans in cancer and ECMreceptor interaction pathways were selected to construct PPI networks Fig a07b c FN1 and ITGB1 were identified as the hub genes in the two networks respectively The mutation landscapes of the genes in these two important pathways are shown in Fig a0 In particular FN1 was altered in of breast cancer samples where missense mutations accounted for The regulatory networks of COL8A1 and enriched genes in the proteoglycans in cancer and ECMreceptor interaction pathways as well as the top two functional modules are displayed in Additional file a0 Figure S15 On the other hand the enrichment results regarding the overlapping CEGs negatively related to COL8A1 and downregulated DEGs showed no statistical significance these data are therefore not shown Additional file a0 Table a0S5DiscussionThe highlight of this study is that it comprehensively explored the upregulation of COL8A1 mRNA in breast cancer from multiple aspects based on breast cancer patients and controls Our study is multicentered because we collected breast cancer patients from Asia American Europe and Oceania covering different countries This is the first study to investigate the protein expression of COL8A1 in breast cancer using immunohistochemistry staining Moreover this study is the first to assess the clinical prognostic value of COL8A1 in breast cancer Furthermore our study sheds light on the biological function and potential molecular mechanisms of COL8A1 underlying breast cancerThis study demonstrates the upregulation of COL8A1 in breast cancer We found higher COL8A1 expression in breast cancer than normal breast tissue based on large platform matrices integrated from data sets Subgroup analysis based on four molecular subtypes of breast cancer showed that elevated COL8A1 expression is independent of subtypes Further analysis also revealed universally higher COL8A1 expression levels in luminal A luminal B HER2 and TNBC patients than in control samples A comparison of COL8A1 expression between nonTNBC and TNBC patients showed no statistically significant difference Immunohistochemistry staining confirmed the upregulation of COL8A1 protein in breast cancer We thus concluded that COL8A1 expression is higher in breast cancer patients than in control samples and that upregulation is independent of molecular subtypes of breast cancer Though the expression of COL8A1 in tissue cannot reflect the early diagnostic value in breast cancer we assume that if COL8A1 is also differentially expressed in the bodily fluid of patients it will be possible to serve as a potential diagnostic marker for breast cancer Nevertheless no previous studies have demonstrated the expression level of COL8A1 in the bodily fluid of breast cancer patients until nowWe determined the clinical value of COL8A1 in breast cancer for the first time Our diagnostic test showed a moderate discriminatory capability of COL8A1 between breast cancer and normal breast tissue Higher COL8A1 expression levels in breast cancer patients correlated with worse survival outcomes Additionally COL8A1 expression was much higher in patients of the white than of the black race Our TCGA cohort analysis showed lower COL8A1 upregulation levels in TNBC than in the luminal A and B subtypes Furthermore high COL8A1 protein levels were related to ER breast cancer Thus COL8A1 might serve as a prognostic marker for breast cancer 0cPeng a0et a0al Cancer Cell Int Page of 0cPeng a0et a0al Cancer Cell Int Page of Fig Functional enrichment based on overlapping genes of upregulated DEGs and COL8A1 positively related CEGs a Kyoto Encyclopedia of Genes and Genomes b Disease Ontology c Reactcome DEGs differentially expressed genes CEGs co‘expressed genes 0cPeng a0et a0al Cancer Cell Int Page of Fig GO enrichment based on overlapping genes of upregulated DEGs and COL8A1 positively related CEGs a GO analysis b Protein‘to‘protein internet based on KEGG pathway proteoglycans in cancer ID hsa05205 c Protein‘to‘protein internet based on KEGG pathway ECM‘receptor interaction ID hsa04512 GO Gene Ontology DEGs differentially expressed genes CEGs co‘expressed genes KEGG Kyoto Encyclopedia of Genes and GenomesMore importantly our study provides important clues about the role of COL8A1 in breast cancer for the first time The intersected CEGs positively related to COL8A1 and upregulated DEGs were significantly aggregated in several cancer pathways such as thyroid cancer colorectal cancer and hepatocellular carcinoma Interestingly we noticed that genes related to COL8A1 WNT2 GADD45B FZD2 CDKN1A KRAS LEF1 WNT7B 0cPeng a0et a0al Cancer Cell Int Page of Fig Mutation landscapes of COL8A1 and co‘expressed genes clustered in two Kyoto Encyclopedia of Genes and Genomes pathways proteoglycans in cancer and ECM‘receptor interactionBAK1 BRCA2 CDK4 GRB2 HRAS PIK3R3 and POLK were associated with the breast cancer pathway ID hsa05224 even though this pathway is not in

Thyroid_Cancer

"SARSCoV2 infection associated respiratory disease COVID19 has evolved into a pandemic but being anew form of virus pathogenesis of disease causation is not fully understood and drugs and vaccinesagainst this virus are still being tested so that no effective drugs or vaccines have been advised byregulatory authority In this context the Ministry of AYUSH Government of India has recommended˜Ayush Kwath™ to improve the immunity and combat the infection Our objective of this literature reviewis to review the role of immunity in pathogenesis of COVID19 and role of Ayush Kwath against the virusand regulation of immunity Current review was conducted using a search of available literature onCOVID19 and immunity Vyadhikshamatwa Ayurveda and COVID19 Rasayana Coronavirus SARSCoV immunomodulatory effects of medicinal plants TulsiHoly BasilOcimum sanctum DalchiniCinnamonCinnamomum zeylanicum SunthiGingerZingiber officinale and MarichBlack PepperPiper nigrum Ayurveda being an ancient science have both medicinal and cultural values and had stimulated our kitchenand ‚uenced what we ate in different seasons and the remedies we used for common ailments Herbssuch as Tulsi Marich Sunthi Dalchini are the most commonly used and easily available drugs in homeThus Ayush Kwath due to its immunemodulatory antiviral antioxidant anti‚ammatory antiplatelet antiatherosclerotic hepatoprotective renoprotective properties seems to be effective inimmunoregulation for controlling viral infections like COVID19 Further preclinical and clinical trialsneed to be done for the evaluation of safety and efficacy of this polyherbal formulation The Authors Published by Elsevier BV on behalf of Institute of Transdisciplinary Health Sciencesand Technology and World Ayurveda Foundation This is an access under the CC BYNCNDlicense httpcreativecommonslicensesbyncnd40 IntroductionCOVID19 also known as severe acute respiratory syndromecorona virus SARSCoV2 is an infectious disease believed to beoriginated from bats and transmitted to human beings [] Being anew form of virus pathogenesis of disease causation is not fullyunderstood and drugs and vaccines against this virus are still beingtested so that no effective drugs or vaccines have been advised byregulatory authority Not only for Coronavirus have many otherviruses also lack preventive vaccines and effective antiviral medications Studies have explored that these viruses can form drugresistant mutants which decrease the existing drug™s efficacy Sothese viruses can be a threat to the mankind for long time [] Corresponding authorEmail shankargautammohpgovnpPeer review under responsibility of Transdisciplinary University BangaloreHigh mortality among immunecompromised and those withsome underlying pathology implies that the factors that improveimmunity can prevent serious manifestations due to COVID19infection [] Many herbal products are found to have immunemodulatory and antiviral property so their discovery can be amilestone in the prevention and control of COVID19 [] In thiscontext the Government of India has recommended to take ˜AyushKwath™ in order to boost the immunity As this is a new formulationthis needs to be validated scientifically We have made an attemptto review the immunepathogenesis of COVID19 and the role ofeach herb over it Immunopathogenesis of COVID19The ˜S™ protein of coronavirus can bind to host cells through theACE2 receptor found in the oral and nasal mucosa [] Other siteswhere ACE2 receptors are found are lungs stomach intestinebladder heart and kidney [] Variable presentation of disease in101016jjaim202008003 The Authors Published by Elsevier BV on behalf of Institute of Transdisciplinary Health Sciences and Technology and World Ayurveda Foundation This isan access under the CC BYNCND license httpcreativecommonslicensesbyncnd40Please cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxdifferent age groups serious manifestations that are seen morecommonly in immunecompromised old aged and in those withunderlying pathology many asymptomatic cases in pediatric agegroup and presence of lymph ia in the majority of the casesthese factors implies that immunity has a vital role in the pathogenesis of COVID19 [16e8] It is assumed that our immune systemhas lack of memory against such a virus that gave it an edge overhumans []Viruses cause cell destruction mainly in two ways direct cytopathic effects ofthe virus and immune response mediateddestruction [] COVID19 cannot lyse the cells directly as the majorpathway of cell destruction is due to immunemediated destruction[] It has been mentioned that unlike adults less vigorous cellmediated immune response in alveoli of children results in beingasymptomatic in the majority of cases []The pathogenesis can be split into two stages Nonsevere andSevere [] Nonsevere stageThe virus fuses with the host cell membrane and enters insidethe host cell through airway epithelium [] The virus propagates and multiplies inside the host cell and can reach lower airwayand alveoli In adults with good innate cellular and humoral immunity propagation of virus can be limited and viral load reachingalveoli can be reduced thus recovery can take place within 2e3weeks with mild symptoms []Humoral immunity prevents the viruses to enter new cells whilecellmediated immunity targets on eradicating virusinfected cells[] In this stage a strong immune system can be helpful inpreventing the propagation of the virus thus reducing the severityof the disease [] Severe stageOnce the immune system is breached the virus propagates andreaches the lower respiratory tract and alveoli Then the virus canpenetrate alveoli and reaches systemic circulation causing viremia[] The virus binds to multiple ans having ACE2 receptor protein During this stage cellmediated immunity becomes robustand starts releasing various pro‚ammatory cytokines IFNaIFNg IL1B IL6 IL12 IL18 IL33 TNFa etc and chemokinesCCL2 CCL3 CCL5 CXCL8 CXCL9 CXCL10 etc causing damage tomultiple ans known as Cytokine storm [] We may need tosuppress the ‚ammation for improvement during this severestage []Tocilizumab and anti‚ammatory interleukin IL10 are proposed to have a therapeutic role in the reduction of severity and mortality of COVID19[] As increased risk of thromboembolic phenomena is alsofound to be associated with COVID19 prophylactic antithromboticmedications are advised during this stage []IL6 receptor antagonist Ayurveda purview Disease conceptIt seems that most early cases had a history of contact with theoriginal market for seafood but the disease has now advanced to betransmitted through human to human contact [] Thus this disease can be considered as Communicableboth contagious and infectious diseases In Ayurveda epidemics are discussed under theterm of Janapadodhwamsa [ CSVi ] by Charaka and Marakaby Sushruta [ SSSoo ] The symptoms like fever coughbreathing difficulty headache and vomiting resemble with clinicalfeatures of SARS [ SSSoo ] Dalhana in his commentary hasmentioned that symptoms like anosmia cough catarrh will occurafter the entry of contaminated air through the nasal ingwhich is similar to typical clinical features of COVID19 [ SSSoo] Furthermore this disease can be classified as Adidaivika BalaPravritta Vyadhi ABPV Sansargaja Upsargaja and Aupasargic RogaABPV are those diseases arising due to causes that cannot becontrolled by human intelligence Upasargaja Vyadhi are thosefeverlike diseases that manifest due to close contact with diseasedpersons [ SSSoo ] whereas Sansargaja Vyadhi resides withpeople who are cursed by almighty god ie due to ‚uence ofinvisible forcesforces behind human control [ SSSoo ]Aupasargic Vyadhi is defined in two different ways by Sushruta oneas a disease which spreads from one person to another person [SSNi ] and another as ˜¦Upadravasangyah™ ie complications or associated diseases that manifest after primary disease [SSSoo ] Susruta mentions the diseases like Jwara Kusthaskin diseases Shosha tuberculosis Netrabhisyandi conjunctivitis and other Aupasargika roga alike communicable diseasescan be spread through Prasanga intimate relationship GatraSansparsha direct contact Nishwasa breathing or airborneSahabhojana eating together Sahashayana sleeping togethersharing and using of others™ clothes ornaments ointments etc [SSNi ]Agantuja Vyadhi Ì´ diseases of exogenous origin occurs due tophysicalexternal factors like Bhuta Visha Vayu Agni and Praharatrauma etc without any involvement of Vataadi Dosha initiallyhowever in later stage dosha are involved in the disease process[ CSSoo1145] Cakrapaṇidatta clarifies that Bhuta meansVisakṛimi or a virulent anism [ CSSa1121] Krimi may beSahaja natural or Vaikarika pathogenic anisms that may bevisible macroscopic or invisible to the naked eye microscopic[ CSVi ]Thus it is difficult to correlate this disease with specific Ayurveda terminology but while interpreting the disease on the basis ofSamprapti by considering the causative agent and the clinical features like fever Jwara cough Kasa anorexia Aruchi fatigueTandra generalized body ache or myalgia Angamarda andTiredness it can be contemplated as an Agantuka Vyadhi whichlater on due to the involvement of dosha develops to Nija Vyadhi asKaphaVatolvana Hina Pitta Sannipataja Jwara Severe Vata andKapha with mild Pitta [ CSSoo CSChi ] Whiletalking about the pathogenesis of fever in Ayurveda Charakamentioned that when Vataadi dosha either singly or in Sansristatwo dosha or in Sannipataja all three dosha got aggravated thenit enters Amashaya and mixed with Rasa Dhatu causing obstructionof Rasavaha and Swedavaha Srotas resulting in the destruction ofAgni Agni then spreads out from its Sthana to whole over the bodycausing the febrile condition [ CSNi120 CSChi3129]Immunity concept in AyurvedaStrength health lifespan and vital breath are dependent on thecondition of Agni [ CSSoo ] Charaka has mentioned theterm Vyadhikshamatwa and states that during certain conditions ordue to certain factors even unwholesome unhealthy food doesnot produce disease immediately all unwholesome diet are notequally harmful all dosha are not equally powerful all persons arenot capable of resisting diseases [ CSSoo ] This suggeststhat the body™s immune system plays a crucial role in diseasedevelopment The equilibrium state of Dhatu is called Swasthya [CSSoo ] The person who is desirous to be healthy should adopthealthy practices related to diet conduct and activities [ CSSooImmunity can be considered in Ayurveda as] ThusPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxVyadhikshamatwa and Oja which depends on the condition of AgniDosha and DhatuThere are three factors Aahara Swapna and Brahmacharya dietsound sleep and celibacy that support the life with which the bodywill be endowed with strength complexion and development tilllife span [ CSSoo ] Bala Ì´ StrengthImmunity is of threetypescongenital time affected and acquired Congenital is thatwhich is developed naturally in the body and mind time affected isdue to seasonal variation and age factor and acquired one is produced by the proper application of diet and exercise [ CSSoo ] Thus not only diet but also performing yoga or exercises withproper methods by giving rest in between exercises as Rasayanatherapy will increase acquired strength [ CSSoo1136] Oja isalso called Bala is the essence of all Sapta Dhatu being located inHridaya combines with Rasa and circulates through the Dhamaniand performs Tarpana or Prinanam of the whole body [ SSSoo CS1774] The equilibrium state of Kapha promotesstrength that™s why normal Kapha is called Oja [ CSSoo ]Normal pure blood promotes strength complexion health andlifespan [ CSSoo ] While dealing with Sannipataja JwaraSusruta in Uttarsthana mentioned Abhinyasa Jwara also called asHataujasa Jwara indicating the loss or deranged condition of Oja[ SSUtt ]The word ˜Rasayana Rasa þ ayana™ refers to nutrition and itstransportation in the body for attaining excellent Rasadi Dhatuswhich leads to gain longevity freedom from disorders optimumstrength of physique and sense ans [ CSChi ]Rasayana promotes nutrition by explicitly enriching the nutritionalvalue of Rasa by enhancing Agni ie digestion metabolism andabsorption by Srotashodhana Consequently any medication thatimproves Rasa™s consistency would enhance the health of all bodytissues Role of Ayurveda and traditional medicineEvery society has its own medical system which is deeplyrooted in its culture and guided by its philosophy of life Beingculturally and linguistically diverse countries there developedseveral types of traditional medicines TM based on practices skilltraditional knowledge based on beliefs theories and experiencesindigenous to different cultures Ayurveda Traditional Chinesemedicine TCM Ancient Egyptian medicine Sowa Rigpa etc system of medicine remain the most ancient yet living traditions inSouth East Asia Western Pacific Eastern Mediterranean Africaregion Up to percent of the population in some Asian and African countries depend on TM for primary health care PHC needs[] Still there is a high trend of using many herbs in religious andcultural works therapeutically for common ailments and as spicesfor foods according to occasion specific and seasonal regimes Ayurveda and TM have made a significant contribution to the prevention and alleviation of various communicable and noncommunicable diseases for thousands of years A long history ofusing many herbal remedies and experiences passed from generation to generation has resulted in people relying on herbal remedies and some simple home remedies for common diseases can beused even by illiterate citizens The selfcare an integral part ofPHC with home remedies using various herbs is the most commontreatment for India Nepal Bhutan and China for different flucommon cold fever GI disorders etc Prevention of smallpox inChina has been an epochmaking effort in the period of mankind™spreventive care One observational study found that the prevalencebetween the total number of COVID19 cases per million populationand the grams of spice supply per capita per day is clearly interrelated Most nations with lower spice intake per capita reportedmore COVID19 cases per million population and vice versa []Nevertheless with the invention of drugs many herbal remediesused historically have become modern medicines Few notableexamples include morphine digoxin artemisinin and colchicine Asmany herbs are found to have immunomodulatory role and possessantiviral activity many people are being optimistic over the traditional system of Medicine Ayurveda and TCM have descriptions ofimmunomodulation along with antiviral treatments even targeted to the coronavirus family []The key factor for COVID19 to occur and evolve is the interaction between the virus and an individual™s immune system [] Asmedicinal plants enhance NK cell activity inhibit activated transcription factor ATF2 downregulate Th17related cytokinesincluding transcription factor RORc IL17A and Th2related cytokines including IL5 IL13 and IL6 inhibit GATA3 IL4 IL6 IL1bRt IL17A TNFa expression and increase the secretions of ILit shows that natural products have potentimmunemodulatory and immuneboosting effects that may behelpful during the infection course by increasing innate immuneresponse to infections []INFg etc Ayush KwathConsidering the importance of immunity boosting measures inthe wake of COVID19 outbreak the Ministry of AYUSH Government of India with the interest of health promotion of the massesrecommends ˜Ayush Kwath™ or ˜Ayush Kudineer™ or ˜Ayush Joshanda™which comprises of four medicinal herbs Table [] Theherbs like holy basil cinnamon ginger black pepper are highlyavailable accessible and widely used in the kitchen and areconvenient to educate and train about its use to community healthworkers community and even to all public that they can have costeffective treatment with herbal home remedies This will help topromote immunity and to lower the gatherings at hospitals andpharmacies in this pandemic This type of public health measurewould eventually promote ˜health for all™ with the theme ˜ourhealth in our own hands™ making responsible to each and everypeople by active involvement in their own health instead of relyingon mass distribution of some medicine As people leave theirhomes to earn a living this herbal decoction will ensure broadaccess to health care The WHO SEARO adopted a resolution torevitalize PHC through health systems strengthening to achievehealth for all with the emphasis on health promotion and diseaseprevention [] This Kwath is not just a mechanical mixtureinvented for the COVID19 pandemic but it is a revival of healthtraditionMethod of preparation and useTake all the ingredients in dry form as per standards laid downin Ayurvedic Pharmacopoeia and make coarse powder Make sachets or tea bags each of g of powder or mg tablet of aqueousextract to be consumed like tea or hot drink by dissolving in mlof boiled water once or twice daily Gud JaggeryDraksha Resinsandor Lemon Juice can be added while consuming the formulation TulsiMany invitro animal and human experimental scientificstudies showed that due to presence of eugenol phenolic compoundslinoleic acid etc compounds Tulsi has antimicrobialincluding antibacterial antiviral antimalarial antidiarrhealantioxidantcardioprotectiveimmunemodulatory properties and is thus recommended as a treatmentfor a range of diseases including features like cough fever asthmaanxiety diarrhea gastric cardiac and genitourinary disorders[32e36] Due to its anti‚ammatory and antioxidant properties itanti‚ammatoryhepatoprotectiverenoprotectiveanalgesicantipyreticPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxTable Contents and properties of Ayush KwathSN NameScientific name Parts used Main chemicalRasaVirya AyurvedicSansthanika KarmaProportion RemarksTulsiOcimumsanctum LinnLeavesconstituentsVolatile oil PhenolAldehyde EugenolAscorbic acid Linoleicacid CaroteneDosha karmaKatu Tikta Ushna KaphavatashamakaPittabardhakaDalchini CinnamomumStem Bark CinnamaldehydezeylanicumBreyncuminaldehydeEugenolKatu TiktaMadhuraUshna KaphavatashamakaPitta vardhakaSunthiZingiberofficinale RoscRhizomeZingiberene Zingiberol KatuUshna KaphavatashamakaVedanahara DeepanaPachana AnulomanaKrimighna HridhyaRaktashodhakaKasahara SwasaharaKshayanashakaMutrala VishaghnaJwaraghna esp useful inVatashlaishmikaVishama and Jirna JwaraDeepana PachanaVajikaranaVataanulomanaYakridutejaka GrahiHriyottejakaOjovardhakaRaktashodhakaShelshmaharaYakshmanashakaMutrajananaDeepana PachanaVrishyaShoolaprashamanaRaktashodhakaHridhyottejakaShothahara KaphaghnaSwasahara JwaraghnaAampachana partsPrabhava Specialaction Krimighna parts partsPrabhava KrimighnaContraindicationsPandu KushthaMutrakriccharaktapitta Grishma andSharada Ritu MarichPiper nigrumLinnFruitPiperine PiperidinePiperettine andChavicineKatuUshna Kaphashamaka Deepana Pachana partYakriduttejakaVatanulomanaKrimighnaHriddhyottejakaKaphaghnaKaphamissarakaJwaraghna espVishamjwarapratibandhakaprotects against toxic chemicalinduced injury enhance the antioxidant enzymes and protect cellular anelles and membranes byclearing damaged free radicals []The compounds such as ursolic acid carnosol rosmarinic acidcirsilineol apigenin eugenol and cirsimaritin present in O sanctumincrease haemoglobin concentration enhance SRBC agglutinin titers decrease cyclooxygenase CoX2 and lipoxygenase LOX5enzymes activity suppress NFkB classical pathway up regulationof IL2 IFNg and TNFa down regulation of IL1b and produce ofSRBC antigenspecific antibodies which represent a major defensemechanism to assess Tcelldependent antibody responses ie Tulsiby enhancing immune response boost the defense mechanismagainst the infection [38e40] Several studies have shown that Tulsiaqueous and methanol extract of leaf and seed oil besidesimproving vital capacity also is an immunemodulator and regulator as it enhances immune response by increasing Thelper andNK cells phagocytic activity and index with the rise in lymphocytecount neutrophil count and antibody titer []In an acute toxicity study it did not produce any hazardoussymptoms or CNS and ANS toxicities or death and did not show anychange in water and food consumption body weight and hematological and biochemical profiles [] DalchiniIt is a potent immune system booster and is used in variousailments like flu indigestion edema cough etc [] Cinnamonbark contains cinnamaldehyde benzaldehyde cuminaldehyde andterpenes [] In one study cinnamon at high dose mgkgshowed immunestimulant activity as it significantly increased thephagocytic index serum immunoglobulin levels and antibody titerand decreased the percentage reductions in neutrophil countCinnamon low dose mgkg increased serum immunoglobulinlevels only This showed that high dose increases both cell mediated and humoral immunity whereas low dose showed effect onlyon humoral immunity [] The studies also suggest that cinnamaldehyde can act as a strong regulator of monocytemacrophagemediated immune responses by inhibition of PI3K PDK1 and NFkBactivation of signaling components In addition to this by theactivation of CD29 and CD43 it blocked cell migration cellecelladhesion induced but not cellfibronectin adhesion and it wasable to suppress both the production of nitric oxide NO and upregulation of surface levels of costimulatory molecules CD69 andCD80 and pattern recognition receptors TLR2 and CR3 []Cinnamon bark decrease systemic levels ofIFNg withoutaltering the levels of IL4 or IL2 inhibit antiCD3 AbstimulatedIFNg and IL4 at the mRNA and secreted protein levels enhance IL protein secretion at the cellular level which helps to decrease celldeath inhibit IL2mRNA expression inhibit antiCD3induced p38JNK ERK12 and STAT4 activation but not IkBa degradation orSTAT6 and ultimately alter the ‚ammatory responses in T cellsThis shows the immunemodulatory effect of cinnamon on cytokine secretion and the involvement of intracellular signaling molecules in activated T cells It also causes a reduction in the subG1Please cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxphase accompanied by an increased ratio of apoptotic cells tonecrotic cells [] The constituents like cinnamaldehyde cinnamophilin etc are found to be a thromboxane A2 receptor antagonistanticoagulative antiatherosclerotic and thus prevents unnecessary clumping of platelets and atherosclerotic CVD []In a systematic review of its adverse events relatively few selflimiting adverse effects were reported like allergic reactions andgastrointestinal disorders on clinical trials case reports and caseseries The evidence available show that cinnamon is safe for use asspice in daily diets or as a medication [] However its use fortherapeutic reasons in high doses or for prolonged periods cancause some adverse effects and should be observed clinically SunthiAn alcohol extract increases the immunological status of micewith increased phagocytosis by macrophages whereas crudeextract was also shown to increase humoral and cellmediatedimmune responses [] The bioactive compounds of ginger suchas nevirapine bsitosterol 6gingediol germacrene methyl6shogaol 6gingerol alinalool 6shogaol gingerdion zingibereneetc are known to inhibit viral replication among these the mostpotent inhibitors of reverse transcriptase RT enzyme is bsitosterol which is predicted to be used as nonnucleoside reversetranscriptase NNRTIs HIV1 inhibitors [] It is reported thatGinger contains TNFa which is also known as an anti‚uenzacytokine [] The rhizome of Ginger and its main componentslike gingerols shogaols etc inhibit prostaglandin and leukotrienebiosynthesis inhibit cyclooxygenase and lipoxygenase activitiesinhibits the synthesis of pro‚ammatory cytokines such as IL1TNFa and IL8 without any significant effect in IL6 levelsinhibit the excessive production of NO PGE TNFa and IL1beta reduce the elevated expression of NFkB and TNFa downregulate ‚ammatory iNOS and COX2 gene expression inhibitthromboxane synthetase raise levels of prostacyclin without aconcomitant rise in PGE or PGE alpha inhibit platelet aggregation decrease agerelated oxidative stress markers and enhancefibrinolysis [53e58]The concentration of IgM and eosinophil count in nonsmokerswas significantly increased in a comparative study of the effect ofginger extract among male smokers and nonsmokers whereas theconcentration of hemoglobin and lymphocyte count in smokerswas strongly increased This indicates that in nonsmokers gingerresults in a stronger antibody response or humoral immunity thanin smokers []According to Ayurveda it is contraindicated to be used in a fewdiseases Kushtha Pandu Mutrakriccha Raktapitta and in Grishmasummer and Sharada autumn Ritu There are few minor adverseeffects recorded that did not need care such as mild gastrointestinal symptoms sleepiness mild diarrhea during prior few days oftreatment It is also explained that ginger has the ability to induceheartburn and as a gastric irritant with doses above g [] Duringpregnancy ginger did not pose a major risk for side effects oradverse events [] MarichIt has been also found to increase bioavailability thus enhancethe therapeutic efficacy of many drugs vaccines and nutrients andhave immunemodulatory antioxidant antiplatelets antihypertensive antiasthmatic antipyretic analgesic anticarcinogenicanti‚ammatory antidiarrheal antispasmodic anxiolytic antidepressants hepatoprotective antiulcer antithyroids antiapoptotic antimetastatic antimutagenic antibacterial antifungal[62e65] The extract and itsand antiamoebic propertiesconstituents like piperine regulate the balance of the cytokinesproduction of Th1 Th2 Th17 and Treg cells reduce the accumulation of ‚ammatory cells inhibit the expressions of GATA3 IL4IL6 IL1b Rt IL17A and TNFa increase INFg and IL10 secretions in BALF Bronchoalveolar lavage fluid and increasemacrophage activation and T and B cell proliferation []Beside this Marich possess cytotoxic activity suppresses thelevels of total IgE antiOVA IgE antiOVA IgG1 and histaminerelease in serum ameliorates fibrosis and ltration of ‚ammatory cells inhibits the allergic responses inhibitsTh2Th17 responses and mast cells activation inhibits NFkB cFos cAMPresponse elementbinding CREB and activated transcription factor ATF2 suppresses PMAinduced MMP9 expression inhibitsPKCaextracellular signalregulated kinase ERK and reducesNFkBAP1 activation In addition piperine also inhibits the Pglycoprotein Pgp and CYP3A4 functions [67e69] Piper nigrum isfound to have dose dependent antifertility effects on mice [] DiscussionAccording to Ayurveda therapeutics is of two types Swasthasyorjaskarawhich promotes strength immunity in the healthyand Roganutwhich alleviates disorders Both of these groupsperform both of these functions but Rasayana and Vajikarana aremostly used for promotive treatment CSChi [] AyushKwath has both immune promoting and disease alleviating properties which can be achieved by various treatment modalities likeRasayana Satwawajaya Yuktivyapashraya Vyadhi Viparitarthakarichikitsa etcThe Katu and Tikta Rasa Usna Virya and Deepana PachanaYakriduttejaka properties of Ayush Kwath help to improve Agni andSrotosodhana improves microcirculation and tissue perforationthus promotes proper digestion metabolism and absorption andacts as Rasayana for the development of preceding Dhatu andfinally form Oja Oja itself acts as immunity to prevent diseaseImmunity is dependent on the condition of Agni Ayush Kwath withits Agni promoting and Kaphashamaka properties balance Kaphaand with Raktashodhaka Hridhya Krimighna properties purify theblood It is already mentioned that natural Kapha and pure bloodpromote Oja and Bala respectively Krimighna is the Prabhavaspecial action of Tulsi and Sunthi which directly acts againstpathogens The properties like Jwaraghna esp VatashlaishmikaVishama Kasahara Swasahara Kshayanashaka ShoolaprashamanaSwothahara Kaphaghna Hridayaottejaka Yakridutejaka have directrole to alleviate various clinical signs symptoms and complicationsAs this disease is considered as KaphaVatolvana Hina PittaSannipataja Jwara the Kapha Vata Shamaka properties of AyushKwath can play a significant role in balancing the vitiated doshasAfter six days of Jwara Charaka suggests the decoction of Pachanadrugs in the case of Amdosha and Shamaniya drugs in Niramadosha[ CSChi ] This shows that Yuktivyapashraya and Vyadhiviparita chikitsa can be done even after the involvement of Dosha inlater stages Ayush Kwath has potential psychoneuroimmunemechanisms via evidence of a reduction in depression anxietyand stress in controlled trials and shows meaning response as it is aspecific remedy for cough and respiratory problems this shows therole of Satvawajaya Chikitsa in its management []Immunity plays a key role in the pathogenesis of COVID19 bothduring the early nonsevere stage and during the severe stage ofthe disease The earlystage strong immune response may preventthe propagation and spread of viruses inside the body thusreducing the severity of cases and early termination of infectionWhile during later stage strong cellmediated immunity of thebody against the virus itself can be a factor responsible for graveconsequences due to cytokine storm The target during the earlyPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxlikesteroidsand IL6 receptorstage should be to reduce viral propagation while at a later stageshould be to reduce the ‚ammatory response of the immunesystem Medicinal herbs with immune booster property can be anoption during the early nonsevere stage while herbs with anti‚ammatory and antithrombotic properties can be an optionduring a later or severe stage Cytokine storm that is believed as amajor factor responsible for complications and death of COVID19patients has been found to be reduced with anti‚ammatorydrugsantagonists Anti‚ammatory interleukins IL10 in modern medicine [] Therole of medicinal herbs with anti‚ammatory property on thecytokine storm is still lacking in research Like anti‚ammatoryinterleukins and IL6 receptor antagonist Tocilizumab IL thatare proposed in modern medicine to have a therapeutic role in thereduction of severity and mortality of COVID19 Cinnamon barkthat is found to decrease INFg and IL4 Its antiatheroscleroticanticoagulative and antiplate

Thyroid_Cancer

Aprospective blinded multicenter studyDear editorThyroid nodules are common and the major point oftheir clinical evaluation rests with the differentiation ofthyroid cancer1“ Herein we present a twostep study todevelop and validate an effective model based on circulating tumor cells CTCs signatures for papillary thyroid cancer PTC diagnosisA total of subjects were clinically evaluated and of which were included in the final analysis A In the first step patients with PTC from a single hospital and healthy subjects formed the test groupMultimarkers reported in the literatures that were relatedto malignancy and thyroid epithelium CK19 SurvivinGalectin3 Tg and TSHR were evaluated and selectedto form a most effective combination for the diagnosticmodel Next the model was evaluated in the validationgroup that was consisted of participants from four different hospitals in China who had one or more thyroidnodules that yielded an ultrasound diagnosis of ThyroidImaging Reporting and Data System45 TIRADS or and measured cm long in diameter Postoperativepathology was considered as the reference standard andonly patients who received surgery and had definite pathological reports were enrolled in the final analysis Thyroidnodule was excluded by ultrasound in healthy subjectsThe process of CTCs separation and enrichmentincluded CD45 negative Dynabeads„¢ M450 CD45 panLeukocyte 11153D Invitrogen by Thermo Fisher Scientific USA and EpCAM positive Invitrogen byThermo Fisher Scientific USA immunomagnetic selections The reliability of CTCs separation and enrichmentwas verified with fluorescence staining in a cell line model PTC cells added into white blood cells separationfrom mL peripheral blood from a healthy subject B mRNA was isolated from the cell fractions Invitrogen by Thermo Fisher Scientific USA and cDNAwas then reverse transcribed with μL of mRNA total μL using the Sensiscript RT Kit QIAGEN Germany mRNA levels of CK19 Survivin Galectin3 Tgand TSHR were analyzed by the QuantiNova SYBR GreenPCR Kit QIAGEN Germany Thermo FisherScientific USA Three independently isolated RNAsamples were measured to determine the threshold cyclevalues CT mRNA level of these markers were measuredwith relative expression level RQ and presented afternormalization against the reference actin and wascalculated with “ΔΔCT method ΔCt Ct target “ Ctactin ΔΔCT ΔCt13 RQ “ΔΔCT The values werepresented as the mean ± standard deviation SDOf all participants recruited in the study the medianage was years range years and were female participants Among patients with thyroid nodules the median size of the nodules was cm range cm and had a tumor ‰ cm According to thepostoperative pathology patients had PTC of which and patients had central compartment and lateral necklymph nodes metastasis respectively In the test group patients and healthy subjects the median mRNA levelsof the markers were significantly higher in patients thanin healthy controls except for TSHR C CK19 andSurvivin mRNA was not detected in any healthy subject sopositive result of the two markers was defined as detectablemRNA level Tg and Galectin3 mRNA were detected in and healthy subjects respectively Based on theROC curves a cutoff value of ngμg was selected falectin3 with a sensitivity of and a specificity of while a cutoff value of ngμg for Tg giving an optimal sensitivity of and a specificity of The areaunder curve AUC of CK19 Survivin Galectin3 and Tgwere and respectivelyAfter evaluating different combinations of the markers Supporting Information CK19 Survivin and Tgwere selected to form a diagnostic model when positiveresult was recognized when any marker was positive itsThis is an access under the terms of the Creative Commons Attribution License which permits use distribution and reproduction in any medium provided theoriginal work is properly cited The Authors Clinical and Translational Medicine published by John Wiley Sons Australia Ltd on behalf of Shanghai Institute of Clinical BioinformaticsClin Transl Med 202010e142101002ctm2142wileyonlinelibrarycomjournalctm2 of 0c of LETTER TO EDITORF I G U R E Flow chart of the study and diagnostic model establishing A Flow chart of the study B Fluorescence staining of EpCAMCD45 and DAPI after immunomagnetic enrichment of a CTC model PTC cells added into white blood cells separation from ml peripheralblood from a healthy subject CTCs white solid arrow white blood cells white dotted arrow C CK19 Survivin Galectin3 Tg and TSHRmRNA levels of healthy subjects n and patients with PTC n in the test group D HE a and immunohistochemical staining forCK19 b Tg c and Survivin d of a PTC patient enrolled in the test group E Expression of markers in cell lines of PTC by western blotpositive predictive value PPV negative predictive valueNPV sensitivity specificity and AUC value were and respectively and the diagnostic model correctly classified of subjects yielding anoverall accuracy of The expression of selected markers CK19 Survivin and Tg was verified with immunohistochemical analysis in formalinfixed paraffinembeddedpostoperative specimen of PTC patients D andwestern blot in two cell lines of PTC E and thenthese markers were evaluated in the validation groupIn the validation cohort n the median mRNAlevels of CK19 Survivin and were significantly higherin patients with PTC than in patients with benign thyroid nodules P A The clinical sensitivity specificity PPV and NPV of the diagnostic model were and respectively with a diagnostic accuracy of and the AUC for predicting malignancy was CI For detailed performance of individual markers the highest sensitivity of single marker Tg was which was significantlylower than the diagnostic model of 3marker combinationAll patients with positive Survivin n had malignant pathology PPV with a sensitivity of which was consistent with the results of the test groupBIn the diagnostic model based on threeCTCs signatures established in the study has a highdiagnostic performance for patients with suspiciousthyroid nodules multimarker analysissignificantlyimproved sensitivity comparing with single marker 0cLETTER TO EDITOR of F I G U R E Validation of the diagnostic model in a prospective cohort n A CK19 Survivin and Tg mRNA levels of benign andmalignant patients in the validation group B Detailed diagnostic performance of the diagnostic model in the validation groupanalysis Moreover it is discovered in this study thatthe CTCs signature Survivin had a potential value forconfirming PTC diagnosis PPV C O N F L I C T O F I N T E R E S TThe authors have no conflicts of interest to disclosureS TAT E M E N T O F E T H I C SThe study was approved by the ethic committee of CancerHospital Chinese Academy of Medical sciencesAU T H O R S ™ C O N T R I B U T I O N SSiyuan Xu and Jingning Cheng participated in the designof the study data collection and paper writing Bojun Weiand Yang Zhang participated in the data collection andhelped to draft the manuscript Yang Li and Rui Zhangparticipated in the data analysis and validation ZongminZhang Yang Liu and Kai Wang participated in the datacollection and quality control of data Ye Zhang and YingHuang participated in the statistical analysis Xiaolei Wangand Shaoyan Liu participated in the manuscript reviewJie Liu and Zhengang Xu participated in the design of thestudy and helped to revise the manuscript All authors readand approved the final manuscriptSiyuan Xu1Jingning Cheng2Bojun Wei3Yang Zhang4Yang Li5Zongmin Zhang1Yang Liu1Ye Zhang6Rui Zhang7Kai Wang1Xiaolei Wang1Shaoyan Liu1Ying Huang1Zhengang Xu1Jie Liu1 Department of Head and Neck Surgical OncologyNational Cancer CenterNational Clinical Research Centerfor CancerCancer Hospital Chinese Academy of MedicalSciences and Peking Union Medical College Beijing P RChina Department of Otorhinolaryngology ChinaJapanFriendship Hospital Beijing P R China Department of Thyroid and Neck Surgery BeijingChaoyang Hospital Capital Medical University Beijing PR China Department of Endocrinology Peking University FirstHospital Beijing P R China Department of General Surgery Hebei Petro ChinaCentral Hospital Langfang P R China Department of Radiation Oncology National CancerCenterNational Clinical Research Center forCancerCancer Hospital Chinese Academy of MedicalSciences and Peking Union Medical College Beijing P RChina Department of Ultrasound National CancerCenterNational Clinical Research Center forCancerCancer Hospital Chinese Academy of MedicalSciences and Peking Union Medical College Beijing P RChinaCorrespondenceJie Liu and Zhengang Xu Postal address No Panjiayuan Nanli Chaoyang District Beijing PRChinaEmail liujcicamsaccn xuzhg06126com 0c of LETTER TO EDITORSiyuan Xu Jingning Cheng Bojun Wei and Yang Zhangcontributed equally to this workTrial Registration Clinicaltrialsgov IdentifierNCT03772496Funding informationCAMS Innovation Fund for Medical Sciences BeijingHope Run Special Fund of Cancer Foundation of China2016I2m1002 Beijing Hope Run Special Fund of CancerFoundation of China LC2018A26 LC2016A01O RC I DJie Liuorcid0000000339757978R E F E R E N C E S Haugen BR Alexander EK Bible KC et al American Thyroid Association Management Guidelines for adult patients withthyroid nodules and differentiated thyroid cancer the American Thyroid Association Guidelines Task Force on thyroidnodules and differentiated thyroid cancer Thyroid Burman KD Wartofsky L Clinical practice Thyroid nodules NEngl J Med Popoveniuc G Jonklaas J Thyroid nodules Med Clin North Am Cheng SP Lee JJ Lin JL Chuang SM Chien MN Liu CL Characterization of thyroid nodules using the proposed thyroid imagingreporting and data system TIRADS Head Neck Zhang J Liu BJ Xu HX et al Prospective validation ofan ultrasoundbased thyroid imaging reporting and data system TIRADS on thyroid nodules Int J Clin Exp MedS U P P O RT I N G I N F O R M AT I O NAdditional supporting information may be found onlinein the Supporting Information section at the end of the 0c'

Thyroid_Cancer