Datasets:

ManuelAlv

/

Cancer

like 0

Decreased expression of the thyroid hormone‘inactivating enzyme type deiodinase is associated with lower survival rates in breast canceriuri Martin Goemann1 Vicente Rodrigues Marczyk15 Mariana Recamonde‘Mendoza23 Simone Magagnin Wajner15 Marcia Silveira Graudenz45 Ana Luiza Maia thyroid hormones tHs are critical regulators of cellular processes while changes in their levels impact all the hallmarks of cancer Disturbed expression of type deiodinase DIO3 the main tH‘inactivating enzyme occurs in several human neoplasms and has been associated with adverse outcomes Here we investigated the patterns of DIO3 expression and its prognostic significance in breast cancer DIO3 expression was evaluated by immunohistochemistry in a primary cohort of patients with breast cancer and validated in a second cohort using RnA sequencing data from the TCGA database DNA methylation data were obtained from the same database DIO3 expression was present in normal and tumoral breast tissue Low levels of DIO3 expression were associated with increased mortality in the primary cohort Accordingly low DIO3 mRnA levels were associated with an increased risk of death in a multivariate model in the validation cohort DnA methylation analysis revealed that the DIO3 gene promoter is hypermethylated in tumors when compared to normal tissue In DIO3 is expressed in normal and tumoral breast tissue while decreased expression relates to poor overall survival in breast cancer patients Finally loss of DIO3 expression is associated with hypermethylation of the gene promoter and might have therapeutic implicationsBreast cancer is the most common cancer in women worldwide accounting for more than two million new cancer cases and of all cancerrelated deaths in women in Despite remarkable advances in the treatment of breast cancer in recent decades not all patients benefit from current therapeutic options and thus will experience relapse23 Genomic tests improve the clinical prediction of patient outcomes and determine the necessity of adjuvant chemotherapy with endocrine therapy34 However it is a highly heterogeneous disease that is diverse in its behavior and responsiveness to the different modalities of treatment56 Breast cancer is characterized based on receptor and gene expression profiles that together with the classic clinicopathological variables guide the treatment and estimate the risk of recurrence34 Gene expression profiling studies have established at least four molecularly distinct types of breast cancer that can be expanded to the œintrinsic subtypes luminal A LumA luminal B LumB HER2enriched basallike and normallike7“Numerous studies have established thyroid hormones THs as critical regulators of multiple cellular processes in normal and tumor cells10 They contribute to cellular proliferation and differentiation during development and adulthood and are finetuned for tissuespecific control1011 Clinical studies associate TH levels with breast 1Thyroid Unit Endocrine Division Hospital de Cl­nicas de Porto Alegre Rua Ramiro Barcelos Porto Alegre RS CEP Brasil 2Institute of Informatics Universidade Federal Do Rio Grande Do Sul Porto Alegre Brazil 3Bioinformatics Core Hospital de Cl­nicas de Porto Alegre Porto Alegre Brazil 4Department of Pathology Hospital de Cl­nicas de Porto Alegre Porto Alegre Brazil 5Faculdade de Medicina Universidade Federal Do Rio Grande Do Sul Porto Alegre Brazil email almaiaufrgsbrScientific RepoRtS 101038s41598020708924Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Patterns of expression of DIO3 in breast samples Immunostaining was performed as described in Materials and Methods From left to right A normal glandular breast tissue B breast carcinoma with low expression overall intensity C breast carcinoma with moderate expression overall intensity and D breast cancinoma with high expression overall intensity of DIO3 protein evaluated through immunohistochemistry The staining intensity level is used to calculate the Hscore combined with the percentage of positive cells see œMethodscancer risk and mortality1213 while in a0vitro models demonstrate the effect of THs on breast cancer cell proliferation apoptosis and migration14“ T4 promotes cell proliferation through the αv3 integrin receptor14 while the proliferative effects of T3 depend at least partially on the presence of estrogen receptors in breast cancer cells1718 Clinically however the effects of THs on specific histopathological and molecular subtypes of breast cancer are still unclear1920Modulation of THs concentrations is orchestrated by a group of selenoproteins called iodothyronine deiodinases which can activate and inactivate thyroid hormones21 Briefly the type deiodinase DIO1 catalyzes both activation and inactivation of thyroxine T4 generating triiodothyronine T3 and reverse triiodothyronine rT3 respectively22 Type deiodinase DIO2 acts locally converting the prohormone T4 into the active T3 Meanwhile type deiodinase DIO3 is the main THinactivating enzyme by degrading T4 and T3 to inactive metabolites rT3 and diiodothyronine respectively21 The DIO3 gene is found in the DLK1DIO3 genomic region which is located on human chromosome 14q3223 DIO3 gene is subject to genomic imprinting an uncommon epigenetic phenomenon that results in the preferential expression of one of the alleles paternal allele in the case2425 DIO3 gene expression is increased in several tissues during embryogenesis but it decreases in most tissues in adulthood2627 Notably DIO3 is expressed in normal and pathological hyperproliferative conditions where it has been implicated in cell proliferation and differentiation20252628 In particular studies have demonstrated that the local control of THs signaling provided by the regulation of DIO3 activity is associated with cancer development progression and recurrence28“ We have previously reported that DIO3 mRNA and activity levels are increased in papillary thyroid cancer PTC which are associated with larger tumor size and the presence of lymph node and distant metastasis at diagnosis30 Others have described hyperexpression of this enzyme in basal cell carcinoma BCC where it modulates intracellular T3 concentrations and thus contributes to the cell tumorigenic potential31 DIO3 exerts a similar function in colon cancer which suggests that attenuation of the TH signal is part of the oncogenic process at least in some types of cancer28Considering the implied role of the DIO3 gene in human neoplasms and the potential effect of TH in breast carcinogenesis13“ we investigated the expression patterns of DIO3 in normal breast tissue and breast cancer Here we demonstrate that DIO3 is expressed in normal breast tissue and breast cancer tissue In breast cancer reduced DIO3 expression is associated with decreased overall survival Interestingly loss of DIO3 expression might be explained at least partially by gene promoter hypermethylationResultsDIO3 in normal breast and fibroadenoma DIO3 immunohistochemistry staining was detected in all samples of normal breast tissue N at an overall moderate intensity Hscore ± DIO3 staining was predominantly cytoplasmatic and more pronounced in the apical extremity in luminal cells in both ducts and acini of the breast Fig a01A DIO3 was markedly positive in myoepithelial cells Fig a01A bottom Benign fibroadenoma lesions N were also positive for DIO3 staining with an intensity comparable to healthy tissue Hscore ± vs ± P Scientific RepoRtS 101038s41598020708924Vol1234567890wwwnaturecomscientificreports 0cCharacteristicMedian age at diagnosis range”yearsTumor size in the largest dimension”mmMedian IQRMean ± SDEstrogen receptor”no PositiveNegativeMissingProgesterone receptor”no PositiveNegativeMissingHER2 status”no PositiveNegativeMissingHistological type of tumor”no Invasive Ductal Carcinoma IDCInvasive Lobular Carcinoma ILCDuctal Carcinoma in a0situ DCISClinicalpathological subtype”no Luminal ALuminal BHER2Triple NegativeNon classifiedLymph node metastasis”no YesNoDistant metastasis”no YesNoTumor staging”no Stage IIIStage IIIIVMissingPretreatment hypothyroidism”no Posttreatment hypothyroidism”no Followup mean ± SD”monthsAllcause mortality”no Mean survival months CIPrimary cohort N Validation cohort N “ “ “ ± AJCC NANA PAM50 ” “ NANA “ “Table Baseline characteristics of patients with breast cancer included in the primary cohort and in the validation cohort NA not available IQR interquatile range SD standard deviation HER2 human epidermal growth factor receptor2 AJCC American Joint Committee on Cancer Classified by the AJCC staging system Classified by PAM50 data available for patientsDIO3 protein in breast cancer the primary cohort To study DIO3 expression in breast cancer we analyzed a cohort of patients who had been seen at our institution primary cohort N and validated the results in the TCGABRCA cohort validation cohort N The clinicopathological characteristics of the patients from both cohorts are summarized in Table a0Patterns of DIO3 staining evaluated through immunohistochemistry in breast cancer samples are shown in Fig a01B“D DIO3 staining in FFPE breast cancer tissues was positive in samples of invasive ductal carcinoma IDC with a mean Hscore of ± When evaluating invasive lobular carcinoma ILC only of samples was positive for DIO3 Hscore A sample of ductal carcinoma in a0situ DCIS was also positive for DIO3 expression Hscore A graph comparing the Hscore for DIO3 in nonmalignant tissues and malignant breast cancer types is presented in Fig a02A Mean DIO3 Hscores of primary tumors were similar to the nontumoral tissues with a marginal decrease in DIO3 seen in invasive lobular carcinoma ILC P Scientific RepoRtS 101038s41598020708924Vol0123456789wwwnaturecomscientificreports 0cType of tissuetumorANormalbreastDFibroadenomaIDCILCLymph node statusPNSerocSHerocSHEstrogen receptor statusHER2 statusCPNSpositivenegativeTNM stagingFPNSPNSpositivenegativeDistant metastasisPNSBEerocSHerocSHerocSHerocSHnegativepositiveabsencepresenceIIIIIIIVGLog Rank p0012liavvrusfoytilibaborPDIO3 positiveDIO3 negativePatients at riskDIO3 posDIO3 negMonthsFigure a0 DIO3 staining and clinicopathological characteristics of patients with breast cancer in the primary cohort A“F Box plots of DIO3 staining in breast tissue samples evaluated through immunohistochemistry and quantified by HScore Samples were divided according to clinicopathological data as follows A type of tissue analyzed B ER status C HER2 status D lymph node status E distant metastasis and F TNM anatomic staging G Kaplan“Meier plot of overall survival in patients with the presence gray or absence black of DIO3 staining in breast cancer evaluated through immunohistochemistry ER estrogen receptor HER2 human epidermal growth factor receptor2 IDC invasive ductal carcinoma ILC invasive lobular carcinoma NS not significant P The mean Hscore of invasive ductal carcinoma was similar to that of normal tissue P No differences were observed between the molecular subtypes of breast cancer P data not shown There was no difference in the Hscore between tumors with ERpositive and ERnegative status P Fig a02B or between tumors with HER2positive and HER2negative status P Fig a02C Among the primary tumors there was no significant correlation between Hscore and Ki67 levels P or between Hscore and histological tumor grade P We found no association of DIO3 positivity negative or positive with tumor size P The mean Hscore in primary tumors of patients without nodal metastases was similar to that observed in patients with lymph node metastasis P Similarly Hscores of primary tumors of patients with distant metastasis did no differ from those without distant metastasis P Fig a02DE There were no differences on DIO3 Hscores when comparing patients with stage III vs stage IIIIV disease P Fig a02F We obtained both primary and lymph node tissues from patients In this subset of patients DIO3 staining was comparable between paired primary tumor and lymph node metastasis P Table a0 shows the variables associated with an increased risk of death in the primary cohort univariate analysis We observed that negative DIO3 staining was associated with poor prognosis HR CI to P Therefore additional studies were performed using Kaplan“Meier analysis and the logrank Scientific RepoRtS 101038s41598020708924Vol1234567890wwwnaturecomscientificreports 0cVariableAge at diagnosis yearsTumor size mmLymph node metastasis pos vs negDistant metastasis pos vs negER status pos vs negP status pos vs negHER2 positivity pos vs negTNM staging IIIIV vs IIIDIO3 status neg vs posHR CI “ “ “ “ “ “ “ “ “P valueTable Univariate Cox regression analysis of overall survival in breast cancer patients in the primary cohort HR hazard ratio CI confidence interval ER estrogen receptor P progesterone HER2 human epidermal growth factor receptor2test Patients with negative DIO3 staining had a worse overall survival than those with positive DIO3 staining The mean overall survival was a0months CI to in the DIO3negative group and a0months CI to in the DIO3positive group Fig a02G logrank P DIO3 mRNA in breast cancer patients validation cohort It has been previously demonstrated that DIO3 protein levels and activity correlate with DIO3 mRNA levels in different contexts303233 Therefore to validate differences of DIO3 expression among patients with breast cancer we analyzed DIO3 mRNA expression in a second cohort using available gene expression data from the TCGABRCA study In this second population DIO3 expression was found to be reduced in primary solid tumors N compared to that observed in normal breast samples N logFC adjusted P value Fig a03A even when the comparison was made only with matched normal tissues logFC adjusted P value Fig a03B The majority of tumor subtypes with the exception of normallike tumors classified according to PAM50 classification system showed reduced DIO3 expression compared to normal tissue Fig a03C On the other hand DIO3 expression was increased in ERpositive samples compared to that in ERnegative samples logFC P Fig a03D There was no significant difference when comparing DIO3 expression between patients with or without lymph node disease logFC adjusted P value or distant metastasis logFC adjusted P value Fig a03E Decreased DIO3 mRNA expression was observed in all tumor stages compared to that seen in normal tissue P However no differences were found between the different tumor stages Fig a03F Interestingly lower DIO3 expression was associated with greater tumor size P and ER negativity P We then evaluated the prognostic value of DIO3 mRNA expression for patient survival We considered patients as having high DIO3 expression when their logCPM values were above the median and as having low DIO3 expression when their logCPM values were below the median Low DIO3 expression was associated with reduced survival with an HR of CI to P in the univariate model Table a0 Additional analysis using a multivariate model adjusted for all variables with a P in the univariate analysis demonstrated that low DIO3 was an independent prognostic factor for death HR IC to P Table a0 Fig a04A The estimated overall survival rate at five years in the Kaplan“Meier analysis was CI to in the high DIO3 group and CI to in the low DIO3 group Fig a04AIn the subgroup analysis of patients with advanced disease stage IV those with low DIO3 expression had reduced overall survival compared to patients with high DIO3 expression P Fig a04B Notably low DIO3 expression was associated with worse overall survival among patients with ERpositive tumors P but not among those with ERnegative tumors P Supplementary Fig a0Methylation of DIO3 gene promoter To further investigate possible factors that could lead to decreased DIO3 expression in breast cancer we performed DNA methylation analysis of a subgroup of patients from TCGABRCA database from whom DNA methylation data were available N Our analysis demonstrated that global DNA methylation levels of breast cancer samples were similar to those of healthy breast tissues Fig a05A However the methylation levels of CpG sites in the DIO3 gene region were increased compared to those from healthy tissue Fig a05B P Figure a0 details the CpG sites that are hypermethylated within the DIO3 gene region The first kbp of ² flanking region red are known to be extremely G C rich of the sequence and this region is highly conserved between mouse and human genome34 Promoter region a0bp of the ² flanking region is composed of several promoter elements Fig a05C enhanced including a TATA box two CAAT boxes and CG rich regions35 We observed a significant increase in DNA methylation levels in CpG sites that are located both at the promoter region and in the ² flanking kbp conserved region of the gene Fig a05CDScientific RepoRtS 101038s41598020708924Vol0123456789wwwnaturecomscientificreports 0cFigure a0 The relationship between DIO3 mRNA expression and clinicopathological parameters in breast cancer samples of patients from the TCGABRCA cohort expressed in Log2 counts per million voomtransformed Comparative expression demonstrates that DIO3 mRNA is decreased in tumoral tissue when compared to normal tissue when analyzing A all samples or B only matched samples C All tumor subtypes have decreased expression of DIO3 mRNA when compared to normal tissue with the exception of normallike tumors compared to normal tissue DIO3 mRNA levels were also reduced in basallike tumors when compared to luminal A logFC adjusted P value and in luminal B when compared to luminal A subtypes logFC adjusted P value and D DIO3 expression is increased in ERpositive samples when compared to ERnegative samples E DIO3 expression is similar in patients with or without metastasis F When samples were separated according to tumor staging all tumor stages had decreased DIO3 expression when compared to normal tissue but there was no difference in expression between the stages ER estrogen receptor Adjusted P value in comparison to normal tissueVariablesAge at diagnosis yearsTumor size ‰¥ a0cm vs ‰¤ a0cmLymph node pos vs negDistant metastasis pos vs negE2 status pos vs negP status pos vs negHER2 positivity pos vs negTNM staging IIIIV vs IIIDIO3 status low vs highUnivariate analysisHR CI “ “ “ ““ “ “ “ “P value Multivariate analysisHR CI “ “ “ “ “ “P value “Table Univariate and multivariate Cox regression and for overall survival in the validation cohort HR hazard ratio CI confidence interval ER estrogen receptor P progesterone HER2 human epidermal growth factor receptor2 All variables with P were included in the multivariate model TNM is not included as it is derived from variables already present in the modelDiscussionDisruption of the iodothyronine deiodinases expression leads to changes in TH concentrations which might contribute to cancer development and progression by impacting virtually all the hallmarks of cancer10 Here we demonstrate that the THinactivating enzyme DIO3 is expressed in normal breast tissue and that its expression is highly prevalent in breast cancer More interestingly our results demonstrated that low DIO3 expression Scientific RepoRtS 101038s41598020708924Vol1234567890wwwnaturecomscientificreports 0cAOverall SurvivalP BOverall Survival Stage IV patientsHighLowDIO3DIO3groupgroupP0011liavvrusfoytilibaborPliavvrusHighLowDIO3DIO3groupgroupHR for death IC to foytilibaborPPatients at riskHigh DIO3Low DIO3MonthsPatients at riskHigh DIO3Low DIO3MonthsFigure a0 Kaplan“Meier estimates of overall survival in patients of the TCGABRCA cohort according to DIO3 mRNA expression Patients were grouped according to the median of DIO3 expression in the population as presenting high DIO3 expression gray lines or low DIO3 expression black lines Plot A shows the overall survival in the entire cohort Plot B refers only to patients with stage IV disease HR hazard ratio CI confidence intervalwas an independent prognostic factor for reduced overall survival in two different populations of patients with breast cancerData on the expression of iodothyronine deiodinases in human breast tissue are scarce Low levels of DIO1 were reported in normal and lactating tissues but DIO2 and DIO3 have not been analyzed thus far36 Here we show that DIO3 is expressed at both the mRNA and protein levels in normal human breast tissue Expression of DIO3 mRNA has been previously described in breast cancer cell lines MCF7 and MDAMB231 cells DIO3 mRNA was found to be upregulated in MCF7 cells and downregulated in MDAMB231 cells when compared to the nontumoral cell line MCF10A cells DIO3mediated T3 deiodination also occurs in MCF7 cells In these cells DIO3 expression is to regulated by retinoids but not by estradiol37“ These findings are consistent with the presence of DIO3 in other tissues of ectodermal origin such as the skin and the nervous system4041The role of thyroid hormone metabolism on human tumorigenesis has been largely debated10 In breast cancer previous studies showed that higher levels of the thyroid hormone receptor alpha were an independent prognostic factor for increased overall survival42 More recently high levels of the thyroid hormone receptor beta in breast tumors were also associated with increased breast cancerspecific survival43In basal cell carcinomas BCC for instance a DIO3mediated decrease in T3 levels relates to increased cell proliferation31 Similarly in colon cancer cells DIO3 knockdown and consequent increases in T3 levels are associated with reduced cell proliferation and induction of differentiation44 High levels of DIO3 expression in primary PTC tumors were associated with advanced disease at the diagnosis30 Some data indeed suggest that T3 can contribute to tumor growth in breast cancer cells in a0vitro17 while a microenvironment with low T3 levels could facilitate invasiveness and dedifferentiation However in agreement with our data in breast cancer similar levels of DIO3 mRNA are observed in glioblastoma and liver carcinomas as compared to respective normal tissues45 These differences could be attributed to the tissue embryological origin since the tissues of ectodermal origin seem to maintain DIO3 expression during adulthood while DIO3 gene is subject to imprinting in other tissues Loss of DIO3 expression was associated with tumor aggressiveness in colon cancer and also in thyroid cancer DIO3 expression is present in papillary and follicular subtypes but not in the most aggressive and dedifferentiated anaplastic subtype30 Taken together these results indicate that although expression of the enzyme is often upregulated in the neoplastic tissue compared to normal tissue loss of DIO3 expression is a common hallmark of dedifferentiation in the neoplastic process which might confer its prognostic significance Alternatively the distinct pattern of expression could be the result of DIO3 regulation or related to the cancertype specific methylation signatureAlthough this was an exploratory study our results point to a prognostic role for DIO3 expression in breast cancer In a primary cohort of patients with breast cancer negative DIO3 staining in the primary tumor was associated with significantly worse prognosis HR CI to P when compared to patients who were DIO3positive More interesting in the second cohort low DIO3 expression was an independent prognostic factor for death in a model adjusted for age tumor size lymph node and distant metastasis estrogen and progesterone status HR IC “ P The prognostic role of DIO3 expression was particularly relevant in the subgroup of patients with advanced diseaseIntriguingly the difference in survival between groups with distinct DIO3 expression was limited to ERpositive patients Previous studies indicate the existence of a crosstalk between estrogen and THdependent regulatory pathways in breast cancer14174647 which might be a potential explanation T3 regulates cell cycle progression and proliferation in breast cancer cells in a0vitro by a common mechanism involving ER and T3 receptormediated pathways46 Moreover T4 can phosphorylate nuclear ERalpha in MCF7 cells via a MAPKdependent pathway promoting proliferation14 Therefore loss of DIO3 expression and the consequent increase in intracellular T3 levels could be specifically detrimental to tumors that express ER as our results suggest Contributing to this Scientific RepoRtS 101038s41598020708924Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Panel A demonstrates mean global DNA methylation levels values in breast cancer tissue compared to healthy breast tissue Panel B demonstrates that the mean DNA methylation of DIO3 gene region is increased in tumor tissue when compared to normal tissue P Panel C is a schematic representation of the location of DIO3 gene in chromosome and the regions that were evaluated by CpG probes The promoter region is composed by several promoter elements including a TATA box two CAAT boxes and CG rich regions C enhanced Significant hypermethylation in several CpG sites is observed in the promoter region of the gene Panel D presents mean values of CpG sites mapped in DIO3 gene region comparing normal and tumoral tissueinterplay previous studies have demonstrated that estrogen progesterone and their receptors regulate DIO3 activity in rat uteri and decidua4849 Therefore we cannot rule out that in the breast DIO3 expression depends partially on the presence of functional estrogen and progesterone receptorsScientific RepoRtS 101038s41598020708924Vol1234567890wwwnaturecomscientificreports 0cThe DIO3 gene is subject to genomic imprinting an uncommon epigenetic phenomenon that results in the preferential expression of one allele the paternal allele in this case2425 The disturbed expression of genes and miRNAs or altered hypermethylation patterns of the DLK1DIO3 genomic region is involved in the pathogenesis of different types of cancer50“ Thus we hypothesized that the loss of DIO3 expression in breast tumors could be a consequence of gene hypermethylation in the tumoral context Indeed our results show that while the mean global methylation in breast tumors is comparable to that of normal tissue the DIO3 genomic region especially its promoter region is significantly hypermethylated in tumors Fig a05C enhanced These findings might explain at least in part the reduced DIO3 expression in breast cancer Of interest the DIO3 gene was also found to be hypermethylated in Bcell Tcell and myeloid malignancies and lung cancer5152Our study has some limitations The absence of data on DIO3 enzymatic activity limits the assumption that the decreases of DIO3 levels cause alterations in intracellular TH homeostasis Alternatively changes in DIO3 expression could simply represent a consequence of broader epigenetic modifications in the tumoral context It is also important to consider that complete clinical data on patient thyroid status was not available which could interfere with deiodinase expression54 Therefore the complex changes on deiodinases and the overall effect on intracellular TH status are still unclear in breast cancer Additionally our analysis is limited to two populations using two different methodology and despite robust supporting data results should be confirmed in other cohortsIn the results of this study demonstrate DIO3 expression in breast tissue and breast cancer Importantly low DIO3 expression is associated with reduced overall survival suggesting that DIO3 might have a prognostic role in this disease Reduced DIO3 expression in breast cancer can be explained at least in part by gene hypermethylation Due to its potential to modulate thyroid hormone intracellular levels and interplay with estrogen metabolism in breast cancer the DIO3 expression might have therapeutic implicationsMethodsPatients and tissues primary cohort Neoplastic tissue from patients diagnosed with breast cancer was retrospectively collected from a consecutive series of unselected patients in the pathology department of Hospital de Cl­nicas de Porto Alegre Tissue samples of the normal breast N and fibroadenomas N were also obtained Histopathological reports containing information on tumor type grade and immunohistochemistry were retrieved clinical data were retrospectively reviewed in medical records Tumors were histologically classified according to the 8th edition of the American Joint Committee on Cancer AJCC staging system56 All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional andor national research committee The study was reviewed and approved by the Institutional Review Board and Research Ethics Committee from the Hospital de Cl­nicas de Porto Alegre with a waiver of informed consent Protocol number Immunohistochemistry studies and DIO3 staining assessment DIO3 protein expression was evaluated by immunohistochemical studies on 6mm sections of formalinfixed paraffinembedded FFPE tissue blocks from normal breast tissues fibroadenomas and primary breast cancers The immunohistochemical technique consists of tissue deparaffinization and rehydration antigenic recovery inactivation of endogenous peroxidase and blockage of unspecific reactions Samples were incubated overnight at a temperature of a0°C with an antiDIO3 rabbit polyclonal antibody Abcam Cambridge UK at a dilution of followed by subsequent incubation with a biotinylated secondary antibody a streptavidin“HRP conjugate LSAB Dako Carpinteria CA USA and diaminobenzidine tetrahydrochloride Kit DAB Dako The slides were examined using an Olympus BX51 microscope The QCapture Pro software Qimaging Surrey BC Canada was used to capture the images DIO3 staining was evaluated by an experienced pathologist blinded to the molecular profile and TNM staging The immunohistochemical results of DIO3 staining were assessed dichotomously negative or positive and semiquantitatively using the Hscore method as described previously5758 The Hscore combines the percentage of positive cells and staining intensity level weak moderate strong and is calculated using the following formula [ — cells — cells — cells ] with results ranging from to Positive epidermis and placenta and epidermal nevus and negative connective and adipose tissue internal controls were assessed for all the evaluated cases Samples from the primary cohort were classified concerning the presence or absence of these receptors and the level of Ki67 expression into the following groups Luminal A LumA luminal B LumB triple negative and HER2 A Ki67 index cut point of was defined to distinguish HER2 negative lumB from lumA tumors5960Differential gene expression and methylation analysis For the validation cohort RNA sequencing RNASeq RSEM gene expression data from The Cancer Genome Atlas TCGA breast cancer BRCA study were obtained from the Genomic Data Commons GDC Data Portal gdcporta lcninihgov using the TCGAbiolinks RBioconductor package61 Raw expression signals for primary solid tumor samples N and solid normal tissue samples N were normalized and analyzed for differential expression of DIO3 using the limmavoom pipeline from the limma RBioconductor package62 P values were adjusted for multiple comparisons using the false discovery rate FDR procedure of Benjamini and Hochberg63 Clinicopathological information for TCGABRCA samples was downloaded through TCGAbiolinks and the Broad GDAC Firehose gdacbread insti tute merged level clinical data For tumors of the TCGABRCA cohort data retrieved from PAM50 classification were used to define tumor subtype classification64 Overall survival OS was estimated by the Kaplan“Meier method and compared by the logrank test using functions provided by TCGABiolinks For the methylation analysis we used the TCGAbiolinks RBioconductor package30 to obtain and analyze Illumina a0K methylation and clinical data for samples from the TCGABRCA study includScientific RepoRtS 101038s41598020708924V

Thyroid_Cancer

The effects of tissue fixation on sequencingand transcript abundance of nucleic acidsfrom microdissected liver samples ofsmallmouth bass Micropterus dolomieuHeather L WalshID Adam J Sperry Vicki S BlazerUS Geological Survey National Fish Health Research Laboratory Leetown Science Center KearneysvilleWest Virginia United States of Americaa1111111111a1111111111a1111111111a1111111111a1111111111 hwalshusgsgovAbstractThere is an increasing emphasis on effectsbased monitoring to document responses associated with exposure to complex mixtures of chemicals climate change pathogens parasitesand other environmental stressors in fish populations For decades aquatic monitoring programs have included the collection of tissues preserved for microscopic pathology Consequently formalinfixed paraffinembedded FFPE tissue can be an important reservoir ofnucleic acids as technologies emerge that utilize molecular endpoints Despite the crosslinking effects of formalin its impact on nucleic acid quality and concentration amplification andsequencing are not well described While freshfrozen tissue is optimal for working withnucleic acids FFPE samples have been shown to be conducive for molecular studies Lasercapture microdissection LCM is one technology which allows for collection of specificregions or cell populations from fresh or preserved specimens with pathological alterationspathogens or parasites In this study smallmouth bass Micropterus dolomieu liver was preserved in three different fixatives including neutral buffered formalin NBF ZFix®ZF and PAXgene® PG for four time periods hr hr seven days and days Controls consisted of pieces of liver preserved in RNALater® or ethanol Smallmouth basswere chosen as they are an economically important sportfish and have been utilized as indicators of exposure to endocrine disruptors and other environmental stressors Small liversections were cut out with laser microdissection and DNA and RNA were purified and analyzed for nucleic acid concentration and quality Sanger sequencing and the NanoStringnCounter® technology were used to assess the suitability of these samples in downstreammolecular techniques The results revealed that of the formalin fixatives NBF samples fixedfor and hr were superior to ZF samples for both Sanger sequencing and the NanostringnCounter® The nonformalin PAXgene® samples were equally successful and they showedgreater stability in nucleic acid quality and concentration over longer fixation times This studydemonstrated that small quantities of preserved tissue from smallmouth bass can be utilizedin downstream molecular techniques however future studies will need to optimize the methods presented here for different tissue types fish species and pathological conditions ACCESSCitation Walsh HL Sperry AJ Blazer VS The effects of tissue fixation on sequencing andtranscript abundance of nucleic acids frommicrodissected liver samples of smallmouth bassMicropterus dolomieu e0236104 101371journalpone0236104Editor Rajakumar Anbazhagan National Instituteof Child Health and Human Development NICHDNIH UNITED STATESReceived April Accepted June Published August Copyright This is an access free of allcopyright and may be freely reproduceddistributed transmitted modified built upon orotherwise used by anyone for any lawful purposeThe work is made available under the CreativeCommons CC0 public domain dedicationData Availability Statement All relevant data arewithin the paper and its Supporting InformationfilesFunding This was fully funded internally by theUSGS Environmental Health Contaminant BiologyProgram and the USGS EcosystemsEnvironments and Fisheries Program MissionAreas There are no actual grant numbersPLOS ONE 101371journalpone0236104 August PLOS ONE 0cCompeting interests The authors have declaredthat no competing interests existEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostIntroductionGlobally environmental monitoring programs are increasingly used to identify adverse effectsof human activities on aquatic resources [“] The recognition that there are numerouschemical contaminants environmental stressors as well as new and emerging pathogensparasites cooccurring has led to an emphasis on biological environmental effectsbased assessments utilizing resident indicator fish species or caged model species [“] Histopathologyhas been used for many years to assess the health of wild fishes both for specific studies as wellas part of effectsbased monitoring programs [“] More recently genomic endpoints arealso being incorporated into environmental monitoring and risk assessment [“] Whenboth histopathology and molecular analyses are part of an assessment pieces of a tissue arecommonly preserved in buffered formalin or a similar preservative and adjacent separatepieces are preserved in RNAlater1 ethanol or frozen for molecular analyses [“]However for alterations not visible the tissue piece chosen for gene expression may not contain the same cellular components or alterations as those within the histology sectionThe use of formalin fixed paraffinembedded FFPE tissue has been regarded as a valuablereservoir of preserved nucleic acids in mammalian studies [“] Although FFPE tissuesprovide a vast source of pathologically diverse types of genetic material there are drawbackscompared to other tissue preservation methods Formalin fixation causes nucleic acids to fragment degrade and crosslink [] Frozen tissues or tissues specifically preserved for downstream nucleic acid applications do not experience the type of degradation observed fromformalin fixation Despite these setbacks nucleic acids extracted from FFPE tissue have provento be suitable for use in endpoint PCR [] realtime qPCR [ ] and Nextgenerationsequencing [ ] Optimization of FFPE tissues for downstream nucleic acid applicationshas been attempted in multiple studies by evaluation of different fixation methods [ ] tissue handling and processing times [ ] and extraction methods [ ]Laser capture microdissection LCM utilizes a microscope equipped with a laser to targetand isolate specific cells from a heterogeneous population of cells [] Single cells foci of cellpopulations within a tissue or pathogens and parasites can be microdissected Hence nucleicacids from specific cell populations of interest can be analyzed for gene expression studiestranscriptome development or molecular identification of pathogens and parasites Thisallows for a more direct connection between the histopathology and molecular analyses LCMhas been previously utilized in fishrelated studies [ “] with frozen sections Snapfrozen tissue is optimal for use with LCM for the downstream recovery of nucleic acids However the use of snapfrozen tissue is not always feasible particularly in wild fish studies whereremoval and fixation of the ans occurs in the field and it can be days before tissues arereturned to the laboratory and processed LCM of FFPE tissue can bridge the gap betweenmicroscopy and molecular analyses [] As with other species there is a vast amount of archival FFPE or similarly preserved fish tissue that could be useful for molecular analysesThe aim of this study was to determine how fixative type and fixation time affects nucleicacids in FFPE smallmouth bass liver tissue dissected with LCM Smallmouth bass Micropterusdolomieu are utilized in ongoing monitoring and assessment studies as an indicator species ofexposure to endocrinedisrupting and other contaminants Additionally they are a nonmodel but economically important species To address the utility of paraffinembedded fishtissue for molecular studies smallmouth bass liver was sampled and preserved for four timeperiods hr hr seven days and days in neutral buffered formalin NBFZFix1 ZF and the nonformalin fixative PAXgene1 PG The PAXgene1 Tissue Systemwas designed to improve tissue quality for parallel molecular and morphological analyses []Similarly ZF a zincbased formalin solution was chosen as it has been shown to producePLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleosthigher yields of DNA and RNA when compared to samples fixed in NBF [] In addition toDNA and RNA quantification downstream molecular techniques including Sanger sequencing and the Nanostring nCounter1 digital multiplexed gene expression assay [] were usedto determine if nucleic acids extracted from LCM tissue sections would have utility in futurestudies To the best of our knowledge this study provides novel research on the optimizationof fixative type and fixation time for the use of fish tissue extracts with Nanostring nCounter1technologyMaterials and methodsEthics statement and smallmouth bass sample collectionAll procedures including the handling and euthanasia of fish were approved by the US Geological Survey™s Leetown Science Center™s Institutional Animal Care and Use CommitteeIACUC protocol Five smallmouth bass approximately years old were sampledfrom a flowthrough tank at the US Geological Survey Fish Health Laboratory in Kearneysville West Virginia Fish were placed in a lethal dosage mgL of tricaine methanesulfonate TricaineS Syndel Ferndale WA for euthanasia An incision from the anus tooperculum was made the liver was excised dissected into five equal pieces and placed into fixatives consisting of NBF ZF Product Anatech Ltd Battle Creek MI and PG Product QIAGEN Valencia CA Pieces of liver from each fish were also placed into RNALater1 Product AM7021 Thermo Fisher Scientific Waltham MA and ethyl alcoholETOH to serve as controls Samples in RNALater1 were stored at ˚C for hr prior tostorage at ˚C and samples in ETOH were stored at room temperature RT until extractionswere completedHistological preparation and laser capture microdissectionSamples were fixed for hrs hrs seven days and days at RT for NBF and ZF Tissues preserved in PG were removed from the PAXgene1 Tissue FIX Product QIAGEN after hrs at RT placed in the PAXgene1 Tissue STABILIZER solution Product QIAGEN and stored at ˚C for hrs hrs seven and days Tissue processing was performed on a Shandon CitadelTM Tissue Processor Thermo FisherScientific as follows hrs in alcohol hr in alcohol hr in alcohol 2x hr in alcohol 3x hr in a solution of alcohol and histoclear 2x Product HS200 National Diagnostics Atlanta GA hr in histoclear 2x and hr in paraffin 2x at ˚C Upon completion tissues were embedded into paraffin wax and cooled tohardenTissues were cut at a thickness of μm using a new sterile razor for each sample and sections placed onto Leica Microsystems UVsterilized polyethylene napthalate PEN membraneslides Product NC0496333 Thermo Fisher Scientific Sterilized diethyl pyrocarbonateDEPC Product Millipore Sigma Burlington MA water was used in the water bathand slides were allowed to air dry after sections were placed on the PEN membrane slideUnstained tissue sections were deparaffinized with Anatech Ltd ProPar Clearant Product NC9537734 Thermo Fisher Scientific for min 2x and allowed to air dry prior to lasermicrodissection Liver sections were cut at 5x magnification with a Leica LMD6500 microscope Leica Microsystems at a pulse rate of “ nm Sections x mm2were cut and dropped into the cap of a sterile microcentrifuge tube by gravity Fig and subsequently extracted for RNA or DNAPLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostFig Laser capture microdissection of a smallmouth liver section A Liver section prior to microdissection BLiver section after microdissection C Liver section floating in buffer in the cap of a microcentrifuge tub prior tonucleic acid extraction101371journalpone0236104g001Nucleic acid extractions and downstream analysesFor RNA purification the EZNA1 FFPE RNA Kit Product R695401 Omega BioTekNorcross GA was used according to manufacturer™s protocols for the xylene extractionmethod Extraction began with the addition of GPL Buffer skipping the beginning of the protocol since the tissues were already deparaffinized Samples were digested with proteinase Kfor min and eluted in μl DEPC water As part of the assay protocol DNA contaminationwas removed with a step involving DNA Clearance Columns that binds genomic DNA andallows RNA to pass through the spin column For the controls preserved in RNALater1approximately “ mg liver was extracted with an EZNA1 Total RNA Kit I Product R683402 Omega BioTek according to manufacturer™s protocols and eluted in μl DEPCwater DNA contamination was also removed from these samples with the use of HiBind1RNA Mini Columns and RNasefree DNase Product E109102 Omega BioTek All samples were quantified with a Qubit1 Fluorometer Invitrogen Carlsbad CA using theQubit1 RNA HS Assay Kit Product Q32852 Thermo Fisher Scientific To analyze degradation RIN values were obtained with the Agilent RNA Pico Kit Product “Agilent Technologies Santa Clara CA on an Agilent Bioanalyzer Agilent Technologies Following quantification samples were stored at ˚C prior to use on the NanostringnCounter1DNA was extracted with a proteinase K digestion buffer 50mM TrisHCl pH mMEDTA Tween mgml proteinase K as described in Lehmann and Kreipe []Each sample was extracted in μl of proteinase K digestion buffer and incubated overnight at˚C The tubes were vortexed centrifuged and incubated at ˚C for min to deactivateproteinase K and stored at ˚C Approximately “ mg of control liver samples preservedin ETOH were extracted with a Qiagen DNeasy Blood Tissue Kit Product QIAGEN according to manufacturer™s protocols It is worth mentioning that in initial trialsfor this study the Qiagen DNeasy Blood Tissue Kit was also used to extract DNA from LCMsamples however no quantifiable DNA could be obtained which was why a single tube extraction method was subsequently utilized DNA was quantified with the Qubit1 dsDNA HSAssay Kit Product Q32851 Thermo Fisher Scientific Samples were analyzed for mean fragment size and distribution on an Agilent Bioanalyzer with the Agilent High SensitivityDNA Kit Product “ Agilent TechnologiesFor all LCM samples the final concentration of purified RNA and DNA was standardizedby dividing the total concentration by the total amount of tissue collected μgmm3 Since agreater amount of tissue was extracted from control samples the concentration of purifiedRNA and DNA was standardized to the amount of tissue collected for LCMTo assess the suitability of LCM samples for downstream molecular analyses Sangersequencing and the NanoString nCounter1 Technology were used For endpoint PCRPLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostprimers EF1α5F ™GAG CCC CCT TAC AGC CAG AAG3™ and EF1α5R ™TTCACC TCA GTG GTC AGG CA3™ were designed with NCBI Primer BLAST [] to amplifya bp amplicon of the smallmouth bass elongation factor alpha EF1α accession HQ4248721 gene This housekeeping transcript was chosen since it has been used in othersmallmouth bass studies [ ] and sequence data was available for both smallmouth bassand the closely related largemouth bass accession KT8277941 PCR amplification was conducted under the following conditions denaturation at ˚C for min followed by cyclesof ˚C for s ˚C for s and ˚C for min s with a final extension at ˚C for min Each reaction consisted of μl clear Go Taq Master Mix Product M7132 PromegaMadison WI μl of each primer at 10μM and μl template for LCM samples approximately “ ng and μl template for ETOH samples approximately “ ng Uponcompletion samples were analyzed on an agarose gel with a bp ladder Amplicons werecleaned with a QIAquick1 PCR purification kit Product QIAGEN and eluted in μl of Buffer EB Purified amplicons were used as template in cycle sequencing reactionswith the Applied Biosystems BigDye Terminator v31 Cycle Sequencing Kit Product Thermo Fisher Scientific for cycles of ˚C for min ˚C for s ˚C for sand ˚C for min Cycle sequencing reactions were purified with an Agencourt CleanSEQKit Product A29151 Beckman Coulter Brea CA and sequenced on an ABI 3130xl GeneticAnalyzer Applied Biosystems Foster City CA Sequences were analyzed with Geneious wwwgeneiouscom and quality was assessed by the percentage of bases with aquality score of or higher Q40 NCBI BLASTn was used to determine sequence similarityto the Micropterus spp EF1α gene HQ4248721 or KT8277941NanoString nCounter1 Technology was used with a Custom CodeSet that targeted transcripts expressed in the liver of smallmouth bass as described in Hahn et al [] The previous establishment and availability of this CodeSet was the reason liver was chosen as the tissue of focus in this study The liver is also the principal an for many chemical detoxificationpathways metabolic pathways and the production of vitellogenin In brief the nCounter1platform provides the capability to quantify up to RNA DNA or protein targets called aCodeSet in a multiplex fashion providing results similar to quantitative PCR qPCR [] Itis a costeffective method to analyze specific mRNA targets unlike RNAsequencing whichproduces a vast amount of data and captures all mRNA in a sample Sample setup for hybridizations was carried out according to manufacturer™s protocols with ng of total RNA forevery sample The limit of detection LOD was calculated as the mean of the negative controls � the standard deviation of the negative controls and was transcriptsStatisticsSignificant differences in nucleic acid concentrations and transcript abundance between fixatives for each fixation time were determined with a nonparametric KruskalWallis onewayANOVA followed by a Dunn™s multiple comparison posthoc analysis with a Bonferroni correction in the statistical program R [] Normalized transcript abundance data was used forthe analysis Transcript abundance data was normalized in nSolver Analysis Software Nanostring Technologies Seattle WA where the geometric mean of the negative controlswas subtracted to estimate background and the normalization factor was computed from thegeometric mean of the positive controls and the housekeeping transcripts Housekeeping transcripts were log2 transformed and analyzed for stability with NormFinder [] in R A KruskalWallis test was also used to identify differences amongst each fixative for each fixationtime and the template concentration used for PCR the Q40 score and sequence lengthobtained with Sanger sequencing Finally Spearman™s rank correlation analyses werePLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostconducted in R to determine if the concentration of DNA samples used for PCR sequencelength and the Q40 score were associated the number of sequences with similarity to theMicropterus spp EF1α gene with Sanger sequencing Pvalues � were considered statistically significantResultsNucleic acid concentrationsBoth RNA and DNA were recovered from samples of all fixatives and fixation times Fig Liver samples preserved in RNALater1 had more than times greater RNA concentrationsthan samples preserved in NBF ZF or PG with a mean concentration of ± ngmm3 mean ± standard error The highest concentration of RNA from LCM samples wasobtained from PG samples at hr ± ngmm3 Fig 2A The lowest concentrationsFig Nucleic acid auantification A Mean standard error of RNA and B DNA concentrations μgmm3 ofmicrodissected smallmouth bass liver samples fixed in neutral buffered formalin NBF ZFix1 ZF andPAXgene1 PG for hours hours and days Samples preserved in alcohol ETOH and RNALater1were included as controls101371journalpone0236104g002PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostwere from NBF preserved tissues at two weeks ± ngmm3 The concentration ofRNA was significantly greater in RNALater1 samples than in NBF Pvalue and PGPvalue at hr NBF Pvalue and ZF Pvalue at hr NBF Pvalue and ZF Pvalue at seven days and NBF Pvalue � ZF Pvalue and PG Pvalue at days Mean concentrations of RNA in samplesfixed in NBF and ZF decreased at seven and days while those fixed in PG remained stablethroughout the time course Fig 2AThe amount of DNA recovered was less than RNA with all mean concentrations of LCMsamples below μgmm3 Samples fixed in ETOH had more than times greater concentrations of DNA than samples fixed for LCM with a mean concentration of ± ngmm3 The concentration of DNA was significantly greater in ETOH samples than in PG at hr Pvalue NBF Pvalue and PG Pvalue at hr NBF Pvalue and PG Pvalue at seven days and NBF Pvalue and PG Pvalue at days There was little variation in DNA concentrations over time for anyof the fixatives although for all fixatives the lowest concentration was at days Fig 2BThe quality of RNA varied among fixatives Mean RIN values of samples fixed in RNALater1 were at least twice as great as samples fixed in NBF ZF and PG The highest RIN valuesfor LCM samples were observed in NBF fixed tissue at hrs seven and days Fig 3A RINvalues were significantly greater in RNALater1 samples than in PG Pvalue and ZFPvalue samples at hr PG Pvalue and ZF Pvalue samples atFig Nucleic acid quality A Mean RIN values of RNA and B fragment size bp of DNA from samples fixed in neutral buffered formalin NBF ZFix ZF and PAXgene PG for hours hours and days Samplespreserved in alcohol ETOH and RNAlater were included as controls101371journalpone0236104g003PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleost hr PG Pvalue and ZF Pvalue samples at seven days and PG Pvalue � and ZF Pvalue samples at days There were no significant differences in RIN values between LCM samplesMean fragment size of DNA varied over time within each fixative group but was not significantly different than samples fixed in ETOH Fig 3B Fragment size was only significantlygreater in NBF samples than in PG samples P value at hrDownstream analysesEndpoint PCR and Sanger sequencing were successful for the amplification and sequencingof the smallmouth bass EF1α gene although differences in sequencing success were apparentWithout trimming the ™ or ™ ends samples preserved in ETOH produced sequences with amean percentage of bases with a Q40 score or greater of while samples fixed for LCMproduced lower quality sequences Fig Of the samples fixed for LCM PG preserved samplesproduced the highest quality sequences At days there were no samples fixed in NBF or ZFthat were successful for sequencing the EF1α gene For NBF and ZF the best quality sequenceswere generated by samples fixed for hr conversely PG had the lowest quality sequencesfrom samples fixed for hr Fig It should be noted that of the five PG samples sequencedat hr two samples had much lower quality sequences than the other three samples whichmay have contributed to the decrease in the mean percentage of high quality sequences at hr Additionally multiple samples failed to sequence These included one of the ETOH samples forward and reverse sequences three NBF day samples forward sequences oneNBF seven day sample forward and reverse sequences two PG seven day samples forwardsequences one PG day sample forward sequence one PG hr sample reversesequence one ZF seven day sample reverse sequence four ZF day samples three forwardand one reverse sequence and one ZF hr sample forward sequence In order to calculatethe percentage of sequences with similarity to the Micropterus spp EF1α gene failed sequenceswere not included ie of sequences with similarity to Micropterus spp EF1α ofsequences with similarity to Micropterus spp EF1α total of sequences that were successfullysequenced � For LCM fixed samples NBF samples fixed for and hr produced theFig Sanger sequencing quality Mean SEM percentage of bases with a Q40 score or above indicative of highquality sequencing Samples preserved in ethanol ETOH were included as controls routinely used for DNApreservation101371journalpone0236104g004PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostgreatest percentage of sequences with similarity to the EF1α gene In ZF samplesthe greatest percentage of samples with similarity to the EF1α gene was at hr and in PG samples it was at seven days Although PG samples produced the leastamount of sequences with similarity to the EF1α gene at and hr it produced the greatestnumber of sequences at seven and daysA Spearman™s rank correlation analysis with all samples revealed that sequence length pvalue � rho and template concentration pvalue rho were significantly associated with the number of sequences with similarity to the EF1α gene Fig Although PCR primers were estimated to produce an amplicon size around bp manysequences were longer than bp This could be due to the high degree of fragmentation inthe samples which may have resulted in the annealing of small fragments to the original template molecules in overlapping regions [] Fragment length and the percentage of bases witha Q40 score or greater were not significantly correlated with the number of sequences withsimilarity to the EF1α gene The correlations were also examined excluding the ETOH controlssince the DNA concentration of the controls was significantly greater than those of many fixedsamples and to examine the differences amongst the fixatives only Sequence length remainedsignificant pvalue � rho however template concentration was not significantpvalue rho Fragment length and the percentage of bases with a Q40 scoreFig Sanger sequencing and NCBI blastn results A Spearman™s rank correlation analysis of sequence length bpand the number of NCBI blastn matches to the Micropterus spp elongation factor alpha EF1α gene B Meansequence length bp of the EF1α gene obtained with Sanger sequencing and the percentage of sequences withsimilarity to the Micropterus spp EF1α gene101371journalpone0236104g005PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable KruskalWallis nonparametric ANOVA results Significant differences in template concentration usedin PCR and Sanger sequencing results for each fixative between fixation timesFixativeNBFZFPGFixativeNBFNBFZFPGFixativeNBFNBFZFZFPGTemplate ConcentrationpvalueZ scoreFixation TimeNo significant differencesNo significant differences hr vs days of Bases with � Q40 ScorepvaluepvalueZ scoreFixation Time hr vs days hr vs hrNo significant differencesNo significant differencesSequence LengthZ scoreFixation Time days vs hr days vs hr days vs hr days vs hrNo significant differencesSignificant differences pvalue � in template concentration of bases with a Q40 score or greater andsequence length between samples fixed in neutral buffered formalin NBF ZFix1 ZF and PAXgene PG101371journalpone0236104t001or greater remained not significantly correlated A KruskalWallis test was used to identify significant differences between fixation times and template concentration percentage of baseswith a Q40 score or greater and sequence length for each fixative Table The Nanostring nCounter1 results revealed multiple occurrences of samples fixed forLCM with similar or greater transcript abundance compared to RNALater1 samples S1Table In samples fixed for LCM there was significantly greater transcript abundance in PGsamples than in NBF andor ZF samples for one transcript at seven days and eight transcriptsat days Interestingly there were multiple significant differences in housekeeping transcriptabundances between samples preserved in RNALater1 and samples fixed for LCM MeanEF1α transcript abundance was significantly greater in RNALater1 samples than in samplesfixed for LCM at all fixation times Conversely at seven and days 40S ribosomal protein S12transcripts were higher in NBF and ZF samples when compared to RNALater1 and ribosomalprotein L8 was higher in the PG samples Table Significant differences were also identified between fixation times for each fixative typeused to preserve LCM samples Table All significant differences were between fixationtimes hr and seven or days and hr and seven or days In some instances sampleswith longer fixation times had transcripts with significantly greater transcript abundance thansamples fixed for or hr Once again significant differences were identified amongsthousekeeping transcripts There were no significant differences in PG samples over timeNormFinder results ranked the housekeeping transcripts according to stability For all fixatives and all fixation times including RNALater1 samples the most stable housekeepingtranscript was Ribosomal Protein L8 stability followed by EF1α stability Eukaryotic Translation Initiation Factor 3D stability and 40S ribosomal protein S12stability PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable KruskalWallis nonparametric ANOVA results Significant differences in transcript abundance betweenfixatives for each fixation timeTranscript NameElongation Factor Alpha�Heat Shock Protein Elongation Factor Alpha�Thyroid Hormone Receptor BetaTranscript NameElongation Factor Alpha�Heat Shock Protein Elongation Factor Alpha�Heat Shock Protein Transcript Name40S Ribosomal Protein S12�ArginaseBetacateninElongation Factor Alpha�Heat Shock Protein Transforming Growth Factor Beta40S ribosomal protein S12�C Reactive ProteinlikeElongation Factor Alpha�Eukaryotic Translation Initiation Factor 3D�Transforming Growth Factor BetaApolipoprotein Elongation Factor Alpha�Ribosomal Protein L8�Superoxide DismutaseThyroid Hormone Receptor BetaThyroid Hormone Receptor BetaTranscript Name40S ribosomal protein S12�ArginaseElongation Factor Alpha�40S ribosomal protein S12�ArginaseC Reactive ProteinlikeElongation Factor Alpha�Ribosomal Protein L8�Transforming Growth Factor BetaApolipoprotein Aryl Hydrocarbon ReceptorElongation Factor Alpha�Heat Shock Protein Ribosomal Protein L8� Hrpvalue Hrpvalue Dayspvalue DayspvalueZ scoreZ scoreZ scoreZ scorePLOS ONE 101371journalpone0236104 August FixativesRNALater vs NBFRNALater vs NBFRNALater vs PGRNALater vs PGFixativesRNALater vs PGRNALater vs NBFRNALater vs ZFRNALater vs ZFFixativesRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGPG vs NBFFixativesRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGContinued PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable ContinuedAryl Hydrocarbon ReceptorEstrogen Receptor Beta Hepcidin Thyroid Hormone Receptor BetaType II Iodothyronine DeiodinaseAryl Hydrocarbon ReceptorHepcidin Thyroid Hormone Receptor BetaType II Iodothyronine DeiodinasePG vs NBFPG vs NBFPG vs NBFPG vs NBFPG vs NBFPG vs ZFPG vs ZFPG vs ZFPG vs ZFSignificant differences pvalue � in transcript abundance between RNALater1 samples and samples fixed in neutral buffered formalin NBF ZFix1 ZF and PAXgene PG� Indicates housekeeping transcripts101371journalpone0236104t002Table KruskalWallis nonparametric ANOVA results Significant differences in transcript abundance betweenfixation times for each fixativeTranscript Name40S ribosomal protein S12�ZFixpvalueCytochrome P450 family subfamily AElongation Factor Alpha�Eukaryotic Translation Initiation Factor 3D�Ribosomal Protein L8�TataBox Binding ProteinThyroid Hormone Receptor BetaTransforming Growth Factor BetaTranscript Name40S ribosomal protein S12�Cytochrome P450 family subfamily ABeta

Thyroid_Cancer

"Handling EditorAdrian CovaciKeywordsNuclear accidentsHealth surveillancePreparednessCommunicationStakeholdersRecommendationsSerious accidents at nuclear power plants have been rare but theirstories can teach us how to prevent or mitigate the eï¬ects of futurenuclear catastrophes The accidents at the Fukushima Daiichi nuclearpower plant and Chernobyl nuclear power plant occurred years and years ago respectively and there are still lessons to learn from themregarding numerous issues including radiation exposure assessmentand medical followup of emergency responders evacuees and residents decisions to lift evacuation orders and communication withresponders and stakeholders Bazyka Callen and Homma Lester Soï¬er Some of the lessons from theseaccidents have been extensively reviewed and taken into considerationby national and international anizations such as the InternationalAtomic Energy Agency the International Commission on RadiologicalProtection and the World Health anisation and are reflected inpublished literature Bennett Carr Clarke IAEA 2015a 2015b Nisbet SGDSN This hasallowed the development of various recommendations and guidancedocuments targeting specific issues of radiation protection training andcommunication and socioeconomic aspects in order to prepare andimprove decision making processes in the early and intermediatephases eg Carr IAEA 2015b Nisbet However the majority of these texts focus on technical issues andare directed towards radiation protection experts rather than for thesupport of aï¬ected populations The traditional approaches of emergency response and recovery including evacuation relocation andhealth surveillance are largely based on dose levels Although manyrecognise the importance of psychosocial or human factors it has beendifficult to adapt the approaches to better address the social economicethical and psychological factors These include the health and welfareeï¬ects that may arise from the accident from the concerns about thepresence of radiation in the environment from the mitigation actionstaken and from the information mixed or absent provided to the population Changes in the ethical and legal requirements for personaldata collection use and storage raise additional challenges particularlyin the area of health surveillance and epidemiologyAbbreviations COVID19 Coronavirus Disease EJP CONCERT European Joint Programme for the Integration of Radiation Protection Research OPERRA Project for the European Radiation Research Area SGDSN Secrtariat gnral de la dfense et de la scurit nationale France SHAMISEN NuclearEmergency Situations Improvement of Medical and Health Surveillance SHAMISEN SINGS SHAMISEN Stakeholder INvolvement in Generating Science UN UnitedNations UNDRR United Nations Office for Disaster Risk Reduction101016jenvint2020106000Received July Accepted July Published by Elsevier Ltd This is an access under the CC BYNCND license httpcreativecommonslicensesBYNCND40 0c Main components of the SHAMISEN projectThe SHAMISEN project started in late at a time when somedeleterious eï¬ects of evacuation and ultrasound thyroid screening inFukushima had started to be reported The project therefore aimed toreview the lessons learned from major nuclear accidents in particularfrom experiences of populations aï¬ected by the Chernobyl andFukushima accidents to develop recommendations for medical andhealth surveillance of populations aï¬ected by previous and future radiation accidents The ultimate motivation was to minimise the negative impacts of the accident and improve the health of aï¬ected populations The holistic WHO definition of health was used in this contextie œa state of complete physical mental and social wellbeing and notmerely the absence of disease or rmity WHO The Recommendations were to address in particular the following complementary aspects dose assessment supporting all phases of an accident including emergency response clinical decisionmaking recoveryactions and health surveillance improvement of living conditions ofaï¬ected populations engaging them and responding to their needs andminimising unnecessary anxiety and health surveillance and wherefeasible improvement of estimates of radiationinduced risk for radiation protection and communication with aï¬ected populationsTo achieve this and recognising the need for a holistic approach toaccident management and health surveillance SHAMISEN brought together a team of researchers from institutions including RadiationProtection Authorities Universities Research Centres and Associationsin Europe and Japan with complementary experience and a long trackrecord in post accidental management dosimetry radiation protectionmedical followup and screening population health surveillance healtheconomics epidemiology ethics and sociology of radiation protectionThe project also drew upon additional expertise from Belarus RussiaUkraine Japan and the UK as well as from outside of the radiationresearch field and established contacts with major international anizations including the World Health anisation and the NuclearEnergy Agency of the anisation for Economic Cooperation andDevelopmentDetails of the SHAMISEN Project are provided in the paper by Ohbaand collaborators Ohba this issue Briefly the approach involved in particular challengingevaluating the eï¬ectiveness of measures taken after Chernobyl or Fukushima accidents in particularcid129 Systematic thyroid screening with ultrasound for early detection ofpotential thyroid cancer casescid129 Criteria for evacuation and their consequencescid129 Measures taken to contribute to the wellbeing of aï¬ected populations and develop a radiological protection culture and resilience inaï¬ected populationscid129 Challenges and good practice in communication and training withthe objective of regaining the trust of the population and engagingthem in retaking control of their livescid129 Role of ethics in disaster preparedness response and health surveillance autonomy and dignity respect of privacy beneficence¦cid129 Role of health professionals in the diï¬erent phases of the accidentmanagementcid129 Cost eï¬ectiveness of the measures takenAll of this was brought together to develop practical recommendations for preparedness and the diï¬erent phases of the accident SHAMISEN results and the way forwardAlthough the SHAMISEN project was developed during a limitedperiod of months in response to the second call of the EuropeanOPERRA project a series of key results has been achieved The mainresults are several topical reports and a set of recommendations dividedinto five main topics eg Evacuation Communication and trainingEnvironment International Dosimetry Health surveillance Epidemiology These topics focusspecifically on the health surveillance of people following a nuclearaccident combining natural and social sciences values and practice tohelp health professionals decisionmakers and local stakeholders to setup protective actions and health programmes responding to the concernof aï¬ected populations Therefore the SHAMISEN recommendationsare not intended to cover all aspects of emergency and recovery response and preparednessThe formulation of the recommendations is generic enough to beapplied in diï¬erent countries recognising that cultural diï¬erences willbe important The structure describes the general context and the mainreasons for developing each recommendation provides explanations onhow to develop it and indicates who would be involved in the development of the recommendation Depending on the context specificarrangements have to be made for the implementation of these recommendations during the diï¬erent phases eg preparedness earlyand intermediate longterm recoveryThe recommendations provide advice on the values to be consideredwhen addressing the issue at stake and what type of tools and protocolsare needed rather than the tools themselves Due to the duration of theproject it was not manageable to develop them However they providea significant input for further developments for practical tools in different domains and identify the main expectations from stakeholderswith regards to health surveillance in postaccident situationsDeveloped as a research project it was not the intention to specifyabsolute dose criteria for the implementation of actions Of coursediscussions on the feedback experience from the management of theChernobyl and Fukushima accidents point out some challenges associated with the use of specific dose criteria but the spirit of the recommendations is to provide indications and guidance for the decisionmakers and health professionals with regard to the choice to be madeon the adoption of dose criteria for the diï¬erent actions to be implementedBesides the management of the direct radiation induced health effects the report underlines the need to develop a multidisciplinaryapproach to identify measure assess and alleviate psychological andother indirect health impacts of socioeconomic and social upheavals ofthe consequences of the accident For this purpose it is recommendedto promote the engagement process of local stakeholders since thepreparedness phase targeting the overall wellbeing of populations withdue considerations of the ethical principles of respect for autonomydignity and justiceThis special issue of Environmental International combines a series ofscientific papers and is an opportunity to emphasize the main analysesdeveloped during the SHAMISEN project combining advanced scientific research analyses of feedback experience from the Chernobyl andFukushima accidents and applying a multidisciplinary approachAlthough the topics presented in this special issue have already beenaddressed in general in several papers the originality of the approachadopted in the SHAMISEN project provides new insights for healthsurveillance issuesIt is worth mentioning that following the SHAMISEN project otherresearch projects have been launched A first series of projects arededicated to the development of Apps with and for citizens as recommended in the SHAMISEN project This has notably been done withthe European research project SHAMISEN SINGS as part of the EJPCONCERT as well as with an ongoing project developed by FukushimaMedical University In addition several projects are currently underdevelopment in diï¬erent countries and at the European level thatpromote a citizen science approach for addressing health and radiological monitoringAs an example of the multidisciplinary approach the SHAMISENproject has identified a series of recommendations calling for furthercooperation with diï¬erent European Research Platforms combininglow doses eï¬ects dosimetry radioecology emergency and recoverymanagement social sciences and humanities and medical research 0cEnvironment International and Thierry SchneideraŽ Deborah Oughtonb Elisabeth Cardiscdea CEPN Nuclear Protection Evaluation Centre Rue de la Redoute FontenayauxRoses Franceb Centre for Environmental Radioactivity CERAD NMBU „«s Norwayc Barcelona Institute for Global Health ISGlobal Barcelona Spaind Pompeu Fabra University Barcelona Spaine Spanish Consortium for Research and Public Health CIBERESP Institutode Salud Carlos III Madrid SpainEmail address thierryschneidercepnassofr T SchneiderReferencesBazyka D Belyi D Chumak A Lessons from chornobyl considerations forstrengthening radiation emergency preparedness in Ukraine Radiat Prot Dosim “ 101093rpdncw196Bennett B Repacholi M Carr Z Eds Health eï¬ects of the Chernobyl accidentand special health care programmes Report of the UN Chernobyl Forum expert groupœHealth WHO Press GenevaCallen J Homma T Lessons learned in protection of the public for the accident atthe Fukushima Daiichi nuclear power plant Health Phys “ 101097HP0000000000000666Carr Z Clarke M Akl EA Schneider R Murith C Li C ParrishSprowl J StenkeL CuiPing L Bertrand S Miller C Using the grade approach to supportthe development of recommendations for public health interventions in radiationemergencies Radiat Prot Dosim “ 101093rpdncw234Clarke R Valentin J International Commission on Radiological Protection Task Group ICRP publication Application of the Commission™s Recommendations forthe protection of people in emergency exposure situations Ann ICRP “101016jicrp200905004Croua¯l P Camps J Raskob W Schneider T NERIS Roadmap on medium andlongterm research on preparedness for nuclear and radiological emergency responseand recovery Version eunerisnetlibrarysra259nerisroadmap2020htmlIAEA 2015a The Fukushima Daiichi Accident No Technical Volume IAEAInternational Atomic Energy Agency Vienna Austria wwwiaeapublications10962thefukushimadaiichiaccidentIAEA 2015b Preparedness and Response for a Nuclear or Radiological Emergency NoGSR7 Safety Standards Series IAEA International Atomic Energy AgencyVienna Austria httpwwwpubiaeaMTCDPublicationsPDFP_1708_webpdfImpens N Salomaa S Second joint roadmap for radiation protection researchDeliverable No EJPCONCERT European Joint Programme for the Integrationof Radiation Protection Research H2020 wwwconcerth2020euDocumentashxdtwebfileListsDeliverablesAttachments206D37_Second20joint20roadmap_draft_reviewed_20052020_approved03062020pdfguid01b5ac77b2ec4cda9c98917dba396f0fLester MS Public information during a nuclear power plant accident lessonslearned from Three Mile Island Bull N Y Acad Med “Nisbet AF Jones A Turcanu C Camps J Andersson KG H¤nninen RRavantaara A Solatie D Kostiainen E Julien T Pupin V Ollagnon HPapachristodoulou C Ioannides K Oughton D Generic Handbook forAssisting in the Management of Contaminated Food Production Systems in Europefollowing a radiological emergency v2 No CAT1TN0901 EURANOS eunerisnetlibraryhandbookshtmlOhba T Liutsko L Schneider T Tanigawa K Fattibene P Laurier D Sarukhan ABarquinero J Kesminiene A Skuterud L Cardis E this issue The SHAMISENProject challenging historical recommendations for preparedness response andfollowup of nuclear accidents lessons learnt from Chernobyl and FukushimaSGDSN National response plan Major nuclear or radiological accident httpwwwgouvernementfrsitesdefaultfilesrisquespdfnational_plan_nuclear_radiological_accidentspdfSoï¬er Y Schwartz D Goldberg A Henenfeld M BarDayan Y Populationevacuations in industrial accidents a review of the literature about four major eventsPrehosp Disaster Med “UNDRR The Sendai Framework for Disaster Risk Reduction “ UnitedNations Office for Disaster Risk Reduction wwwundrrpublicationsendaiframeworkdisasterriskreduction20152030WHO WHO Definition of Health World Health anisation Geneva httpwwwwhointaboutdefinitionenprinthtmlThese recommendations have already been considered in the development of the European joint roadmap for radiation protection researchImpens and Salomaa and of the strategic research agenda ofdiï¬erent European platforms notably NERIS on emergency and recovery Croua¯l The results of the SHAMISEN project have been presented and discussed in several national and international workshops and meetingsRecommendations are being disseminated to decision makers and radiation protection authorities for translation into strategy and policy aswell as to scientific medical and nonexpert audiences They are nowreferred and used as basis of the reflections and the initiatives of national and international anizations for both preparedness NuclearEnergy Agency World Health anisation International Commissionon Radiological Protection ICRP National committee for postaccident management CODIRPA in France and the management of theFukushima situation with a key role of the Japanese partners involvedin the SHAMISEN project Fukushima Medical University NagasakiUniversity Hiroshima UniversityMore broadly the approach adopted in the SHAMISEN project andits results may contribute to address other hazards including naturaldisasters industrial accidents or even pandemic crisis Similarities canbe emphasized with the Sendai Framework for Disaster Risk Reduction“ UNDRR adopted at the 3rd UN World Conference in This framework underlines the importance of improving theunderstanding of disaster risk better addressing vulnerability and hazard characteristics strengthening risk governance reinforcing accountability for risk management with development of preparednessinvolvement of stakeholders and due considerations of resilience ofhealth infrastructureFinally the pandemic crisis of COVID19 highlights a series of issuesquite similar to those addressed in the SHAMISEN project confinementversus evacuation psychosocial aspects communication and dialogueanisation of the transition phases and of course the preparation ofhealth surveillance strategies and structures of epidemiological studiesThese diï¬erent issues would benefit from crosscomparison analysisand the s presented in this special issue could certainly contributeto the reflectionDeclaration of Competing InterestThe authors declare that they have no known competing financialinterests or personal relationships that could have appeared to ‚uence the work reported in this paperAcknowledgmentsSHAMISEN was supported by the EURATOM European AtomicEnergy Community program of the European Commission in the framework of the OPERRA Project for the European RadiationResearch Area project FP7 grant agreement No The authors are grateful to all partners experts and stakeholderscontributed to theandorwho participated in the projectRecommendationsISGlobal acknowledges supportfrom the Spanish Ministry ofScience Innovation and Universities through the œCentro de ExcelenciaSevero Ochoa “ Program CEX2018000806S and supportfrom the Generalitat de Catalunya through the CERCA Program NMBUacknowledges the support of the Research Council of Norway RCNthrough its Centres of Excellence funding scheme project numberŽ Corresponding author 0c"

Thyroid_Cancer

"Handling EditorAdrian CovaciKeywordsNuclear accidentsHealth surveillancePreparednessCommunicationStakeholdersRecommendationsSerious accidents at nuclear power plants have been rare but theirstories can teach us how to prevent or mitigate the eï¬ects of futurenuclear catastrophes The accidents at the Fukushima Daiichi nuclearpower plant and Chernobyl nuclear power plant occurred years and years ago respectively and there are still lessons to learn from themregarding numerous issues including radiation exposure assessmentand medical followup of emergency responders evacuees and residents decisions to lift evacuation orders and communication withresponders and stakeholders Bazyka Callen and Homma Lester Soï¬er Some of the lessons from theseaccidents have been extensively reviewed and taken into considerationby national and international anizations such as the InternationalAtomic Energy Agency the International Commission on RadiologicalProtection and the World Health anisation and are reflected inpublished literature Bennett Carr Clarke IAEA 2015a 2015b Nisbet SGDSN This hasallowed the development of various recommendations and guidancedocuments targeting specific issues of radiation protection training andcommunication and socioeconomic aspects in order to prepare andimprove decision making processes in the early and intermediatephases eg Carr IAEA 2015b Nisbet However the majority of these texts focus on technical issues andare directed towards radiation protection experts rather than for thesupport of aï¬ected populations The traditional approaches of emergency response and recovery including evacuation relocation andhealth surveillance are largely based on dose levels Although manyrecognise the importance of psychosocial or human factors it has beendifficult to adapt the approaches to better address the social economicethical and psychological factors These include the health and welfareeï¬ects that may arise from the accident from the concerns about thepresence of radiation in the environment from the mitigation actionstaken and from the information mixed or absent provided to the population Changes in the ethical and legal requirements for personaldata collection use and storage raise additional challenges particularlyin the area of health surveillance and epidemiologyAbbreviations COVID19 Coronavirus Disease EJP CONCERT European Joint Programme for the Integration of Radiation Protection Research OPERRA Project for the European Radiation Research Area SGDSN Secrtariat gnral de la dfense et de la scurit nationale France SHAMISEN NuclearEmergency Situations Improvement of Medical and Health Surveillance SHAMISEN SINGS SHAMISEN Stakeholder INvolvement in Generating Science UN UnitedNations UNDRR United Nations Office for Disaster Risk Reduction101016jenvint2020106000Received July Accepted July Published by Elsevier Ltd This is an access under the CC BYNCND license httpcreativecommonslicensesBYNCND40 0c Main components of the SHAMISEN projectThe SHAMISEN project started in late at a time when somedeleterious eï¬ects of evacuation and ultrasound thyroid screening inFukushima had started to be reported The project therefore aimed toreview the lessons learned from major nuclear accidents in particularfrom experiences of populations aï¬ected by the Chernobyl andFukushima accidents to develop recommendations for medical andhealth surveillance of populations aï¬ected by previous and future radiation accidents The ultimate motivation was to minimise the negative impacts of the accident and improve the health of aï¬ected populations The holistic WHO definition of health was used in this contextie œa state of complete physical mental and social wellbeing and notmerely the absence of disease or rmity WHO The Recommendations were to address in particular the following complementary aspects dose assessment supporting all phases of an accident including emergency response clinical decisionmaking recoveryactions and health surveillance improvement of living conditions ofaï¬ected populations engaging them and responding to their needs andminimising unnecessary anxiety and health surveillance and wherefeasible improvement of estimates of radiationinduced risk for radiation protection and communication with aï¬ected populationsTo achieve this and recognising the need for a holistic approach toaccident management and health surveillance SHAMISEN brought together a team of researchers from institutions including RadiationProtection Authorities Universities Research Centres and Associationsin Europe and Japan with complementary experience and a long trackrecord in post accidental management dosimetry radiation protectionmedical followup and screening population health surveillance healtheconomics epidemiology ethics and sociology of radiation protectionThe project also drew upon additional expertise from Belarus RussiaUkraine Japan and the UK as well as from outside of the radiationresearch field and established contacts with major international anizations including the World Health anisation and the NuclearEnergy Agency of the anisation for Economic Cooperation andDevelopmentDetails of the SHAMISEN Project are provided in the paper by Ohbaand collaborators Ohba this issue Briefly the approach involved in particular challengingevaluating the eï¬ectiveness of measures taken after Chernobyl or Fukushima accidents in particularcid129 Systematic thyroid screening with ultrasound for early detection ofpotential thyroid cancer casescid129 Criteria for evacuation and their consequencescid129 Measures taken to contribute to the wellbeing of aï¬ected populations and develop a radiological protection culture and resilience inaï¬ected populationscid129 Challenges and good practice in communication and training withthe objective of regaining the trust of the population and engagingthem in retaking control of their livescid129 Role of ethics in disaster preparedness response and health surveillance autonomy and dignity respect of privacy beneficence¦cid129 Role of health professionals in the diï¬erent phases of the accidentmanagementcid129 Cost eï¬ectiveness of the measures takenAll of this was brought together to develop practical recommendations for preparedness and the diï¬erent phases of the accident SHAMISEN results and the way forwardAlthough the SHAMISEN project was developed during a limitedperiod of months in response to the second call of the EuropeanOPERRA project a series of key results has been achieved The mainresults are several topical reports and a set of recommendations dividedinto five main topics eg Evacuation Communication and trainingEnvironment International Dosimetry Health surveillance Epidemiology These topics focusspecifically on the health surveillance of people following a nuclearaccident combining natural and social sciences values and practice tohelp health professionals decisionmakers and local stakeholders to setup protective actions and health programmes responding to the concernof aï¬ected populations Therefore the SHAMISEN recommendationsare not intended to cover all aspects of emergency and recovery response and preparednessThe formulation of the recommendations is generic enough to beapplied in diï¬erent countries recognising that cultural diï¬erences willbe important The structure describes the general context and the mainreasons for developing each recommendation provides explanations onhow to develop it and indicates who would be involved in the development of the recommendation Depending on the context specificarrangements have to be made for the implementation of these recommendations during the diï¬erent phases eg preparedness earlyand intermediate longterm recoveryThe recommendations provide advice on the values to be consideredwhen addressing the issue at stake and what type of tools and protocolsare needed rather than the tools themselves Due to the duration of theproject it was not manageable to develop them However they providea significant input for further developments for practical tools in different domains and identify the main expectations from stakeholderswith regards to health surveillance in postaccident situationsDeveloped as a research project it was not the intention to specifyabsolute dose criteria for the implementation of actions Of coursediscussions on the feedback experience from the management of theChernobyl and Fukushima accidents point out some challenges associated with the use of specific dose criteria but the spirit of the recommendations is to provide indications and guidance for the decisionmakers and health professionals with regard to the choice to be madeon the adoption of dose criteria for the diï¬erent actions to be implementedBesides the management of the direct radiation induced health effects the report underlines the need to develop a multidisciplinaryapproach to identify measure assess and alleviate psychological andother indirect health impacts of socioeconomic and social upheavals ofthe consequences of the accident For this purpose it is recommendedto promote the engagement process of local stakeholders since thepreparedness phase targeting the overall wellbeing of populations withdue considerations of the ethical principles of respect for autonomydignity and justiceThis special issue of Environmental International combines a series ofscientific papers and is an opportunity to emphasize the main analysesdeveloped during the SHAMISEN project combining advanced scientific research analyses of feedback experience from the Chernobyl andFukushima accidents and applying a multidisciplinary approachAlthough the topics presented in this special issue have already beenaddressed in general in several papers the originality of the approachadopted in the SHAMISEN project provides new insights for healthsurveillance issuesIt is worth mentioning that following the SHAMISEN project otherresearch projects have been launched A first series of projects arededicated to the development of Apps with and for citizens as recommended in the SHAMISEN project This has notably been done withthe European research project SHAMISEN SINGS as part of the EJPCONCERT as well as with an ongoing project developed by FukushimaMedical University In addition several projects are currently underdevelopment in diï¬erent countries and at the European level thatpromote a citizen science approach for addressing health and radiological monitoringAs an example of the multidisciplinary approach the SHAMISENproject has identified a series of recommendations calling for furthercooperation with diï¬erent European Research Platforms combininglow doses eï¬ects dosimetry radioecology emergency and recoverymanagement social sciences and humanities and medical research 0cEnvironment International and Thierry SchneideraŽ Deborah Oughtonb Elisabeth Cardiscdea CEPN Nuclear Protection Evaluation Centre Rue de la Redoute FontenayauxRoses Franceb Centre for Environmental Radioactivity CERAD NMBU „«s Norwayc Barcelona Institute for Global Health ISGlobal Barcelona Spaind Pompeu Fabra University Barcelona Spaine Spanish Consortium for Research and Public Health CIBERESP Institutode Salud Carlos III Madrid SpainEmail address thierryschneidercepnassofr T SchneiderReferencesBazyka D Belyi D Chumak A Lessons from chornobyl considerations forstrengthening radiation emergency preparedness in Ukraine Radiat Prot Dosim “ 101093rpdncw196Bennett B Repacholi M Carr Z Eds Health eï¬ects of the Chernobyl accidentand special health care programmes Report of the UN Chernobyl Forum expert groupœHealth WHO Press GenevaCallen J Homma T Lessons learned in protection of the public for the accident atthe Fukushima Daiichi nuclear power plant Health Phys “ 101097HP0000000000000666Carr Z Clarke M Akl EA Schneider R Murith C Li C ParrishSprowl J StenkeL CuiPing L Bertrand S Miller C Using the grade approach to supportthe development of recommendations for public health interventions in radiationemergencies Radiat Prot Dosim “ 101093rpdncw234Clarke R Valentin J International Commission on Radiological Protection Task Group ICRP publication Application of the Commission™s Recommendations forthe protection of people in emergency exposure situations Ann ICRP “101016jicrp200905004Croua¯l P Camps J Raskob W Schneider T NERIS Roadmap on medium andlongterm research on preparedness for nuclear and radiological emergency responseand recovery Version eunerisnetlibrarysra259nerisroadmap2020htmlIAEA 2015a The Fukushima Daiichi Accident No Technical Volume IAEAInternational Atomic Energy Agency Vienna Austria wwwiaeapublications10962thefukushimadaiichiaccidentIAEA 2015b Preparedness and Response for a Nuclear or Radiological Emergency NoGSR7 Safety Standards Series IAEA International Atomic Energy AgencyVienna Austria httpwwwpubiaeaMTCDPublicationsPDFP_1708_webpdfImpens N Salomaa S Second joint roadmap for radiation protection researchDeliverable No EJPCONCERT European Joint Programme for the Integrationof Radiation Protection Research H2020 wwwconcerth2020euDocumentashxdtwebfileListsDeliverablesAttachments206D37_Second20joint20roadmap_draft_reviewed_20052020_approved03062020pdfguid01b5ac77b2ec4cda9c98917dba396f0fLester MS Public information during a nuclear power plant accident lessonslearned from Three Mile Island Bull N Y Acad Med “Nisbet AF Jones A Turcanu C Camps J Andersson KG H¤nninen RRavantaara A Solatie D Kostiainen E Julien T Pupin V Ollagnon HPapachristodoulou C Ioannides K Oughton D Generic Handbook forAssisting in the Management of Contaminated Food Production Systems in Europefollowing a radiological emergency v2 No CAT1TN0901 EURANOS eunerisnetlibraryhandbookshtmlOhba T Liutsko L Schneider T Tanigawa K Fattibene P Laurier D Sarukhan ABarquinero J Kesminiene A Skuterud L Cardis E this issue The SHAMISENProject challenging historical recommendations for preparedness response andfollowup of nuclear accidents lessons learnt from Chernobyl and FukushimaSGDSN National response plan Major nuclear or radiological accident httpwwwgouvernementfrsitesdefaultfilesrisquespdfnational_plan_nuclear_radiological_accidentspdfSoï¬er Y Schwartz D Goldberg A Henenfeld M BarDayan Y Populationevacuations in industrial accidents a review of the literature about four major eventsPrehosp Disaster Med “UNDRR The Sendai Framework for Disaster Risk Reduction “ UnitedNations Office for Disaster Risk Reduction wwwundrrpublicationsendaiframeworkdisasterriskreduction20152030WHO WHO Definition of Health World Health anisation Geneva httpwwwwhointaboutdefinitionenprinthtmlThese recommendations have already been considered in the development of the European joint roadmap for radiation protection researchImpens and Salomaa and of the strategic research agenda ofdiï¬erent European platforms notably NERIS on emergency and recovery Croua¯l The results of the SHAMISEN project have been presented and discussed in several national and international workshops and meetingsRecommendations are being disseminated to decision makers and radiation protection authorities for translation into strategy and policy aswell as to scientific medical and nonexpert audiences They are nowreferred and used as basis of the reflections and the initiatives of national and international anizations for both preparedness NuclearEnergy Agency World Health anisation International Commissionon Radiological Protection ICRP National committee for postaccident management CODIRPA in France and the management of theFukushima situation with a key role of the Japanese partners involvedin the SHAMISEN project Fukushima Medical University NagasakiUniversity Hiroshima UniversityMore broadly the approach adopted in the SHAMISEN project andits results may contribute to address other hazards including naturaldisasters industrial accidents or even pandemic crisis Similarities canbe emphasized with the Sendai Framework for Disaster Risk Reduction“ UNDRR adopted at the 3rd UN World Conference in This framework underlines the importance of improving theunderstanding of disaster risk better addressing vulnerability and hazard characteristics strengthening risk governance reinforcing accountability for risk management with development of preparednessinvolvement of stakeholders and due considerations of resilience ofhealth infrastructureFinally the pandemic crisis of COVID19 highlights a series of issuesquite similar to those addressed in the SHAMISEN project confinementversus evacuation psychosocial aspects communication and dialogueanisation of the transition phases and of course the preparation ofhealth surveillance strategies and structures of epidemiological studiesThese diï¬erent issues would benefit from crosscomparison analysisand the s presented in this special issue could certainly contributeto the reflectionDeclaration of Competing InterestThe authors declare that they have no known competing financialinterests or personal relationships that could have appeared to ‚uence the work reported in this paperAcknowledgmentsSHAMISEN was supported by the EURATOM European AtomicEnergy Community program of the European Commission in the framework of the OPERRA Project for the European RadiationResearch Area project FP7 grant agreement No The authors are grateful to all partners experts and stakeholderscontributed to theandorwho participated in the projectRecommendationsISGlobal acknowledges supportfrom the Spanish Ministry ofScience Innovation and Universities through the œCentro de ExcelenciaSevero Ochoa “ Program CEX2018000806S and supportfrom the Generalitat de Catalunya through the CERCA Program NMBUacknowledges the support of the Research Council of Norway RCNthrough its Centres of Excellence funding scheme project numberŽ Corresponding author 0c"

Thyroid_Cancer

A prediction model of outcome of a0SARS‘CoV‘ a0pneumonia a0based a0on a0laboratory a0findingsGang a0Wu1 a0Shuchang a0Zhou1 a0Yujin a0Wang1 a0Wenzhi a0Lv2 a0Shili a0Wang3 a0Ting a0Wang4 Xiaoming a0Li1The a0severe a0acute a0respiratory a0syndrome a0coronavirus a0 a0SARS‘CoV‘ a0has a0resulted a0in a0thousands a0of a0deaths a0in a0the a0world a0Information a0about a0prediction a0model a0of a0prognosis a0of a0SARS‘CoV‘ a0infection a0is a0scarce a0We a0used a0machine a0learning a0for a0processing a0laboratory a0findings a0of a0 a0patients a0with a0SARS‘CoV‘ a0pneumonia a0including a0 a0non‘survivors a0and a0 a0discharged a0patients a0The a0maximum a0relevance a0minimum a0redundancy a0mRMR a0algorithm a0and a0the a0least a0absolute a0shrinkage a0and a0selection a0operator a0logistic a0regression a0model a0were a0used a0for a0selection a0of a0laboratory a0features a0Seven a0laboratory a0features a0selected a0in a0the a0model a0were a0prothrombin a0activity a0urea a0white a0blood a0cell a0interleukin‘ a0receptor a0indirect a0bilirubin a0myoglobin a0and a0fibrinogen a0degradation a0products a0The a0signature a0constructed a0using a0the a0seven a0features a0had a0 a0[ a0] a0sensitivity a0and a0 a0[ a0] a0specificity a0in a0predicting a0outcome a0of a0SARS‘CoV‘ a0pneumonia a0Thus a0it a0is a0feasible a0to a0establish a0an a0accurate a0prediction a0model a0of a0outcome a0of a0SARS‘CoV‘ a0pneumonia a0based a0on a0laboratory a0findingsMost human coronavirus infections are mild However several betacoronaviruses can cause serious diseases or even death12 The mortality rates of severe acute respiratory syndrome coronavirus SARSCoV and Middle East respiratory syndrome coronavirus MERSCoV were and respectively SARSCoV2 is the pathogen for novel coronavirus disease COVID1934 which has resulted in thousands of deaths in the world since the beginning of The diagnosis of SARSCoV2 infection must be confirmed by the realtime reverse transcriptase polymerase chainreaction RTPCR or gene sequencing of specimens of patients56 Chest radiograph and laboratory findings are both important for accessing the severity of the disease7“ Critical patients should be admitted to Intensive Care Unit ICU of infectious disease hospital while mild patients could be kept and treated at isolation It is very important to effectively prioritize resources for patients with the highest risk because of the large number of infected people10ICU patients and nonICU patients differed significantly in some blood parameters including leukocytes neutrophils prothrombin time Ddimer total bilirubin TB lactate dehydrogenase high sensitivity cardiac troponin I and procalcitonin5711 Ruan et a0al12 retrospectively analyzed laboratory findings of nonsurvivors and discharged patients and found significant differences in lymphocytes platelets albumin TB urea nitrogen creatinine myoglobin Creactive protein and interleukin6 between the two groups These laboratory findings seemed useful in predicting outcome of SARSCoV2 infection However an advanced prediction model involving multiple laboratory parameters is urgently required to be applied in a clinicaldecision support system to improve the predictive and prognostic accuracyAs a branch of artificial intelligence machine learning ML helps establish accurate prediction model13“ However there are few publications reporting prediction of the outcome of SARSCoV2 pneumonia using ML methods based on laboratory findings Thus we retrospectively collected laboratory findings of discharged patients and nonsurvivors These data were dealt with a ML method similar to radiomics1617 We aim to establish a prediction model of outcome of SARSCoV2 pneumonia based on laboratory data1Department of Radiology Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Wuhan China 2Julei Technology Wuhan China 3Computational Biology Carnegie Mellon University Pittsburgh USA 4Department of Medical Ultrasound Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Wuhan China email 751884926qqcom lilyboston2002qqcomScientific RepoRtS 101038s41598020711147Vol0123456789wwwnaturecomscientificreports 0cMethodsAll methods were carried out in accordance with relevant guidelines and regulationsStudy a0design a0and a0participants a0 This study was approved by the Ethics Commission of Hospital TJ Written informed consent was waived by the Ethics Commission of hospitalThe author™s center was the designated hospital for severe and critical SARSCoV2 pneumonia Patients underwent repeated RTPCR tests to confirm SARSCoV2 Laboratory tests for SARSCoV2 pneumonia included blood routine test serum biochemical including glucose renal and liver function creatine kinase lactate dehydrogenase and electrolytes coagulation profile cytokine test markers of myocardial injury infectionrelated makers and other enzymes Repeated tests were done every “ a0days for monitoring the patient™s conditionOxygen support from nasal cannula to invasive mechanical ventilation was administered to patients according to the severity of hypoxaemia All patients were administered with empirical antibiotic treatment and received antiviral therapy Most of patients improved after treatment However a few critical patients continued to deteriorate and eventually diedData a0collection a0 fatal cases of SARSCoV2 pneumonia male median age a0years were collected by the electronic medical record system discharged patients with SARSCoV2 pneumonia whose age and gender matched the nonsurvivors were selected male median age a0years The admission date of these patients was from Feb to Mar We reviewed all laboratory findings for each patient Results of repeated tests were carefully compared to find the greatest deviation from normal value In general the greatest number in series of values was recorded However for platelets red blood cell lymphocytes hemoglobin calcium total protein albumin estimated glomerular filtration rate eGFR and prothrombin activity PTA the minimum was recorded Laboratory findings at the day of mortality were not used These recorded laboratory findings were considered as lab features of a patient A initial data set of patients nonsurvivor discharge was thus builtThere were patients who did not have the entire group of laboratory features thus their data were deleted from the dataset The remaining data of patients nonsurvivor discharge were analyzed by machine learningStatistical a0analysis a0and a0modeling a0 First all the variables were compared between nonsurvivors and discharged patients using the Mann“Whitney U test for nonnormally distributed features or the independent t test for normally distributed features1617 Features with P were considered significant variables and selected1617 Second Spearman™s correlation coefficient was used to compute the relevance and redundancy of the features1617 Third we applied the maximum relevance minimum redundancy mRMR algorithm to assess the relevance and redundancy of the features1617 The features were ranked according to their mRMR scores1617 Fourth the top features with highrelevance and lowredundancy were selected for least absolute shrinkage and selection operator LASSO logistic regression model The LASSO logistic regression model was adopted for further features selection1617 Some candidate features coefficients were shrunk to zero and the remaining variables with nonzero coefficients were finally selected1617 The model was used for calculating signature for each patient Mann“Whitney U test was used for comparing signature between two groups1617 Receiver operator characteristic ROC precision recall curve PRC analysis and Hosmer“Lemeshow test were used for further evaluation of modelThe statistical analyses were performed using R software version wwwrproje ct1617 The following R packages were used the œcorrplot package was used to calculate Spearman™s correlation coefficient the œmRMRe package was used to implement the mRMR algorithm the œglmnet was used to perform the LASSO logistic regression model and the œpROC package was used to construct the ROC curve1617ResultsNine laboratory features were eliminated in the first step of feature selection because of nonsignificance The remaining thirtyeight lab features were significantly different between two groups P and then mRMR scores were obtained for them There were seven features having nonzero coefficients after LASSO algorithm and were selected for the model Table a0 shows the fifteen features with the highest mRMR scores Figure a0 shows the correlation matrix heatmap of the thirtyeight significant features Figure a0 shows the feature selection process with LASSO algorithm Figure a0 shows the contribution of the seven features to the model Figure a0 shows the signatures of all patients as well as ROC Figure a0 shows the PRC for the modelNonsurvivors and discharged patients differed significantly in the signature derived from the model P The AUC was [ CI ] The sensitivity and specificity in predicting outcome of SARSCoV2 pneumonia were [ ] and [ ] respectively The area under precision recall curve AUPRC was Hosmer“Lemeshow test showed good calibration P for the modelThe seven features included in the prediction model were as follows PTA urea white blood cell WBC interleukin2 receptor IL2r indirect bilirubin IB myoglobin and fibrinogen degradation products FgDP All features had coefficients of positive number except PTA PTA and FgDP are from coagulation profile Urea and IB are from renal and liver function respectively WBC is from blood routine test Myoglobin is a marker of myocardial injury IL2r is related to immune response The signatures derived from the model could be positive or negative numbersScientific RepoRtS 101038s41598020711147Vol1234567890wwwnaturecomscientificreports 0cRankFeaturesPTAWBCUreaIL2rIBMyoglobinTBFgDPhsCRPFerritinLDHDdimereGFRNeutrophilsSodiummRMR scoreCoefficient after LASSOˆ’ Table The fifteen features with higher mRMR scores were selected for the step of LASSO logistic regression Some candidate features coefficients were shrunk to zero and the remaining variables with nonzero coefficients were selected mRMR maximum relevance minimum redundancy LASSO least absolute shrinkage and selection operator PTA prothrombin activity WBC white blood cell IL2r interleukin2 receptor IB indirect bilirubin TB total bilirubin FgDP fibrinogen degradation products hsCRP hypersensitive Creactive protein LDH lactate dehydrogenase eGFR estimated glomerular filtration rateFigure a0 Correlation matrix heatmap of significant features Spearman™s correlation coefficient was used to compute the relevance and redundancy of the featuresNonsurvivors and discharged patients did not differ in age or gender median age vs P percentage of males vs P The comparisons of laboratory findings between nonsurvivors and discharged patients are shown in Table a0Blood a0 routine a0 test a0 WBC and neutrophils were significantly higher in nonsurvivor group versus discharge group Lymphocyte platelets and red blood cells were significantly lower in nonsurvivors AUC for them were “Scientific RepoRtS 101038s41598020711147Vol0123456789wwwnaturecomscientificreports 0cFigure a0 The fivefold crossvalidation A of the least absolute shrinkage and selection operator algorithm for feature selection process A vertical line was drawn at the optimal value Some candidate features coefficients were shrunk to zero B and the remaining seven variables with nonzero coefficients were finally selectedElectrolyte a0 Potassium chlorine and sodium were significantly higher in nonsurvivor group versus discharge group Calcium was significantly lower in nonsurvivors AUC for them were “Serum a0biochemical a0test a0 Glucose and globulin were significantly higher in nonsurvivor group versus discharge group Albumin and total protein were significantly lower in nonsurvivors AUC for them were “Renal a0 function a0 Urea and creatinine were significantly higher in nonsurvivor group versus discharge group The eGFR was significantly lower in nonsurvivors AUC for them were “Liver a0function a0 Total bilirubin direct bilirubin IB and glutamic oxaloacetic transaminase were significantly higher in nonsurvivor group versus discharge group AUC for them were “Scientific RepoRtS 101038s41598020711147Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Contribution of the features to the model The histogram shows the contribution of the seven features with nonzero coefficients The features are plotted on the yaxis and their coefficients are plotted on the xaxisFigure a0 Bar charts of the signature for patients The red bars indicate the signatures of discharged patients while the light green bars indicate the signatures of nonsurvivors The AUC was for the signatureCoagulation a0 profile a0 Prothrombin time activated partial thromboplastin time Ddimer international normalized ratio INR fibrinogen and FgDP were significantly higher in nonsurvivor group versus discharge group PTA was significantly lower in nonsurvivors AUC for them were “Cytokine a0them were “IL2r and IL6 were significantly higher in nonsurvivor group versus discharge group AUC for Infection‘related a0markers a0and a0myocardial a0injury a0markers a0 Procalcitonin high sensitive Creactive protein ferritin and Nterminal probrain natriuretic peptide NTproBNP were significantly higher in nonScientific RepoRtS 101038s41598020711147Vol0123456789wwwnaturecomscientificreports 0cFigure a0 The precision recall curve for the model The area under precision recall curve was survivor group versus discharge group Myoglobin MB isoenzyme of creatine kinase and high sensitive cardiac troponin I were significantly higher in nonsurvivors AUC for them were “DiscussionNonsurvivors and discharged patients with SARSCoV2 pneumonia differed significantly in thirtyeight laboratory findings By using machine learning method we established a prediction model involving seven laboratory features The model was found highly accurate in distinguishing nonsurvivors from discharged patients The seven features selected by artificial intelligence also indicated that dysfunction of multiple ans or systems correlated with the prognosis of SARSCoV2 pneumoniaThe SARSCoV2 triggers a series of immune responses and induces cytokine storm resulting in changes in immune components518 When immune response is dysregulated it will result in an excessive inflammation even cause death719 Excessive neutrophils may contribute to acute lung damage and are associated with fatality20 Higher serum level of IL2r was found in nonsurvivors indicating excessive immune response In addition high leukocyte count in SARSCoV2 patients may be also due to secondary bacterial infection2122Liver injury has been reported to occur during the course of the disease2324 and is associated with the severity of diseases Increased serum bilirubin level was observed in fatal cases Acute kidney injury could have been related to direct effects of the virus hypoxia or shock2526 Blood urea level continued to increase in some cases Nonsurvivors had higher blood urea compared to survivors Myocardial injury was seen in nonsurvivors which was suggested by elevated level of myoglobin The mechanism of multiple an dysfunction or failure may be associated with the death of patients with SARSCoV2 pneumonia Some patients with SARSCoV2 infection progressed rapidly with sepsis shock which is well established as one of the most common causes of disseminated intravascular coagulation DIC27 The nonsurvivors in our cohort revealed significantly lower PTA compared to survivors At the late stages of SARSCoV2 infection level of fibrinrelated markers FgDP markedly elevated in most cases suggesting a secondary hyperfibrinolysis conditionA number of laboratory features were compared between nonsurvivors and discharged patients with SARSCoV2 pneumonia The two groups differed significantly in as many as thirtyeight lab features However none of the futures provided adequate accuracy in predicting the outcome of SARSCoV2 pneumonia Thus a novel prediction model involving multiple features was established in the study With machine learning methods previously used in radiomics a prediction model combining seven out of thirtyeight laboratory features was built for predicting the outcome of SARSCoV2 pneumoniaThe mRMR algorithm was used for assessing significant features to avoid redundancy between features The mRMR score of a feature is defined as the mutual information between the status of the patients and this feature minus the average mutual information of previously selected features and this feature172829 The top fifteen features with high mRMR scores were selected for the next step of modeling The least absolute shrinkage and selection operator logistic regression model was used to processing the features selected by mRMR algorithm LASSO is actually a regression analysis method that improves the model prediction accuracy and interpretability30 The signature calculated with the model can be positive or negative number corresponding with poor and good prognosis respectively Our results showed that the AUC of the signature was “ higher than that of a single featureThe modeling process is a black box however the choice of variables seems reasonable PTA can more accurately reflect the coagulation function compared to prothrombin time and can also reflect the degree of liver injury Urea is a good index to reflect the degree of renal function damage WBC can not only reflect immune Scientific RepoRtS 101038s41598020711147Vol1234567890wwwnaturecomscientificreports 0cLeucocyte 109LPlatelet 109LErythrocyte 1012LNeutrophils 109LLymphocyte 109LHemoglobin gLPotassium mmolLCalcium mmolLChlorine mmolLSodium mmolLGlucose mmolLTotal protein gLGlobulin gLAlbumin gLCreatinine μmolLUric acid μmolLTotal bilirubin μmolLDirect bilirubin μmolLIndirect bilirubin μmolLUrea mmolLEstimated glomerular filtration rate mlmin173 a0m2Glutamic oxaloacetic transaminase ULGlutamicpyruvic transaminase ULMyoglobin ngmLHigh sensitive cardiac troponin I pgmLMB isoenzyme of creatine kinase ngmLLactate dehydrogenase ULGlutamate dehydrogenase ULCreatine kinase ULProthrombin time sFibrinogen gLActivated partial thromboplastin time sThrombin time sD“D dimer μgmLProthrombin activityInternational standardized ratioFibrinogen degradation products μgmLProcalcitonin ngmLNterminal probrain natriuretic peptide pgmLFerritin μgLHypersensitive Creactive protein mgLInterleukin1β pgmLInterleukin2 receptor UmLInterleukin6 pgmLInterleukin10 pgmLNonsurvivors [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ]Discharged patients [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ]P Table Medians [interquartile range] of laboratory findings of patients with SARSCoV2 pneumonia were provided in the table Features were compared between nonsurvivors and discharged patients using the Mann“Whitney U test for nonnormally distributed features or the independent t test for normally distributed features SARSCoV2 severe acute respiratory syndrome coronavirus status but also secondary infection IL2r is an indicator of inflammation and immune response20 IB is related to liver function and possible hemolysis Myoglobin reflects the degree of myocardial injury The increase of FgDP is related to coagulation disorders including DIC Thus the current model involves multiple important systems related to prognosis In consideration of the high accuracy of the model it can be concluded that liver kidney myocardial damage coagulation disorder and excess immune response all contribute to the outcome of SARSCoV2 pneumoniaScientific RepoRtS 101038s41598020711147Vol0123456789wwwnaturecomscientificreports 0cIt is suitable to start to use this model after three repeated laboratory tests about a0weeks after admission because doctors may have enough data at that time Lots of laboratory findings are generated in hospitalization Which are most important for predicting outcome Our study at least answered such a problem Seven laboratory features could be used to construct a new signature with the model The new signature seems more useful than any single feature We encourage such a simpletouse model widely used in clinical practiceMost of clinical factors are not continuous variables such as underlying disease We used a machine learning method similar to radiomics which mainly deals with continuous features Our study focused on continuous laboratory variables We had to exclude noncontinuous clinical factor with the current machine learning method By using other methods a model that involves both continuous variables and category variables can be established Thus clinical factors raised as significant predictive factors such as respiratory status or radiological features could be included in the models However there are more than forty laboratory findings in our study making establishment of model difficult We felt it necessary to simplify laboratory features Thus we establish a submodel based on lab findings A new lab signature is thus created and is proved highly valuable In future study the signature may be combined with clinical factors to establish a more complex modelOur study has some limitations First this is a singlecenter retrospective study Multicenter largesample studies are required to validate our prediction model Second our model may not be directly used in other centers However they could easily establish a prediction model using their own data with machine learning method Third some patients who did not have all the lab findings were excluded Selection bias must be present due to patients exclusion Other studies with more strict design were thus required to reveal the bias Fourth statistical approach conducted in this study is not perfect As LASSO was used for variables or more patients were needed More patients should be collected in future studyIn conclusion it is feasible to establish a accurate prediction model of outcome of SARSCoV2 pneumonia based on laboratory findings Injury of liver kidney and myocardium coagulation disorder and excess immune response all correlate with the outcome of SARSCoV2 pneumoniaData a0availabilityAfter publication the data will be made available to others on reasonable requests to the corresponding authorReceived March Accepted August References Drosten C et al Identification of a novel coronavirus in patients with severe acuterespiratory syndrome N Engl J Med “ “ Zaki A M Boheemen S Bestebroer T M Osterhaus A D Fouchier R A Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia N Engl J Med “ Phelan A L Katz R Gostin L O The novel coronavirus originating in Wuhan China challenges for global health governance JAMA 101001jama20201097 Li Q et al Early transmission dynamics in Wuhan China of novel coronavirusinfected pneumonia N Engl J Med Huang C et al Clinical features of patients infected with novel coronavirus in Wuhan China Lancet “ Zhu N et al A novel coronavirus from patients with pneumonia in China N Engl J Med “ Wang D et al Clinical characteristics of hospitalized patients with novel coronavirusinfected pneumonia in Wuhan China JAMA “ Bernheim A et al Chest CT findings in coronavirus disease19 COVID19 relationship to duration of infection Radiology 101148radio l20202 Fang Y et al Sensitivity of chest CT for COVID19 comparison to RTPCR Radiology 101148radio l20202 General Office of the National Health Commission of China Diagnosis and treatment protocol for 2019nCoV 5th ed Beijing China National Health Commission of China Yang X et al Clinical course and outcomes of critically ill patients with SARSCoV2 pneumonia in Wuhan China a singlecentered retrospective observational study Lancet Respir Med “ Ruan Q Yang K Wang W Jiang L Song J Clinical predictors of mortality due to COVID19 based on an analysis of data of patients from Wuhan China Intensive Care Med “ Shiri I et al Nextgeneration radiogenomics sequencing for prediction of EGFR and KRAS mutation status in NSCLC patients using multimodal imaging and machine learning algorithms Mol Imaging Biol 101007s1130 Matsuzaka Y et al Prediction model of aryl hydrocarbon receptor activation by a novel QSAR approach deepSnapdeep learning Molecules Katić K Li R Zeiler W Machine learning algorithms applied to a prediction of personal overall thermal comfort using skin temperatures and occupants™ heating behavior Appl Ergon Jiang M et al Nomogram based on shearwave elastography radiomics can improve preoperative cervical lymph node staging for papillary thyroid carcinoma Thyroid “ Zhang P et al T2weighted imagebased radiomics signature for discriminating between seminomas and nonseminoma Front Qin C et al Dysregulation of immune response in patients with COVID19 in Wuhan China Clin Infect Dis Oncol 101093cidciaa2 Mahallawi W H Khabour O F Zhang Q Makhdoum H M Suliman B A MERSCoV infection in humans is associated with a proinflammatory Th1 and Th17 cytokine profile Cytokine “ Channappanavar R Perlman S Pathogenic human coronavirus infections causes and consequences of cytokine storm and immunopathology Semin Immunopathol “ Chen N et al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet “ Guan W et al Clinical characteristics of novel coronavirus infection in China N Engl J Med “ Scientific RepoRtS 101038s41598020711147Vol1234567890wwwnaturecomscientificreports 0c Tang N Li D Wang X Sun Z Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia J Thromb Haemost “ Xu L Liu J Lu M Yang D Zheng X Liver injury during highly pathogenic human coronavirus infections Liver Int Estenssoro E et al Pandemic influenza A in Argentina a study of patients on mechanical ventilation Am J Respir Crit Li K et al The clinical and chest CT features associated with severe and critical COVID19 pneumonia Investig Radiol “ Care Med “ “ Abe T et al Complement activation in human sepsis is related to sepsisinduced disseminated intravascular coagulation Shock Lin X Li C Ren W Luo X Qi Y A new feature selection method based on symmetrical uncertainty and interaction gain 101097SHK00000 Comput Biol Chem Wang J et al Machine learningbased analysis of MR radiomics can help to improve the diagnostic performance of PIRADS v2 in clinically relevant prostate cancer Eur Radiol “ Sauerbrei W Royston P Binder H Selection of important variables and determination of functional form for continuous predictors in multivariable model building Stat Med “ AcknowledgementsWe thank all patients and their families involved in the studyAuthor a0contributionsGW SZ and YW collected the epidemiological and clinical data TW WL and SW summarized all data GW XL drafted the manuscript TW and XL revised the final manuscriptCompeting a0interests The authors declare no competing interestsAdditional a0informationCorrespondence and requests for materials should be addressed to TW a0or a0XLReprints 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 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 licen sesby40 The Authors Scientific RepoRtS 101038s41598020711147Vol0123456789wwwnaturecomscientificreports 0c'

Thyroid_Cancer

This study was performed assess the clinical outcomes of elderly patients withosteoporotic femoral neck fractures FNFs AOOTA 31BC treated by initial uncementedtotal hip arthroplasty UTA or cemented total hip arthroplasty CTAMethods This study involved consecutive elderly patients with osteoporotic FNFs AOOTA31BC treated by initial UTA or CTA in our medical centre from to The primaryoutcomes were the Harris hip score HHS and the rates of revision loosening periprostheticfracture and dislocationResults In total patients were included in the final analysis UTA n¼ CTA n¼ The mean followup duration was months range “ months The mean HHS was1Department of Microsurgery Trauma and Hand SurgeryThe First Affiliated Hospital Sun Yatsen UniversityGuangzhou China2Department of Pediatrics The First Affiliated HospitalSun Yatsen University Guangzhou China3Department of Orthopaedics The First AffiliatedHospital Sun Yatsen University Guangzhou China4Department of Orthopaedics The Third People™sHospital of Wuxi Jiangsu Province The Affiliated Hospitalof Jiangnan University Wuxi China5Department of Urology The First Affiliated Hospital SunYatsen University Guangzhou ChinaThese authors contributed equally to this workCorresponding authorsJunxing Ye Department of Orthopeadics The ThirdPeople™s Hospital of Wuxi Jiangsu Province The AffiliatedHospital of Jiangnan University No Xingyuan NorthRoad Liangxi District Wuxi Jiangsu ChinaEmail yejunxing0514163comJintao Zhuang Department of Urology The First AffiliatedHospital Sun Yatsen University No Zhongshan 2ndRoad Yuexiu District Guangzhou ChinaEmail brianzg86163comCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cJournal of International Medical Research 06 for UTA and 06 for CTA Significant dissimilarities were detected in therates of revision loosening and periprosthetic fracture between UTA and CTA vs vs and vs respectively A significant difference was also detected inthe probability of revision between the two groupsConclusion Elderly patients with osteoporotic FNFs AOOTA 31BC treated with CTA showgreater improvements in functional outcomes and key orthopaedic complications than thosetreated with UTAKeywordsFemoral neck fracture arthroplasty outcome complication retrospective osteoporosisDate received December accepted July Introduction31BCAOOTAfemoral neck fracturesManagement ofin elderlyFNFspatients is still undergoing considerableresearch12 Uncemented total hip arthroplasty UTA or cemented total hip arthroplasty CTA for displaced FNFs tendsto be a recognised surgical strategy3“Comparisons between UTA and CTA forelderly individuals with an FNF generallyfavour CTA this is primarily attributed tothe exceptional clinical outcomes of CTA interms of relieving pain and improving dailyactivities as well as the higher rate of majororthopaedic complications ie revisionloosening periprosthetic fracture and dislocation associated with UTA4 Howeverrecent studies of UTA in elderly individualshave demonstrated encouraging shorttermclinical outcomes67 Moreover cementedprosthesis syndrome tends to occur morefrequently in CTA than UTA8 Cementedprosthesis syndrome theoretically poses asignificantlifealthough the specific probability of thisthreat has not been calculated89 Hencewhether to utilise CTA for elderly individuals may present the clinician with a dilemma9 The lack of consensus regarding whichtechnique UTA or CTA is preferable fortreating FNFs AOOTA 31BC in elderlyindividuals is related to the remarkableto the patient™sthreatdistinction in clinical outcomes betweenthe two types of implants610Most previousconcentrated medicalstudies have involvedhighlycentresand several brands of prostheses356Additionally shortterm followup is commonplace in these studies To overcomethese drawbacks of previous studies and tocompare the midterm results of the twoprostheses we assessed the clinical outcomesof elderly patients with osteoporotic FNFsAOOTA 31BC treated with initial UTAor CTA with a mean followup of yearsMaterials and methodsStudy populationtheandrequirementEthical approval was obtained from theFirst Affiliated Hospital of Sun YatsenUniversityforinformed consent was waived by theInvestigational Review Board Consecutiveelderly individuals with the principle diagnosis of an FNF AOOTA 31BC whounderwentinitial UTA or CTA from March to March and forwhom detailed information was availablethroughoutidentifiedfrom the orthopaedics department of theFirst Affiliated Hospital Sun YatsenUniversity The manufacturer details offollowup were 0cMao et alTable Manufacturer details of stems and cupsemployed in the arthroplasty proceduresProcedureStemCupCORAIL1Exeter3Exeter3REFLECTIONUncemented2UTA n¼ CTA n¼ 1DePuy Synthes Warsaw IN USA2Smith Nephew London UK3Stryker Corporation Kalamazoo MI USAUTA uncemented total hip arthroplasty CTA cementedtotal hip arthroplastyclosed FNFsthe stems and cups employed in the arthroplasty are shown in Table The surgicalprocedure and postoperative rehabilitationprotocol were described in our previouslypublished study11 The inclusion criteriawereAOOTA 31BCactive and cognitively intact patients ageof 15 years independently mobile priorto the injury and a bone mineral densityTscore of at the femoral neck Themajor exclusion criteria were multiple fractures or contralateral limb fractures pathological FNFs lower limb dyskinesia priorto surgery cancer planned surgery polytrauma severe comorbidities eg thyroiddisorder with calcium and phosphorusmetabolism disorderwithcomplications drug abuse affecting bonehealing or bone metabolism early interruption of followup months and cognitive impairment Clinical and radiographicassessments were performed at and months after surgery and every monthsthereafter The primary outcomes were theHarris hip score HHS and the rates ofrevision loosening periprosthetic fractureand dislocationdiabetesStatistical analysisRevision was defined as partial or completereplacement of the prosthesis12 Looseningof the acetabulum andor stem componentstomographycompared usingas well as dislocation were defined based ona previous description13 Periprostheticconfirmed by Xray orfracture wascomputedexaminationContinuous data ie age bone mineraldensity body mass index followup timeand HHS wereanindependentsamples t test and categoricalvariables ie sex side [leftright] fracturetype comorbidities mechanism of injuryAmerican Society of Anesthesiologists classification and major orthopaedic complications were compared using the chisquaretest or the Mann“Whitney test A Kaplan“Meier survival curve was used to assess theprobability of revision Hazard ratios werecalculated using a Cox proportional hazards model The significance threshold wasset at p The statistical analysis wasperformed using SPSS IBM CorpArmonk NY USAResultsIn total consecutive patientsarthroplasties with an FNF AOOTA31BC who underwentinitial UTA orCTA met our inclusion criteria and wereincluded for analysis Figure The meanfollowup duration was months range“ months The patients™ mean age was 06 years for UTA and 06 for CTA The mean body mass indexfor UTA andwas 06 kgm2 for CTA The patients™baselinesimilarbetween the two groups Table 06 kgm2characteristics werePrimary outcomesImproved functional outcomes were notedin both groups as indicated by the HHSUTA 06 prior to surgery vs 06 at final analysis p CTA 06 prior to surgery vs 06 at final analysis p At the end of followup the HHS was 0cJournal of International Medical ResearchFigure Flow diagram exhibiting methods for identifying patients with FNFs AOOTA 31BC whounderwent an initial UTA or CTAFNFs femoral neck fractures UTA uncemented total hip arthroplasty CTA cemented total hiparthroplastysignificantly different between the twoUTA 06 vs CTAgroups 06 p¼ and patients whounderwent CTA had higherfunctionalscores than those who underwent UTANo distinct betweengroup differenceswere observed at any time point before months postoperatively Table No early year postoperative complications were detectedincluding revisionloosening periprosthetic fracture or dislocation The rate of key orthopaedic complications wasfor UTAand for CTA p Table In the UTA group patients underwent revision UTA developed prosthesis loosening developed periprosthetic fracturesand developed prosthesis dislocation In the CTA group patientsunderwent revision UTA developed prosthesis loosening developed periprosthetic fractures and developed prosthesis dislocation The average time interval from the initial surgery torevision UTA was months range “months for UTA and months rangefor CTA p¼ “ monthsSignificant differences in revision looseningand periprosthetic fracture were observedbetween the UTA and CTA groups revision vs p¼ loosening 0cMao et alTable Patient demographics and outcomesVariableSex femalemaleAge yearsBMI kgm2BMDSide leftrightFNFs AOOTA type31B31CComorbiditiesDiabetes mellitusHypertensionCerebrovascular diseaseMechanism of FNFsTraffic accidentFallingTamping accidentASA classificationUTA n¼ 06 06 06 06 HHS prior to surgeryData are presented as n n or mean 06 standard deviationpvalueCTA n¼ 06 06 06 06 UTA uncemented total hip arthroplasty CTA cemented total hip arthroplasty HHS Harris hip score ASA AmericanSociety of Anesthesiologists BMI body mass index BMD bone mineral density FNFs femoral neck fracturesTable Comparison of hip functional scoresMonths postoperativelyFinal followupData are presented as mean 06 standard deviationUTA n¼ 06 06 06 06 06 06 06 06 CTA n¼ 06 06 06 06 06 06 06 06 pvalueStatistically significantUTA uncemented total hip arthroplasty CTA cemented total hip arthroplasty vs p¼ and periprosthetic fracture vs p¼ respectively A significant difference in theprobability of revision was also detectedbetween the groups hazard ratio interval confidence“p¼ Figure No significant difference was found in the rate of prosthesis dislocation between the UTA and CTA groups vs respectively 0cJournal of International Medical ResearchTable Rates of key orthopaedic complicationsComplicationsProsthesis revisionProsthesis looseningPeriprosthetic fractureDislocationUTA n¼ CTA n¼ pvalueData are presented as n Statistically significantUTA uncemented total hip arthroplasty CTA cemented total hip arthroplastyFigure Kaplan“Meier curves showing probability of revision after primary surgery HR was calculatedper the Cox proportional hazards model with age sex American Society of Anesthesiologists classificationbody mass index bone mineral density and femoral neck fracture type as covariates and surgery as the timedependent factorHR hazard ratio CI confidence interval UTA uncemented total hip arthroplasty CTA cemented total hiparthroplastysurgeryProbableDiscussionThis review characterised the outcomes of asolitary brand of a total hip arthroplastyimplant during a mean followup of years in elderly patients with osteoporoticFNFs AOOTA 31BC The data demonstrated that patients treated with CTAshowed better improvements in functionaloutcomes and key orthopaedic complications than those treated with UTAThe current findings are consistent withprevious studies361415 Although the betterfunctional outcomes and lower rates ofrevision loosening periprosthetic fractureand dislocation are apt to favour CTA nosignificant betweengroup differences in theHHS were detected during the initial yearsafterexplanationsinclude the timedependent clinical efficacyof the implants and the properties of theprostheses34616 Whether UTA or CTA ispreferable in elderly patients with a discontroversial6917placed FNF remainsA recent retrospective study involving patients with an FNF AOOTA 31B whounderwent primary unilateral UTA or CTAshowed that the mean HHS was 06 for CTA and 06 for UTAp¼ A singleblinded randomisedcontrolled trial CHANCEtrial involving individuals treated with an uncementedor cemented tapered hydroxyapatitecoatedfemoral stem and a cemented cup demonstratedtaperedhydroxyapatitecoated femoral stem andcementedthatthe 0cMao et alcemented cup provided better functionalresultsthan the uncemented taperedhydroxyapatitecoated femoral stem andcemented cup16ratesrevisionthe bone microstructureIn the present study the Kaplan“Meiersurvival curve demonstrated that at the2year analysis neither group showed evidence of a target event and no significantbetweengroup differences were found inofthelooseningfracture or dislocationperiprostheticNeverthelessit would be interesting toexplore whether the prosthesis materialinfluencesthepeak effect and the duration of the effectand if so what mechanisms affect the bonemicrostructure and whether there is a wayto change the outcome by blocking thiseffect during a 2year followup We currently have one option for prevention oravoidance of adverse events and thesechanges in treatment strategies may play akey role in improving the clinical resultsif the effects of the prosthesis materialitself cannot be blocked18 There is still alack of consensus on standards for prosthesis revision in this context19When assessing the impact of CTA onthe target events we did not observe anincreased incidence of severe orthopaediccomplications other than the complicationsmentioned in this study In one systematicreview the authors presumed that CTA wassuperior to UTA with respect to functionalscores and tolerable orthopaedic complications20 We obtained analogous results interms of hiprelated complications andfunctional scores Multicentre hip arthroplasty data indicate that UTA remains ahighrisk factor for late revision looseningand periprosthetic fractures810 The notabledissimilarities in the results of these variousstudies on hiprelated complications may belargely attributed to the design of the prosthesis prosthesis size and material selection and the surgeon™s experience46affected theThis study has several limitations It hada small sample and its retrospective designis association with some inherent disadvantages We did not stratify the patientsaccording to fracture type or sex In addition the potential comorbidities betweengroups were not well exposed and compared The statistical power used to addressdifferences between the groups was insufficient Differences in the patients™ baselinedata may haveresultsFurthermore our analysis did not determine whether the deaths were instigatedby bone cement The risk of hiprelatedcomplications was not analysed The survivalcurve of other prosthesisrelatedcomplications was estimated using theKaplan“Meier method and competitiverisks ie death could have affected thesurvival of the prosthesis Patients whodied lostrevisionHence the revision rate might have beenunderestimated during this long followupwith a fairly high mortality ratethe opportunity forevidenceIn conclusion the findings described inthe current review uphold an increasingbody ofthat CTA provideshigher functional scores and lower rates ofhiprelated complications than does UTAin elderly patients with osteoporotic displaced FNFs AOOTA 31BC In thiscontext we recommend CTA for the treatment of such FNFs Our findings may beconducive to alleviating continuing debateregarding which prosthesis UTA or CTAis more suitable for the elderly populationA future prospective study may be essentialto confirm whether our conclusion continues to be acceptable as the followup timeincreasesDeclaration of conflicting interestThe authors declare that there is no conflict ofinterest 0cFundingThis research received no specific grant from anyfunding agency in the public commercial ornotforprofit sectorsORCID iDsWeiguang YuGuowei HanReferencesorcid00000001orcid00000003 Chammout G Muren O Laurencikas Eet al More complications with uncementedthan cemented femoral stems in total hipreplacement for displaced femoral neck fractures in the elderly a singleblinded randomized controlled trial with patientsActa Orthop “ Gjertsen JE Lie SA Fevang JM et al Totalhip replacement after femoral neck fracturesin elderly patients results of fracturesreported to the Norwegian ArthroplastyRegister Acta Orthop “ Hailer NP Garellick G and Karrholm JUncemented and cemented primary totalhip arthroplastyin the Swedish HipArthroplasty Registerof operations Acta Orthop “evaluation Makela KT Eskelinen A Paavolainen Pet al Cementless total hip arthroplasty forprimary osteoarthritis in patients aged years and older results ofthe mostcommon cementless designs compared tocemented reference implants in the FinnishArthroplasty Register Acta Orthop “ YliKyyny T Sund R Heinanen M et alCemented or uncemented hemiarthroplastyfor the treatment of femoral neck fracturesActa Orthop “ Yang C Han XL Wang J et al Cementedversus uncemented femoral component totalhip arthroplasty in elderly patients with primary osteoporosisretrospective analysiswith 5year followup J Int Med Res “Journal of International Medical Research Solarino G Zagra L Piazzolla A et alceramiconResults of consecutiveceramic cementless hip arthroplastiesinpatients up to years of age a yearsof followup study J Arthroplasty S232“S237 Hanly RJ Whitehouse SL Lorimer MFet al The outcome of cemented acetabularcomponents in total hip arthroplasty forosteoarthritis defines a proficiency thresholdresults of cases from the AustralianOrthopaedic Association NationalJointReplacement Registry J Arthroplasty “ Imam MA Shehata MSA Elsehili A et alContemporary cemented versus uncementedhemiarthroplasty for the treatment of displaced intracapsular hip fractures a metaanalysis offortytwo thousand fortysixhips Int Orthop “ Jameson SS Baker PN Mason J et al Thedesign of the acetabular component and sizeof the femoral head influence the risk of revision following singlebrand cementedhip replacements a retrospective cohortstudy of mediumterm data from a nationaljoint registry J Bone Joint Surg Br 94B “ Zeng XS Zhan K Zhang LL et alConversion to total hip arthroplasty afterfailed proximal femoral nail antirotationsor dynamic hip screw fixations for stableintertrochanteric femur fractures a retrospective study with a minimum followupof years BMC Musculoskelet Disord Johnson RL Abdel MP Frank RD et alImpact of frailty on outcomes after primaryandarthroplastyJ Arthroplasty “64e5revisiontotalhip Chen KH Tsai SW Wu PK et al Partialcomponentretained twostage reconstruction for chronic infection after uncementedtotal hip arthroplasty results of sixteen casesafter five years of followup Int Orthop “ DeAngelis JP Ademi A Staff I et alCemented versus uncemented hemiarthroplasty for displaced femoral neck fracturesa prospective randomized trial with early 0cMao et alfollowup J Orthop Trauma “ Rolfson O Donahue GS Hallsten M et alPatientreported outcomes in cemented anduncemented total hip replacements Hip Int “ Chammout G Muren O Boden H et alCemented compared to uncemented femoralstems in total hip replacement for displacedfemoral neck fractures in the elderly studyprotocol for a singleblinded randomizedCHANCEtrial BMCcontrolled trialMusculoskelet Disord Liu T Hua X Yu W et al Longtermfollowup outcomes for patients undergoingprimary total hip arthroplasty with uncemented versus cemented femoral components a retrospective observational studywith5year minimum followupJ Orthop Surg Res a Engesaeter LB Espehaug B Lie SA et alDoes cement increase the risk of infection inprimary total hip arthroplasty Revisionrates in cemented and uncementedprimary THAs followed for years inthe Norwegian Arthroplasty Register ActaOrthop “ Schmale GA Lachiewicz PF and Kelley SSEarly failure of revision total hip arthroplasty with cemented precoated femoralcomponents Comparison with uncementedcomponents at to years J Arthroplasty “ Azegami S Gurusamy KS and Parker MJCemented versus uncemented hemiarthroplasty for hip fractures a systematic reviewof randomised controlled trials Hip Int “ 0c'

Thyroid_Cancer

progression of breast cancerare greatly affected by the immune environment Neutrophils are the most abundantleucocytes in circulation and act as the spearhead in ‚ammationincluding inbreast cancer Circulating neutrophils are closely related to the prognosis of breastcancer patients and tumorltrating neutrophils have varied functions at differentstages of breast cancer such as antitumor or tumorpromoting neutrophils which aretermed N1 and N2 neutrophils respectively In this review we will discuss the utilityof circulating neutrophils for predicting prognosis and therapeutic efficacy and theunderlying mechanisms of their chemotaxis the dynamic regulation of their antitumoror protumor functions and their different spatial distributions in tumor microenvironmentFinally we also discuss the possibility of targeting neutrophils as a therapeutic strategyin breast cancerKeywords breast cancer immunotherapy neutrophils neutrophiltolymphocyte ratio tumor microenvironmentSpecialty sectionThis was submitted toCancer Immunity and ImmunotherapyINTRODUCTIONa section of the journalFrontiers in ImmunologyReceived May Accepted July Published August CitationZhang W Shen Y Huang H Pan SJiang J Chen W Zhang T Zhang Cand Ni C A Rosetta Stone forBreast Cancer Prognostic Value andDynamic Regulation of Neutrophil inTumor MicroenvironmentFront Immunol 103389fimmu202001779Breast cancer BC is the most common malignancy in women worldwide Although BCis classified as a malignant disease with low immunogenicity recent evidence has revealeda promising outcome of therapies with blocking immune checkpoints in both early andadvanced stages “ The efficacy of immunotherapy is closely related to the tumor immunemicroenvironment especially to ltrating immune cells To date macrophages and Tcells are the most wellstudied immune cells in BC whereas increasing evidence has indicatedthat neutrophils are also key in the oncogenesis and metastasis of BC in addition circulatingneutrophils have been reported to have great prognostic prediction value Neutrophils are themost abundant leucocytes in blood and usually act as the first line of host defense against pathogens However due to their short life span an average of “ h in blood it is difficult to employthis subset of cells for experiments which has resulted in a poor understanding of their role in solidtumors In addition some contradictory results reported in vitro studies or animal experimentshave suggested a dual eï¬ect of neutrophils in tumor developmentFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerNeutrophils can present both antitumorigenic œN1 andprotumorigenic œN2 phenotypesin various cancers orspecific circumstances The term neutrophil in several studiesalso includes both mature neutrophils and myeloidderivedsuppressor cells MDSCs MDSCs are described as a subsetof neutrophils with immunosuppressive functions that expressCD11b and Gr1 and can be divided into monocyticM CD11bLy6C MDSCs and GPMN CD11bLy6GMDSCs and GPMN MDSCs usually share a commonset of markers and similar morphologicalfeatures withneutrophils To avoid confusion we mainly focus on the biologicalfunction of mature neutrophils and related therapeutic strategiesfor targeting them in BC We provide a comprehensive reviewofthe prognostic value of circulating neutrophils and themechanisms of how tumorassociated neutrophils TANs exertantitumor or tumorpromoting functions in BC and in theend we also discuss the potential of targeting neutrophils as atherapeutic strategy in cancerPROGNOSTIC VALUE OF THENEUTROPHILTOLYMPHOCYTE RATIONLRTumors can be thought of as wounds that will not heal andare characterized by chronic ‚ammation Neutrophils are themost rapidly responding immune cells to ‚ammation andmany studies have found that the NLR is closely related to theprognosis and treatment response in patients bearing BC A recent metaanalysis of studies including patientswith both early and advanced BC revealed that patients witha higher NLR before treatment had poorer diseasefree survivalDFS than those with a lower NLR before treatment but theNLR was not related to overall survival OS the subgroupanalysis found that the NLR was associated with prognosisonly in earlystage patients but not in patients with metastasisAbbreviations BC Breast cancer MDSCs Myeloidderived suppressor cellsTANs Tumorassociated neutrophils NLR Neutrophiltolymphocyte ratio DFSDiseasefree survival OS Overall survival ALC Absolute lymphocyte count NCTNeoadjuvant chemotherapy pCR Pathological complete response PLR Platelettolymphocyte ratio TAMs Tumorassociated macrophages CTCs Circulatingtumor cells NETs Neutrophil extracellular traps MPO MyeloperoxidaseGCSF Granulocyte colonystimulating factor ECs Endothelial cells PMNsPolymorphonuclear neutrophils ICAM1 Intercellular adhesion molecule MMP9 Matrix metalloproteinases9 ROS Reactive oxygen species HMGB1Highmobility group box TLR4 Tolllike receptor TNBC Triplenegativebreast cancer MES Macrophageenriched subtype NES Neutrophilenrichedsubtype H2O2 Hydrogen peroxide TNFα Tumor necrosis factorα HOCIHypochlorous acid TRPM2Transientcation channelsubfamily M member ADCC Antibodydependent cellular cytotoxicityNK Natural killer NE Neutrophil elastase NRP1 Neuropilin1 IRS1 Insulinreceptorsubstrate1 PI3K Phosphatidylinositol 3kinase VEGF Vascularendothelial growth factor TIMP1 Tissue inhibitor of matrix metalloproteaseTGF Transforming growth factor 27HC 27hydroxycholesterol PAD4Peptidyl arginine deiminase TINs Tumorltrating neutrophils CRTConventionalradiotherapy MRT Microbeam radiation therapy DAMPsDamageassociated molecular patterns ICB Immune checkpoint blockadeLDNs Lowdensity neutrophils HDNs Highdensity neutrophils NAMPTNicotinamideadeninedinucleotide GTX granulocyte transfusiontransferase NAD Nicotinamidereceptor potentialphosphoribosyl Since similar metaanalyses were not based on individualpatient data which may cause significant bias we reviewed andcompared the individual reports and found some issues worthdiscussing here Widmann first reported the correlationbetween the NLR and BC prognosis in patients and itwas found that a higher NLR ‰¥ before treatment was anadverse factor for both short and longterm mortality Themajority of retrospective studies thereafter have drawn similars “ and the NLR was found to be consistentamong diï¬erent BC subtypes at baseline However aprospective substudy of GEICAM9906 which comprised patients did not find any prognostic value of the NLR afteradjustment for clinicopathological factors in addition a highNLR was independently associated with worse DFS in only highrisk patients the hormone receptornegativeHER2 populationand in patients with ‰¥ lymph node metastases Anotherstudy with early BC patients also found that the NLR beforesurgery was not associated with DFS indicating that thepresurgery NLR may be valuable only in patients with a hightumor burdenseveralIn addition to the above studiesstudies alsoexplored the prognostic value of the NLR posttreatment or withcontinuous assessment A retrospective study comparing theabsolute lymphocyte count ALC and the NLR eight consecutivetimes before and after chemotherapy found that patients whodied had lower ALC and higher NLR values than patients whoremained alive throughout the treatment course additionallyamong the patients who died a steady increase in the NLRover the baseline measurement was observed at subsequenttime points Another retrospective study included BCpatients with DFS values of more than years and it interestinglyfound that NLR sampled during followup rather than beforeany treatment was an independent prognostic factor for laterecurrence However there is still no compelling explanationfor the abovementioned inconsistent results In addition sincelymphocytes are critical in cancer immune surveillance andneutrophils have been reported to play a protumor role in moststudies low lymphocytes and high neutrophils in circulationmay also suggest immunosuppression status and studiesfocused on the relationship between neoadjuvant chemotherapyNCT and the NLR might support the above hypothesis Acomprehensive review of the existing reports shows that moststudies have found that a low NLR indicates a higher NCTresponse and pathological complete response pCR rate “in addition the NLR has showed predictive value not only inall molecular types of BC but also in both operable and locallyadvanced BC Interestingly although Suppan et aldid not find a significant correlation between the initial NLR andprognosis the same cohort revealed a low NLR as a significantparameter for predicting chemotherapy response p A low NLR was also reported to be associated with a higherresponse rate to primary endocrine therapy for locally advancedor metastatic BC Although increasing evidence suggests a close associationbetween the NLR and prognosis in BC several issues remain thatmake clinical application difficult One of the most importantreasons is the lack of a consensus cutoï¬ value As we listhere Table the cutoï¬ values for the NLR in the publishedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerTABLE Characteristics of the studies related to neutrophiltolymphocyte ratioReferencesCountry Study periodCancer typeMedianage ysNo patientslowhigh NLRTreatmentFollowupSignificance of NLRNR ysHigh NLR indicates lowersurvival rate p Surgery NCT moSurgery ysNR moUnivariate analysis indicateshigh NLR related to lower RFSp and OS p High NLR indicates lower5year OSMultivariate analysis indicateshigh pretreatment NLR iscorrelated with poor DFS p and OS p Adjuvanttranstuzumab moHigh pretreatment NLRindicates shorter DFSNoh Korea“Koh Korea“Yao China“Luminal ABHER2enrichedTNBCERPRpositiveHER2enrichedLuminal ABERPRpositiveHER2enrichedTNBCPistelli Italy“TNBCUlas Turkey“HER2enrichedJia China“ER PRpositiveHER2enrichedTNBCBozkurt Turkey“TNBCAsano Japan“TNBCn NLR n ‰¤ NLR ‰¥ n n NLR ‰¤ n NLR n n NLR NLR n NLR ‰¤ n NLR n n NLR n NLR n n NLR n NLR ‰¤ n n NLR ‰¤ n NLR n n NLR n NLR n NCT surgery mo moSurgeryadjuvantchemotherapyandradiotherapyNCT ysMultivariate analysis indicateslow NLR is related to superiorDFS p and p Multivariate analysis indicateshigh pretreatment NLR iscorrelated with poor DFS p and OS p Univariate analysis indicateslow NLR is related to favorableprognosis in TNBC patientswho achieved pCR p hazard ratio High pretreatment NLRindicates poor allcausemortality with a multivariableHR of CI“High NLR upon recurrenceindicates shorter OSrecurrence rates p Low NLR indicates higher OSp Rimando USA“Nonmetastatic BC n NLR ‰¤ n NLR n Radiotherapychemotherapy moIwase Japan“TNBCn NRL n NLR ChemotherapyNRNCT surgery moHernandez et alSpain“Miyagawa Japan“Ferroni Italy“Luminal ABERPRpositiveHER2enrichedTNBCLocally Advanced orMetastatic BCLuminal ABHER2enrichedTNBCn NLR n NLR ‰¥ n n NLR ‰¤ n NLR n Qiu China“NonmetastaticTNBCn NLR n NLR ‰¥ n EribulinNRLow NLR indicates better PFSp NCTchemotherapyendocrinetherapytrastuzumabregimensSurgery NCTchemotherapy moHigh pretreatment NLRindicates worse DFS HR and OS HR moLow NLR indicates higher OSp and DFS p ContinuedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alTABLE ContinuedNeutrophils in Breast CancerReferencesCountry Study periodCancer typeMedianage ysNo patientslowhigh NLRTreatmentFollowupSignificance of NLRIimori Japan“Luminal ABHER2enrichedTNBCMando Argentina “Early stage BCLee Korea“TNBCXuan China“TNBCFujmoto Japan“With high counts oflymphocytesImamura Japan“HER2enrichedn NLR n NLR ‰¥ n n NRL n NLR ‰¤ n NLR n n NLR n NLR ‰¥ 293n n NLR n NLR n n NLR n NLR ‰¥ n Endocrinetherapy moSurgery moNCTNRSurgeryNRNRSurgeryadjuvantchemotherapiesendocrinetherapiesTrastuzumabemtansineNRLow NLR indicates aprolongation of PFS p and OS p High NLR indicates lower DFSp Low NLR indicates superiorOS p and DFS p Low NLR indicates longer DFSp Low NLR indicates better RFSp Low NLR at baseline indicatesbetter PFS p andOS p NLR Neutrophiltolymphocyte ratio ER Estrogen receptor PR Progesterone receptor HER2 Human epidermal growth factor receptor Mo Months Ys Years DFS Diseasefreesurvival OS Overall survival PFS Progressionfree survival RFS Relapse free survival pCR Pathological complete response TNBC Triplenegative breast cancer NCT Neoadjuvantchemotherapy NR Not recordedstudies were between and In addition based on individualstudies the sensitivity of the NLR fluctuates greatly “and the specificity is much lower “ Therefore some researchers have tried to determine a betteralternative parameter In addition to the NLR the platelettolymphocyte PLR ratio has also been investigated and comparedwith the NLR in BC A single central retrospective study with hormone receptornegative nonmetastatic BC patients reportedthat both elevated NLR and PLR were associated with poor OShowever the multivariate analysis revealed that only the NLR p but not the PLR p was a significant indicatorfor both DFS and OS Additionally since the absolutelymphocyte count has also been reported as a prognostic factorthe predictive values of the PLR and NLR were evaluated afteradjusting for the total lymphocyte count The results showed thatthe PLR was no longer a significant predictor for 5year mortalityand the NLR remained a significant predictor irrespective ofthe lymphocyte count Furthermore it was revealed thatthe combination of the NLR and PLR could further improvethe predictive value Two retrospective studies found that thehighest rate of pCR was in the group of patients withan NLRlowPLRlow profile and the lowest rate was inthe group with an NLRhighPLRhigh profile in additionwhen the cutoï¬ values for the NLR and PLR were applied thespecificity of predicting a pCR increased from to Howeverthe causal relationship between the NLR andpoor prognosis in malignant disease has yet to be illuminatedAccording to an assessment with paired peripheral bloodand pancreatic cancer specimens Takakura found thata high NLR was associated with increased tumorassociatedTAMsanditseemsdecreased Thereforetumorassociatedmacrophageslymphocytes but was not significantly related to CD66bltrating neutrophilsthat anincrease in neutrophils in peripheral blood is not necessarilyrelated to the number of TANs Several basic studies havesuggested a unique mechanism ofthe protumor functionof circulating neutrophils protecting circulating tumor cellsCTCs Circulating neutrophils can cluster around tumor cellsand induce tumor cell aggregation aiding tumor cell survivalby hiding them from immune surveillance Neutrophilextracellular traps NETs are webs of decondensed chromatbers conjugated together with histones myeloperoxidaseMPO elastase and other cytoplasmic proteins Recentstudies also found that neutrophils could form many NETs bothin circulation and in tumor lesions and could coordinate withplatelets to capture CTCs and facilitate cancer metastasis In addition neutropenia is very common in cancer patientsundergoing chemotherapy and supportive treatment withgranulocyte colonystimulating factor GCSF can inducea neutrophilic response as a consequence neutrophils areprimed toward a proNETotic phenotype and may suppress thecytotoxic activity of T cells as well as impair immune surveillance On the other hand lymphocytes have the propensityto mount an adaptive antitumor response in malignant disease and decreased lymphocyte numbers are considered to berelated to an insufficient immunologic reaction which mayincrease the risk of tumor relapse or metastasis Clearlya general association between prognosis and the NLR exists inBC but large prospective studies and rigorous research are stillrequired to determine its clinical significanceFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerMECHANISM OF NEUTROPHILCHEMOTAXIS TO THE TUMORMICROENVIRONMENTNeutrophils are considered the main immune cells that provideprotection against invading pathogens which can be induced bytrauma infection and malignant disease The recruitmentof neutrophils is greatly dependent on certain chemokinesincluding interleukin IL8 also known as CXCL8 CXCL and CXCL2 IL8 is a pro‚ammatory cytokineand acknowledged as the most important chemoattractant forneutrophils in the tumor microenvironment IL8 mainlycomes from endothelial cells ECs and monocytes in the tumormicroenvironment upon certain stimulation such as physicalinjury hypoxia chemotherapy or radiotherapy and other celltypes including fibroblasts and keratinocytes can secrete IL8 aswell In addition to its chemotactic eï¬ect it was revealedthat IL8 could provoke neutrophils to release NETs to assistcancer cell migration By livecell fluorescence microscopyGupta confirmed that activated ECs could induceNETosis characterized by typical extracellular DNA latticeswhen cocultured with polymorphonuclear neutrophils PMNsand activated ECs In addition activated ECs produceother ‚ammatory cytokines such as Pselectin Eselectinand intercellular adhesion molecule ICAM1 to facilitateneutrophil adhesion to ECs and migration Furthermoretumorpromoting neutrophils in BC cells are also characterizedby high expression of matrix metalloproteinases9 MMP9 which was found to cleave CXCL5 potentiating itsaction in neutrophil recruitment as a positive feedback functionin tumors IL17 was also found to control neutrophilrecruitmentin lung metastasis of BC in a mouse modelCD3CD4 and γδ T cells were the major sources of IL17 and it was interesting to find that the absence of γδT cells or neutrophils markedly reduced pulmonary and lymphnode metastases without ‚uencing primary tumor progressionwhich suggested a collaborative relationship between γδ T cellsand neutrophils in promoting BC lung metastasis Howeverin an orthotopic hepatocellular carcinoma model Sofia et alreported that TANs exert an overt antitumor role by suppressingγδ T17 cells via reactive oxygen species ROS contraryto the phenomenon that within the 4T1derived BC modelCD11bLy6G neutrophils that ltrate and surround livermetastases were found to be tumor promoting Thesecontroversial results suggest both promoting and suppressiveroles of TANs in diï¬erent circumstancesHighmobility group box HMGB1 usually acts as adamageassociated molecular pattern that is released by dyingcells or stressed cells to initiate ‚ammation and was laterfound to be an important chemoattractant for neutrophils Epithelial cellderived HMGB1 was found to recruit neutrophilsto the necrotic site through its receptor RAGE Enrichmentof platelets has been reported in the microenvironment ofmultiple cancers including BC and ltrating plateletscould be activated by the large amounts of adenosine phosphatereleased by necrotic cells as a result of chemotherapy Activated plateletderived HMGB1 known as the major mediatorof injuryinduced thrombosis in vivo can also stimulateNETosis through Tolllike receptor TLR4 and RAGE onneutrophils and as a positive feedback mechanism releasedNETs strongly induce a prothrombotic state and activate platelets Meanwhile tumor cellderived exosomal HMGB1 wasalso found to activate neutrophils through the TLR4NFκB pathway which promotes its survival by increasing theautophagic response and polarizing TANs to a protumor type It is noteworthy that various reports imply the coreposition of the NFκB pathway in the activation and recruitmentof neutrophils In addition to HMGB1 tumor cellsincluding BC cells have been reported to secrete other peptidessuch as a2 isoform VATPase a2V to activate the NFκBpathway in neutrophils thereby promoting their recruitment andinhibiting their apoptosis Additionally breast involutionafter weaning is characterized by acute ‚ammation and anincrease in estrogen It was found that estrogen could inducethe mammary ltration of neutrophils and upregulate theexpression of protumor cytokineschemokines such as COX2and MMPs in mammary ltrating neutrophils similarIn additionto lymphocytes and macrophagesneutrophils are more likely to localize in tumors of triplenegativebreast cancer TNBC than to tumors of other BC subtypes Recently Zhang identified neutrophils and macrophages asthe most frequent ltrating immune cells in various BC murinemodels and BC could be classified into a macrophageenrichedsubtype MES and a neutrophilenriched subtype NES It wasinteresting to find that there were only a few neutrophils inthe MES but a large number of macrophages in the NES This mutual repelling phenomenon in the MES and NES mayresult in spatial segregation within the same tumor The authorsspeculated that a possible mechanism could be the factors derivedfrom macrophages that inhibit the IL8dependent chemotaxis ofneutrophils ANTITUMOR FUNCTION OF TANs IN BCThe polarization of neutrophils can be diï¬erentially regulatedin the tumor microenvironment In a mouse model Fridlender found that TANs from the early tumor stage were liketumorkilling cells which produce high levels of hydrogenperoxide H2O2 tumor necrosis factor TNFα and NOand that TANs are more likely to obtain a protumorigenicphenotype with tumor progression Although few studieshave directly compared the phenotype and function of TANsbetween early and latestage tumors there are still some clues tosupport this hypothesis A phenotypical and functional analysisof TANs in earlystage lung cancer found an activated phenotypeCD62lowCD54high that was able to stimulate T cell proliferationand IFNγ release which suggested a pro‚ammatory ratherthan immunosuppressive state of TANs in earlystage lungcancer MPO is an enzyme characteristic of mature œN1type neutrophils which are able to convert H2O2 to cytotoxichypochlorous acid HOCI Recently a retrospectivestudy of BC cases revealed that MPOpositive neutrophilsFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast Cancerin additiondefined as ‰¥ cellstissue punch were found in of evaluablecases while the luminal ERPR and Her2 Her2enriched andtriplenegative types had positive rates of and respectivelyltrationby MPOpositive neutrophils was a significant independentfavorable indicator for both OS and DFS Notably almost all ofthe patients included in this study had earlystage disease T1 N01 and the data suggested that MPOpositiveneutrophils were much more abundant in BC cases with low Tand N stages than in advanced cases in univariate analysesIn addition a direct tumor killing function of neutrophils hasalso been reported One of the classical factors working againsttumor cells is ROS Recent research in mouse BC models revealedthat ROSmediated cell lysis was dependent on Ca2 channelsand mediated by transient receptor potential cation channelsubfamily M member TRPM2 expression on tumor cells Although TCGA analysis revealed a high expression ofTRPM2 in BC cells httpgepia2cancerpkucnindex activeNOX1 catalase and SOD were also increased in the membraneof cancer cells forming a complex mechanism by which tumorcell apoptosis induced by ROS is prevented In additiontumor cells are characterized by enhanced metabolic activity andhigh levels of intracellular ROS which indicates that directcytotoxic eï¬ects of neutrophilproduced ROS are not sufficientIn addition to the direct cytotoxic eï¬ect TANs containing ROShave been found to strongly suppress IL17producing γδ Tcells which are critical for shaping the immune suppressivemicroenvironment in various solid tumors “ and have alsobeen reported to promote BC cell extravasation and metastasis In addition neutrophils could also express Fc receptors andexert antibodydependent cellular cytotoxicity ADCC eï¬ectssimilar to those of T cells and macrophagesleading to atrogocytosis eï¬ect to destroy cancer cells However somestudies have indicated that neutrophils are more likely to bedistributed at the periphery of tumors at the initiation stage which may make controlling tumor growth with thesecellcell contactdependent mechanisms ineï¬ectivePROTUMOR EFFECTS OF TANsMore studies suggest that neutrophils facilitate tumor promotionand metastasis in BC than antitumor eï¬ect Overexpression ofthe chemokines CCL2 and CCL17 is a recognized feature ofN2 neutrophils Richmond found that exogenousCCL2 enhances the killing eï¬ect of neutrophils against BCcells in vitro while this antitumor activity was not observedin vivo Instead intranasal delivery of CCL2 to BALBc micemarkedly enhanced lung metastasis of BC cells and increasedthe recruitment of CD4 T cells and CD8 central memory Tcells CCL17 secretion from TANs was found to support tumrowth by recruiting CD4 Treg cells and macrophages In addition to recruiting immunesuppressive cells TANs werereported to promote the accumulation of BC cells in the lungand directly inhibit natural killer NK cellmediated clearanceof tumor cells Human NK cells can be divided intoCD56dim antitumor and CD56bright protumor subsets andCD56bright NK cells are enriched in the tumor microenvironmentand draining lymph nodes Early reports revealed thatROS and arginase1 from neutrophils impair the maturation andcytotoxic function of NK cells but CD56brightCD16ˆ’ NKcell are resistant to neutrophilderived ROS perhaps due to theirhigh antioxidative capacity Meanwhile NK cells couldbe recruited by TANs via CCL2 and CCL5 which may explainthe preferential accumulation of CD56bright NK cells in tumormicroenvironments with high ROS levels Extracellular arginine is crucial to signal local CD8 cellsand increase their CD3ζ expression which is key for T cellsto survey antigens presented on MHC class I molecules andit was also found to be necessary for T cell activation andsurvival Tumor cellderived IL8 could lead to TANdegranulation resulting in arginase1 release and conversion ofextracellular arginine to ornithine and urea thereby dampeningthe survival and cytotoxic eï¬ect of CD8 T cells Neutrophil elastase NE is also released by TANs and canbe endocytosed by tumor cells via neuropilin1 NRP1 thisresults in the crosspresentation of PR1 which is an NEderivedHLAA2restricted peptide that may be an immunotherapeutictarget Besides upon endocytosis NE is to bind insulinreceptor substrate1 IRS1 which removes the inhibitory eï¬ectof IRS1 on phosphatidylinositol 3kinase PI3K to enhance theproliferation of cancer cells Recentleukocytesreports highlighted thethe important characteristics of malignantespeciallyneutrophils preferentially uptake tumor derived extracellularvesicles or named exosomes Hypercoagulability is oneoftumors andhas been reported associated with NETs Breast cancer cell4T1derived exosomes induced NETs formation in neutrophilsbesides tumorderived exosomes also interacted with NETsto significantly accelerate venous thrombosis in vivo Furthermore several reports also indicated the cancer derivedexosomes prolonged lifespan of neutrophils and also polarizedneutrophils toward protumor type increasingevidence hasIn addition to direct modulation ofthe protumormicroenvironmentfound thatneutrophils promote tumor cell migration and the formation ofa metastatic niche Tumor angiogenesis is regardedas a prerequisite for tumor metastasis and TANs have beenrecognized as an important source of vascular endothelialgrowth factor VEGF upon specific stimulation in the tumormicroenvironment Neutrophils were also found to beone of the main sources of MMP9 and the link betweenMMP9 and VEGF has been reported previously The absenceof MMP9 has been reported to have a similar function as theinhibition of VEGF signaling indicating that MMP9 serves asan angiogenic switch during tumorigenesis by inducing VEGFrelease from the matrix “ In addition Gabriele et alalso found that MMP9 was expressed by a small number ofcells in close proximity to the vasculature such as ltrating‚ammatory cells rather than tumor cells In additionseveral serine proteases are also produced by TANs such asNE cathepsin G and proteinase3 which have been reported toactivate MMP2 to promote tumor invasion and proliferation In addition although neutrophils were reportedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast Cancerto produce little tissue inhibitor of matrix metalloproteaseTIMP1 Wang observed that BC cells with CD90positiveexpression could induce the TIMP1 secretion by TANs and asa reciprocal eï¬ect TIMP1 induced EMT and metastasis in BC Other neutrophilderived cytokines such as IL1 IL6and IL17α have been reported to initiate EMT of cancer cells byactivating JAK2STAT3 and ERK signaling Inadditiontheprimaryto modulatingtumormicroenvironment neutrophils can also assist the formationof the cancer premetastatic niche in distant ans CTCsare precursors for metastatic lesion formationintravascularNETs were found to protect CTCs from attack by circulatingimmune cells and dysregulated NETs were found to induce‚ammatory vascular injury EC shrinkage and tissue damage “ Moreover in vitro and in vivo experiments foundthat activated neutrophils promote the adherence of CTCs toECs and facilitate their lung and liver metastasis RecentlyAceto provided strong evidence that neutrophils escortCTCs in BC to assist metastasis With detection of cellsurface markers and Wright Giemsa staining they identified thatmost CTCassociated white blood cells were N2like neutrophilsIn addition singlecell RNA sequencing revealed higher Ki67expression in disseminated tumor cells from CTC neutrophilclusters than in standalone CTCs In the same study TNFαoncostatin M IL1 and IL6 were frequently expressed byCTCassociated neutrophils and matched by the receptors oncorresponding CTCs on the other hand CTCs from the CTCneutrophil clusters expressed high gene levels encoding GCSFtransforming growth factor TGF3 and IL15 which havebeen reported to activate neutrophils “ illuminating amechanism of neutrophilCTC cluster formationIn addition to escorting CTCs in circulation several studieshave found that neutrophil accumulation is a prerequisitefor cancer metastasis For both orthotopic transplantationand spontaneous BC models neutrophils were suggestedto accumulate in the distant an before cancer cellsltration Obesity and elevated cholesterol arerisk factors for BC development and poor prognosis Interestingly 27hydroxycholesterol 27HC increased thenumber of polymorphonuclearneutrophils and γδ T cells atdistal metastatic sites and neutrophils were required for themetastatic eï¬ects of 27HC Egeblad developeda confocal intravital lung imaging system and found that NETswere formed early in the lung and continued to form forthe next few days after tail vein injection of BC cells Inaddition based on immunofluorescence staining of humanprimary BC and matched metastatic lung lesions they foundthat the abundance of NETs was highest in TNBC but NETswere absent or very rare in luminal BC samples which mayexplain the higher metastatic ability of TNBCs than luminalBCs In ovarian cancer an ‚ux of neutrophils in the omentumwas also observed before metastasis and blockade of NETformation with peptidyl arginine deiminase PAD4 an enzymethat is essentia

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

"unrestricted use distribution and reproduction in any medium provided the original work is properly citedPurpose The present study was aimed at determining the serum levels of actinin4 ACTN4 in cervical cancer CC andinvestigating the diagnostic and prognostic value of serum ACTN4 in CC Materials and Methods We included CC patients cervical intraepithelial neoplasia CIN patients and healthy women Serum ACTN4 levels were assessed using an ELISAmethod A receiver operating characteristic ROC curve was performed to evaluate the diagnostic value of serum ACTN4 Thesurvival curves were used to display the overall survival distributions Results Serum ACTN4 levels in CC patients were ± pgmL which is significantly higher than those in CIN patients ± pgmL P and those in healthycontrols ± pgmL P The ROC analysis demonstrated that the area under the curve AUC of ACTN4 was 95CI “ with sensitivity of and specificity of Serum ACTN4 levels were associated with theFIGO stage lymph node metastasis and lymphovascular space invasion of CC all P The survival curve suggested thathigh serum ACTN4 levels were related to poor prognosis Conclusion Our findings suggest that serum ACTN4 levels may bevaluable diagnostic and prognostic biomarkers for CC IntroductionCervical cancer CC is the second most common femalemalignancy globally and it is the most common femalemalignancy in developing countries which has high morbidity and mortality rates [] In recent years the incidence ofCC has increased greatly in young women under the age of [] Despite great advances in surgical and adjuvant therapy the overall survival of CC patients especially that ofadvanced patients is still very poor [] At present a Papsmear combined with an HPV test has been used for the earlyscreening of cervicalthe screeningmethods are invasive and costly leading to lower screeningcoverage in China [] Previous studies have reported thatthe human papillomavirus HPV screening results have arelatively high falsepositive rate and a relatively low specificity [ ] In addition the results of TCT interpretation byfilmreading doctors are uneven which might cause somelesions Howevermisleadingness in the choices of prevention measures andtreatment for CC [] Noteworthily when applying the sametreatment plan to patients with similar pathological types theefficacy and prognosis are quite diï¬erent Therefore it is necessary to identify new biomarkers directly related to the progression and prognosis of CCAlphaactinins ACTNs are actinbinding proteins inthe spectrin gene superfamily [] which are known to becrosslinked with filamentous actin Factin to maintainthe integrity of cytoskeleton and to control cell motility []The ACTN family has four members numbered ACTN1“which are present in humans and other mammals [“]ACTN4 is encoded by the ACTN4 gene and is widelyexpressed in many tissues especially in glomerular podocytes[] ACTN4 has an actinbinding domain at the Nterminus and ACTN4 monomers can form a homodimer throughreverse binding forming a dumbbellshaped structure []As an actinbinding protein ACTN4 is closely related to 0cDisease Markersenhancing cell viability and tumor invasion and metastasis[] Recent researches have reported that the expression ofACTN4 is significantly elevated in multiple cancers including breast cancer [] pancreatic cancer [] ovarian cancer[] and lung cancer [] In addition the ACTN4 levels aremarkedly associated with the poor prognosis of lung cancer[ ] thyroid cancer [] and salivary gland carcinoma[] An [] have found that the expression level ofACTN4 in human cervical tumors is dramatically higherthan that in normal cervical tissues Their finding demonstratedepithelialtomesenchymal transition and tumorigenesis by regulatingSnail expression and the Akt pathway in CC [] Thereforethe expression of ACTN4 in cervical tissues may be used inthe clinical diagnosis and prognosis prediction of CCthat ACTN4promotestheHowever up to now the significance of the serumACTN4 levels in CC has not been evaluated Hence in thecurrent study the serum levels of ACTN4 in patients withCC were measured In addition we estimated the potentialdiagnostic and prognostic value of serum ACTN4 expressionin CC Materials and Methods Study Population A retrospective study was designed toevaluate serum actinin4 as a biomarker for CC Between July and June newly diagnosed female CC patientsand newly diagnosed female cervical intraepithelial neoplasia CIN patients who received treatment at Huai™anMaternal and Child Health Care Hospital Huai™an JiangsuChina were recruited The diagnoses of all patients were verified by the histopathological examination The patients withother types of tumor or autoimmune atherosclerotic andhematologic diseases were excluded The mean age of CCpatients was years with a range of years Meanwhile healthy women with no evidence of neoplasmsand other serious diseases were enrolled from the physicalexamination center in the same hospital There was no significant diï¬erence in age among the CC CIN and healthy control groups This study was consistent with the Helsinkideclaration and was authorized by the Ethics Committee ofHuai™an Maternal and Child Health Care Hospital approvalnumber H20130504 All participantssigned writteninformed consent Clinicopathologic Feature Collection and FollowUp Byreviewing the medical records we collected the clinicopathologic characteristics of the patients including age at diagnosis pathological type FIGO stage tumor diï¬erentiationpelvic lymph node metastasis tumor size and lymphovascular space invasion The CC patients were classified based onthe revised FIGO staging system for CC in The tumorsize was the maximum tumor diameter determined by agynecologic oncologist during pelvic examination Thepatients in stage 1A1 received hysterectomy the patients instages IB1 and IIB received radical hysterectomy and pelviclymph node dissection the patients with ‰¥stage IIB receivedradiotherapy or radiotherapy combined with chemotherapyA regular telephone followup was conducted after treatmentto obtain the overall survival OS time of CC patients andthe OS was defined as the time from diagnosis to death orthe last followup The followup was in accordance withthe FIGO guidelines Blood Sample Collection and Detection of Serum Actinin and SCCA A mL peripheral blood sample from eachpatient was collected before receiving any treatment Afterstanding at room temperature for minutes the blood samples were centrifugated at gmin for min and then°the supernatant was stored at ˆ’C until further usageThe serum actinin4 concentration was measured by a quantitativeELISAmethod Uscn Life Science Inc Wuhan China The levelsof SCCA in serum were determined using an ELISA kitRD Systems Minneapolis MN The detection of all samples was strictly in accordance with the instructions providedby the manufacturer and was performed in duplicatesenzymelinked immunosorbentassay Statistical Analysis All statistical analyses were conducted by using SPSS and GraphPad Prism The continuous data following normal distribution were expressed asthe mean ± standard deviation°SDž A ttest was used tocompare serum ACTN4 levels between the two subgroupsof each clinicopathological parameters and the serumACTN4 levels of CC patients CIN patients and healthy controls were compared by the SNKq test Receiver operatingcharacteristic ROC curves were performed to assess thediagnostic value of serum ACTN4 levels for diï¬erentiatingCC patients from CIN patients and healthy controls TheKaplanMeier method and logrank test were used to plotsurvival curves The Cox proportional hazards models in univariate and multivariate analyses were used for evaluating theprognostic value of serum ACTN4 expression A twotailed Pvalue was considered to be statistically significant Results Serum ACTN4 Levels Are Higher in Patients with CCSerum concentrations of ACTN4 were detected to rangefrom to pgmL with a mean ±SD of ± pgmL for CC patientsto range from to ngmL with a mean ±SD of ± pgmL forCIN patients and to range from to ngmL witha mean ±SD of ± pgmL for healthy controlsSerum ACTN4 levels in CC patients were significantly higherthan those in CIN patients and healthy controls P However no significant diï¬erence in serum ACTN4 wasfound between CIN patientscontrolsP as shown in Figure and healthy The Diagnostic Value of Serum ACTN4 Levels for CC Wenext used ROC curve analysis to estimate the diagnostic valueof serum ACTN4 expression for CC The ROC curve showedthat the serum levels of ACTN4 were robust for discriminating CC patients from benign and healthy control subjectswith an area under the curve AUC value of 95CI “ as demonstrated in Figure index we usedAccordingto maximum Youden™s 0cDisease MarkersŽŽlymph node metastasis were the independent prognostic factors for CC all P Table Lmgp NTCAnsCCCINCON DiscussionCervical cancer is a heterogeneous disease with complicatedetiology Genetic and environmental factors play a crucialrole in the pathogenesis of CC [] Although the diagnosisand prognosis of CC have improved greatly over the pastfew decades it is necessary to improve early detection andscreening methods to determine additional promising circulating biomarkers for better patient selection and more personalized treatments [] As far as we know this studyrepresented the first eï¬ort to evaluate the serum expressionof ACTN4 as a new biomarker for CCAs an actinbinding protein ACTN4 can participate inregulating cell migration invasion and metastasis via regulating the actin filament flexibility at the leading edge ofinvading cancer cells [ ] ACTN4overexpressing cancercells have the potential to metastasize because the overexpression of ACTN4 protein in cancer cells can stimulate thedynamic reconstruction of the actin cytoskeleton [] Upto now numerous studies have reported the associationbetween ACTN4 and multiple cancers Okamoto []observed that ACTN4 is expressed in smallcell lung cancerNSCLC and it had a significant correlation with invasionand distant metastasis Additionally ACTN4 was reportedto be a potential predictive biomarker for the efficacy of adjuvant chemotherapy in patients with NSCLC [] Watabe [] revealed that the copy number increase of ACTN4is a novel indicator for poor overall survival of patients withsalivary gland carcinoma and the copy number variationwould aï¬ect the expression of protein A recent study demonstrated that serum ACTN4 levels were dramatically elevated in patients with breast cancer when compared tohealthy controls and serum ACTN4 may be an eï¬ective clinical indicator for diagnosing or predicting the clinical outcomes of breast cancer patients [] In addition ACTN4was proven to be associated with the pathogenesis of CCAn [] proposed a novel mechanism for epithelialtomesenchymal transition and tumorigenesis in CC whichcould be induced by ACTN4 through regulating Snail expression and βcatenin stabilization Hence it is significant toinvestigate the role of serum ACTN4 in CCIn the current study we observed that serum levels ofACTN4 in CC patients were statistically higher than thosein CIN patients and those in healthy controls Howeverserum ACTN4 levels were not significantly diï¬erent betweenthe CIN group and the control group It was shown thatserum ACTN4 expression could strongly diï¬erentiate CCpatients from CIN patients and healthy controls The ROCanalysis demonstrated that the AUC of ACTN4 was and at the optimal cutoï¬ of pgmL the sensitivity andspecificity were respectively and suggestingthat serum ACTN4 might be a potential diagnostic biomarker for CC In a recent study which included Chinesewomen Hu [] reported that the sensitivity and specificity of HPV screening in the diagnosis of CC were and The sensitivity of the HPV test was a litter higherFigure The serum ACTN4 levels in CC patients CIN patientsand healthy controls ˆ—P pgmL as the cutoï¬ value and the sensitivity and specificity were and respectively Association between Serum ACTN4 Levels andClinicopathological Parameters of CC Patients We furtherinvestigated the correlations between serum levels of ACTN4and clinical pathological data of CC patients and theresults are demonstrated in Table We observed that serumACTN4 levels were related to the FIGO stage lymph nodemetastasis and lymphovascular space invasion all P Nevertheless no significant association was found betweenserum ACTN4 levels and age pathological type diï¬erentiation degree and tumor size in CC patients all P Survival Analysis of Serum ACTN4 Levels in CC Duringthe followup period nine CC patients were lost and thefollowed up rate is Finally the prognostic value ofserum ACTN4 was assessed in patients The patients werefollowed up to December The range of followup timewas to months with the median time of months andmean time of months According to the median serumlevels of ACTN4 in CC patients pgmL the CCpatients were divided into the high ACTN4 level group pgmL N and low ACTN4 level group‰¥ pgmL N The estimated 5year OS of patientswith high serum ACTN4 levels and low serum ACTN4 levelswere and respectively The KaplanMeier survival curve and logrank test indicated that CC patients withhigh serum ACTN4 levels had a worse prognosis than thosewith low serum ACTN4 levels P Figure Univariate Cox regression analyses showed that theserum ACTN4 levels P FIGO stage P diï¬erentiation degree P lymph node metastasisP and lymphovascular space invasion P had significant prognostic value for OS Multivariate analysiswas further performed to evaluate the prognostic value ofserum ACTN4 as an independent factor for CC All the statistically significant factors from univariate analyses wereincluded and the results indicated that the FIGO stage and 0cDisease MarkersytivitisneS ˆ’ specificityFigure ROC curve analysis assessed the diagnostic performance of serum ACTN4 in CC The AUC was P Table Serum ACTN4 levels in CC patients according toclinicopathological parametersParametersAge years‰¤Pathological typeSquamous cell carcinomaAdenocarcinomaFIGO stageIA1IB1‰¥IB2Diï¬erentiationWell and moderatelydiï¬erentiatedPoorly diï¬erentiatedLymph node involvementNegativePositiveTumor size‰¤Lymphovascular space invasionNegativePositiveN ACTN4pgmLP ± ± ± ± ± ± ± ± ± ± ± ± ± ± than that of serum ACTN4 detection though the specificityof serum ACTN4 detection was well above that of the HPVtest Hence comparing with the HPV test in diagnosingCC detecting serum ACTN4 has some advantages Furthermore serum ACTN4 levels have been indicated to be a greatbiomarker for diagnosing multiple cancers Fang [] intheir study reported that serum ACTN4 was a promisingindicator for diagnosing breast cancer with the AUC of Wang [] used ACTN4 expression in peripheralblood to diï¬erentiate NSCLC patients from healthy individuals in two groups of participants and they obtained bothsatisfactory eï¬ects Furthermore we investigated the correlation between serum ACTN4 and clinical characteristics ofCC patients The serum ACTN4 levels were significantlyassociated with the FIGO stage lymph node metastasis andlymphovascular space invasion of CC which suggests thatACTN4 could contribute to the development invasion andmetastasis of CC In addition our results indicated that highACTN4 levels were associated with the poor survival of CCpatients In the multivariate analysis although ACTN4 levelsdid not reach the statistical significance it still seems to beable to influence the OSHowever several limitations in the present study should betaken into consideration First the sample size was relativelysmall which was likely to reduce the statistical power of ourresults Second we only explored the relationship betweenserum ACTN4 and OS and other prognostic indicators werenot examined due to the incomplete data which needs to beimproved in the future Third this study was a primary studyto determine the clinical significance of serum ACTN4 levelsfor the diagnosis and prognosis of CC but the specific molecular mechanisms remain unclear Hence further experimentsshould be conducted to elucidate the mechanismsIn conclusion our study showed that serum ACTN4levels were increased in CC patients and were related to the 0cDisease Markers lavivrus muCLog rank P Overall survival monthsLow ACTN4 groupHigh ACTN4 groupFigure KaplanMeier curve compared OS of CC patients with high serum ACTN4 levels versus those with low serum ACTN4 levelsTable Univariate and multivariate Cox regression analysis of OS in CC patientsUnivariate CIVariablesAge vs ‰¤ yearsPathological type squamous cell carcinoma vs adenocarcinomaFIGO stage ‰¥IB2 vs IA1IB1Diï¬erentiation poorly diï¬erentiated vs well and moderately diï¬erentiated “Lymph node involvement positive vs negativeTumor size vs ‰¤ cmLymphovascular space invasion positive vs negativeSerum ACTN4 levels high vs low levelsHR “ “ “ “ “ “ “ “ “ “ “ “P”””Multivariate CIPHRFIGO stage lymph node metastasis and lymphovascularspace invasion of CC patients In addition serum levels ofACTN4 have great diagnostic and prognostic value in CCNevertheless further studies with a larger sample size shouldbe carried out to confirm our resultsAcknowledgmentsWe thank all the patients and blood donors who participatedin our study This study was funded by grants from the Science and Technology Project of Traditional Chinese Medicine Bureau of Jiangsu province China YB2015128Data AvailabilityReferencesThe datasets used andor analyzed during the present studyare available from the corresponding author on reasonablerequestConflicts of InterestAll authors declare that they have no conflicts of interestAuthors™ ContributionsXigui Ma and Huiying Xue contributed equally to this workand should be considered as cofirst authors[] M H Forouzanfar K J Foreman A M Delossantos et alœBreast and cervical cancer in countries between and a systematic analysis The Lancet vol no pp “ [] E Pelkofski J Stine N A Wages P A Gehrig K H Kimand L A Cantrell œCervical cancer in women aged yearsand younger Clinical Therapeutics vol no pp “ [] Y Zhou W Wang R Wei œSerum bradykinin levels as adiagnostic marker in cervical cancer with a potential mechanism to promote VEGF expression via BDKRB2 International Journal of Oncology vol pp “ [] Y J Hu H P Zhang B Zhu H Y Chen L H Ma andY Wang œThe role of FH detection combined with HPV 0cDisease Markers[] N Miura M Kamita T Kakuya œEfficacy of adjuvantchemotherapy for nonsmall cell lung cancer assessed by metastatic potential associated with ACTN4 Oncotarget vol no pp “ [] N Tanaka T Yamashita S Yamamoto œHistologicalgrowth pattern of and alphaactinin4 expression in thyroidcancer Anticancer Research vol no pp “[] Y Watabe T Mori S Yoshimoto œCopy numberincrease of ACTN4 is a prognostic indicator in salivary glandcarcinoma Cancer Medicine vol no pp “ [] HT An S Yoo and J Ko œÎ±Actinin4 induces theepithelialtomesenchymal transition and tumorigenesis viaregulation of Snail expression and βcatenin stabilization incervical cancer Oncogene vol no pp “[] F Niu T Wang J Li œThe impact of genetic variants inIL1R2 on cervical cancer risk among Uygur females fromChina a casecontrol study Molecular Genetics GenomicMedicine vol no article e00516 [] W Li Y Zhao L Ren and X Wu œSerum human kallikrein represents a new marker for cervical cancer Medical Oncology vol no p [] H Shao J HC Wang M R Pollak and A Wells œÎ±Actinin4 is essential for maintaining the spreading motility andcontractility of fibroblasts PLoS One vol no articlee13921 [] K Honda T Yamada Y Hayashida œActinin4 increasescell motility and promotes lymph node metastasis of colorectalcancer Gastroenterology vol no pp “ [] D G Thomas and D N Robinson œThe fifth sense mechanosensory regulation of alphaactinin4 and its relevance forcancer metastasis Seminars in Cell Developmental Biologyvol pp “ screening on the diagnostic significance of cervical cancer andprecancerous lesions European Review for Medical and Pharmacological Sciences vol no pp “ [] KH Wang C J Lin C J Liu œGlobal methylationsilencing of clustered protocadherin genes in cervical cancerserving as diagnostic markers comparable to HPV CancerMedicine vol no pp “ [] T Li Y Li G X Yang œDiagnostic value of combination of HPV testing and cytology as compared to isolatedcytology in screening cervical cancer a metaanalysis Journal of Cancer Research and Therapeutics vol no pp “ [] K Honda T Yamada R Endo œActinin4 a novel actinbundling protein associated with cell motility and cancer invasion The Journal of Cell Biology vol no pp “ [] E de Almeida Ribeiro N Pinotsis A Ghisleni œThestructure and regulation of human muscle αactinin Cellvol no pp “ [] D Wang X W Li X Wang œAlphaactinin4 is a possible target protein for aristolochic acid I in human kidneycellsin vitro The American Journal of Chinese Medicinevol no pp “ [] I V Ogneva N S Biryukov T A Leinsoo and I M Larina œPossible role of nonmuscle alphaactinins in musclecell mechanosensitivity PLoS One vol no articlee96395 [] K Honda œThe biological role of actinin4 ACTN4 in malignant phenotypes of cancer Cell Bioscience vol no p [] X Zhao K S Hsu and J H Lim œÎ±Actinin potentiatesnuclear factor κlightchainenhancer of activated BcellNFκB activity in podocytes independent of its cytoplasmic actin binding function The Journal of BiologicalChemistry vol no pp “ [] H Shams J Golji K Garakani and M R Mofrad œDynamicRegulation of α Actinin's Calponin Homology Domains on FActin Biophysical Journal vol no pp “[] C Fang J J Li T Deng B H Li P L Geng and X TZeng œActinin4 as a diagnostic biomarker in serum ofbreast cancer patients Medical Science Monitor vol pp “ [] T Watanabe H Ueno Y Watabe œACTN4 copynumber increase as a predictive biomarker for chemoradiotherapy of locally advanced pancreatic cancer British Journal of Cancer vol no pp “ [] S Yamamoto H Tsuda K Honda œACTN4 gene amplification and actinin4 protein overexpression drive tumourdevelopment and histological progression in a highgrade subset of ovarian clearcell adenocarcinomas Histopathologyvol no pp “ [] M C Wang Y H Chang C C Wu œAlphaactinin is associated with cancer cell motility and is a potential biomarker in nonsmall cell lung cancer Journal of ThoracicOncology vol no pp “ [] N Okamoto H Suzuki K Kawahara œThe alternativelyspliced actinin4 variant as a prognostic marker for metastasisin smallcell lung cancer Anticancer Research vol no pp “ 0c"

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

complex disease caused by coordinated alterations of multiple signaling pathways TheRasRAFMEKERK MAPK signaling is one of the bestdefined pathways in cancer biology and its hyperactivation isresponsible for over human cancer cases To drive carcinogenesis this signaling promotes cellular overgrowthby turning on proliferative genes and simultaneously enables cells to overcome metabolic stress by inhibitingAMPK signaling a key singular node of cellular metabolism Recent studies have shown that AMPK signaling canalso reversibly regulate hyperactive MAPK signaling in cancer cells by phosphorylating its key components RAFKSRfamily kinases which affects not only carcinogenesis but also the outcomes of targeted cancer therapies againstthe MAPK signaling In this review we will summarize the current proceedings of how MAPKAMPK signalingsinterplay with each other in cancer biology as well as its implications in clinic cancer treatment with MAPKinhibition and AMPK modulators and discuss the exploitation of combinatory therapies targeting both MAPK andAMPK as a novel therapeutic interventionKeywords RasRAFMEKERK signaling AMPK signaling Interplay Tumorigenesis Cellular metabolism RAFMEKERKinhibitors AMPK inhibitors AMPK activators Autophagy Targeted therapyIntroductionThe RasRAFMEKERK MAPK signaling is a fundamental pathway in cell biology and its alteration causeshuman cancers or developmental disorders Given its crucial roles in physiology and pathology this pathway hasbeen extensively studied for over two decades Unfortunately the regulation of MAPK signaling remains ambiguous till now by virtue of its intrinsic complexity anddiverse crosstalks with other signalings Here we focus onthe complicated interplays between the MAPK and theAMPK signalings in cellular carcinogenesis and their implications in current targeted cancer therapies We hopethis review would provide a conceptual framework for Correspondence yuanjiminszhospitalcom hujianchengnccscomsg1Department of Urology Shenzhen People™s Hospital The Second ClinicalMedical College Jinan University The First Affiliated Hospital SouthernUniversity of Science and Technology Shenzhen Guangdong China4Cancer and Stem Cell Program DukeNUS Medical School College RoadSingapore SingaporeFull list of author information is available at the end of the developing more effective therapeutic approaches againsthyperactive MAPK signalingdriven cancersThe RasRAFMEKERK MAPK signaling and itsaberrant activation in cancersThe RasRAFMEKERK MAPK signalingThe RasRAFMEKERK MAPK mitogenactivated protein kinase signaling is a central pathway that regulatescellular proliferation differentiation and survival Thissignaling pathway was discovered in the 1970s“1980swhen Ras small GTPases were identified as first oncogenes from sarcoma viruses [“] Later studies on viraloncogenes had also led to the discovery of a Nterminaltruncated version of RAF SerThr kinase RAF1 or CRAF[“] In contrast the other two components of this signaling pathway MEK mitogenactivated protein kinasekinase and ERK mitogenactivated protein kinase wereidentified as cytoplasmic protein kinases activated by mitogens in the 1990s [“] Following these discoveries 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 0cYuan Journal of Hematology Oncology Page of RAF was identified as the upstream kinase of MEK in and the first direct effector of Ras in [ ]resulting in the delineation of the whole MAPK signalingpathway which is considered as a milestone in our understanding of how cell senses external stimuliThe first component of MAPK signaling Ras smallGTPases have three gene isoforms Hras Kras and Nras that encode four proteins with splicing isoforms ofKras giving rise to Kras4A and Kras4B Although allRas proteins possess highly homologous sequences theyhave quite different activities tissue expression patternsand effector preferences which lead to their differentialphysiological and pathological functions [“]The downstream of Ras small GTPases is the RAFMEKERK kinase cascade [] The first kinases in thiscascade RAFKSR kinase suppressor of Ras family kinases include three RAF isoforms ie CRAF BRAF andARAF and two close pseudokinases ie KSR1 and KSR2All RAF isoforms have highly homologous sequences andsimilar structures with three conserved regions conservedregion CR1 contains RASbinding domain RBD anda Cysrich domain [ ] conserved region CR2 ischaracterized by a SerThrrich sequence conserved region CR3 comprises of a putative kinase domain with aNterminal acidic motif NTA [“] and a Cterminalregulatory tail [“] Nevertheless RAF isoforms havevariable kinase activities with an order as BRAFCRAFARAF likely by virtue of their distinct NTA motifs andAPE motifs that contribute to the dimerizationdriventransactivation of RAFs [“] In contrast to RAF isoforms KSR proteins replace the RBD at the Nterminusfused sterile αmotif and Prorichwith a coiledcoilstretch that are responsible for recruiting proteins to theplasma membrane upon stimulation and lack the catalyticlysine in VAIK motif of kinase domain which impairs theircatalytic activity [ ] Given their associations withMEK and ERK as well as low kinase activity KSR proteinshave been thought as scaffold proteins in a long termHowever recent studies have indicated that KSR proteinscan also function as allosteric activators to stimulate thecatalytic activity of RAF proteins through dimerization[ “] The sidetoside dimerization of RAFKSRfamily kinases is critical not only for their activation butalso for their catalytic activity towards downstream kinases [ “] MEKs MEK1 and MEK2 are the second kinases of the RAFMEKERK kinase cascade whichhave both redundant and nonredundant functions [] These two dualspecific kinases comprise a shortregulatory Nterminus and a canonic kinase domain TheNterminal regulatory region of MEK12 contains a docking site for substrate ERKs a nuclear export sequence thatcontrols the cytoplasmicnuclear shuttling of proteins anda negative regulatory sequence that forms a helix andlocks kinase in an inactive conformation [ ]Further through its kinase domain MEK12 forms a facetoface heterodimer with RAFKSR or a homodimerheterodimer with itself which is indispensable for its activation stimulated by RAF and for its activity towards ERKs[ ] Like MEKs the terminal kinases of MAPKsignaling ERKs also include two highly homologousmembers ERK1 and ERK2 which have a central kinasedomain flanked by short N and Cterminal tails Thesetwo isoforms also have redundant functions albeit different expression patterns [“] However unlike RAFs andMEKs that have very limited substrates ERKs recognizeand phosphorylate numerous substrates that include transcription factors protein kinases and phosphatases andother functional proteins [“]It should be noted that active Ras also turns on othersignaling pathways such as PI3KAKTmTORC whichregulate different cellular functions [] In this reviewwe focus only on the MAPK signaling given its dominant role in cancer biologyHyperactive RasRAFMEKERK MAPK signaling incancersThe MAPK signaling plays a crucial role in cell biologyand is tightly regulated in normal cells Upon engagement of receptor tyrosine kinases RTKs or other stimulations Ras small GTPases are activated by GTPGDPexchange factors GEFs which in turn recruit RAFMEK complexes to the plasma membrane and triggerthe RAFMEKERK kinase cascade through facilitatingRAFRAF or KSR RAFMEK and MEKMEK interactions as well as subsequent phosphorylations [] ActiveERKs are further translocated into the nuclei or stay inthe cytoplasm where they phosphorylate a number ofsubstrates that regulate cell functions [“ ]On the other hand active MAPK signaling also turns onsome negative feedback loops which help cells return toquiescent status [“] An aberrant activation ofMAPK signaling frequently induces human cancers ordevelopmental disordersthough an extremely highMAPK signaling may induce cell death or senescenceunder some conditions [“]its upstream activators or componentsHyperactive MAPK signaling exists in over ofcancers which is caused directly by genetic alterationsofincludingRTKs Ras and BRAF or indirectly by those independent of Ras or RAF [“] and significantly promotesdisease progression [] Since genetic alterations ofRTKs in cancers have been extensively reviewed in recent years [“] here we focus on oncogenic mutations of Ras and BRAF As a small GTPase Ras cyclesbetween active GTPbound status and inactive GDPbound status which is regulated by GEFs and GTPaseactivating proteins GAPs Oncogenic Ras mutationscan be mainly classified into two groups mutations 0cYuan Journal of Hematology Oncology Page of on glycine or G1213 that impair GAP associations and mutations on glutamine Q61 that diminish the intrinsic GTPase activity of Ras [] both ofwhich lead to an extended halflife of GTPloaded RasOncogenic Ras mutations have both isoform andcancertype preferences Kras is mostly mutated in allcancers followed by Nras and Hras and its mutations prevailin pancreatic cancers whilethose of Nras in myeloma and melanomas and Hrasin adrenal gland cancers [ ] This phenomenon mayreflect underlying fundamental signaling landscapes andRAS mutants interplay with these landscapes As thedownstream effector of Ras RAF is another dominanttarget of oncogenic mutations in the MAPK signalingpathway Similarly RAF mutations have isoform preference in cancers as Ras mutations with BRAF CRAF ARAF which may arise from their different basal activities Overall a single point mutation that converts Val into Glu in the activation loop of BRAF accounts for cases [] Although BRAF V600E exists only in of all cancers it is highly prevalent in some tissuespecific cancers such as melanoma thyroid cancer and histiocytosis [“] albeit theunderlying molecular mechanisms remains unknown Incontrast to Ras and RAF MEK and ERK have rare mutations in cancers though their mutations have been shownto be responsible for some RAF inhibitor RAFiresistantcases in current cancer therapies [“]Targeting the RasRAFMEKERK MAPK signalingpathway for cancer therapy promising but challengingGiven their high prevalence in cancers great efforts havebeen made to develop specific inhibitors against oncogenicRas and RAF mutants in the last decades These inhibitorsthat have been approved for clinic treatment of RasRAFmutated cancers or under clinical trials are listed in Table However none of these inhibitors can effectively target thelarge portion of Ras mutants in cancers Since having no attractive docking sites suitable for designing highaffinity andselective small molecule inhibitors Ras mutants have beenthought as œundruggable cancer drivers in a long term Untilrecently a group of covalent small inhibitors that are dockedinto a previously unknown pocket of GDPbound Ras andare linked to the adventive cysteine of RasG12C have beendeveloped and achieved encouraging outcomes for treatingRasG12Cdriven cancers as a single agent in clinical trials[“] Fig To further enhance their efficacy theseRasG12C inhibitors are also undergoing clinical evaluationwhen combined with SHP2 Src homology region domaincontaining phosphatase2 inhibitors that block the pathwayreactivation caused by the relief of negative feedback loops[ ] Clinical Trial NCT04330664 In addition these inhibitors have also been further developed into RasG12C degraders by conjugating with ligands of ubiquitin E3 ligaseswhich effectively deplete Ras mutant proteins in cancer cells[ ] though their efficacy in vivo remains unknown Unlike RasG12C the majority of Ras mutants remain œundruggable at present []It has been shown that Ras activates downstream effectors through direct interactions Therefore disruptingRaseffector interactions might be an alternative approach that can effectively block cancer growth drivenby Ras mutations Such a type of small moleculeblockers include rigosertib sulindac and MCP110 andamong which the therapeutic efficacy of rigosertib combined with nivolumab for Rasmutated cancers is beingdetermined by phase III clinical trials currently []Clinical Trial NCT04263090 Howeverit has to benoted that these inhibitors impair the MAPK signalingin both Rasmutated cancers and normal tissues andthereby their therapeutic index may not be highGenetic studies have revealed that the ablation of theRAFMEKERK kinase cascade but not other effectorpathways is a most efficient approach to inhibit thegrowth of Rasmutated cancers [] which leads to extensive developments of specific inhibitors against thiskinase cascade for treating Rasmutated cancers Moreover these inhibitors should be also effective for treatingRAFmutated cancers Indeed a number of RAFMEKERK inhibitors have been developed and applied to clinical trials for treating RasRAFmutated cancers [ “] At present three RAF inhibitors and three MEKinhibitors have been approved to treatlatestageBRAFV600Eharboring cancers as a single agent or incombination with other chemotherapeutics and exhibited excellent efficacies [ “] Fig HoweverRasmutated cancers possess intrinsic resistance to bothRAF and MEK inhibitors [] and even BRAF V600Eharboring cancers develop acquired resistance after “months treatment [ ] Mechanistic studies haveshown that active Ras facilitates the RAF dimerizationon plasma membrane which leads to both intrinsic andacquired resistance to RAF inhibitors [“] Toovercome the drug resistance arising from enhancedRAF dimerization the secondgeneration RAF inhibitorssuch as PLX8394 BGB283 TAK580 and CCT3833have been developed and are undergoing clinical evaluations Clinical Trials NCT02428712 NCT02610361NCT03905148 NCT02327169 NCT02437227 Thesenovel RAF inhibitors reduce the RAF dimerizationdriven resistance through distinct mechanismsPLX8394 and BGB283 impair RAF dimerization uponloading on RAF proteins [“] TAK580 bindsto and inhibits both protomers in RAF dimers [] CCT3833 inhibits both RAF and upstream kinases ofRas and thereby prevents the activation of Ras by the relief of negative feedback loops [ ] Besides thesesecondgeneration RAF inhibitors a unique RAFMEK 0cYuan Journal of Hematology Oncology Page of Table Summary of small molecule inhibitors approved and under clinical trials for treating RasRAFmutated cancersTargetKRasG12CDescriptionPhase I results showed ORR of nonsmall cell lung cancer NSCLC harboring KRas G12CCompoundAMG510Development stagesPhase IIINCT04303780MRTX849JNJRigosertibRasPhase IIINCT03785249Phase IIINCT04330664Evaluation of clinical activity of MRTX849 alone and combined with TNO155SHP2 inhibitor in KRas G12C mutated cancersPhase I NCT04006301 Safety and PK of JNJ74699157Phase IIINCT04263090Evaluation of safety and clinical efficacy of Rigosertib plus Nivolumab PD1 Abin KRas mutated NSCLCBRAFVemurafenib ApprovedLatestage or unresectable melanoma expressing BRAF V600E in ErdheimChester disease ECD with BRAF V600E mutation in DabrafenibApprovedEncorafenibApprovedLatestage or unresectable melanoma expressing BRAF V600E in Combination with trametinib for the treatment of unresectable or metastatic melanoma withBRAF V600EK in Combination with trametinib for the treatment of metastatic NSCLC with BRAF V600E in Combination with trametinib for the adjuvant treatment of melanoma with BRAF V600EK in Combination with trametinib for the treatment of anaplastic thyroid cancer ATC that cannotbe removed by surgery or has spread to other parts of the body with BRAF V600E in Combination with binimetinib for the treatment of patients with unresectable or metastaticmelanoma with BRAF V600EK in Combination with cetuximab EGFR Ab for the treatment of metastatic colorectal cancerwith BRAF V600E in PLX8394BGB283TAK580Phase IIINCT02428712PLX8394 with cobicistat CYP3A inhibitor was well tolerated and showed promising activityin BRAFmutated refractory cancersPhase I NCT02610361Phase IIINCT03905148Evaluation of safety and PK of BGB283 alone and combination with mirdametinibPhase I NCT02327169Phase I NCT03429803TAK580 is the inhibitor of BRAF V600E and dimersTreatment in pediatric lowgrade gliomaCCT3833Phase I NCT02437227 CCT3833 is a panRAF inhibitor of mutant BRAF CRAF and SRC kinasesRAFMEKRO5126766Phase I NCT00773526Phase I NCT03681483Phase I NCT03875820Phase I NCT02407509RO5126766 is a dual inhibitor for both RAF and MEKTreatment of advanced KRasmutant lung adenocarcinomasEvaluation of safety and PK of RO5126766 with VS6063 FAK inhibitor or everolimusmTOR inhibitorRO5126766 showed activity across Ras and RAFmutated malignancies with significantresponse in lung and gynecological cancersMEK12TrametinibApprovedCobimetinib ApprovedPhase IIINCT03989115BinimetinibApprovedSelumetinibApprovedMirdametinib Phase IINCT03962543Phase IINCT02022982Phase IIINCT03905148A singleagent oral treatment for unresectable or metastatic melanoma with BRAFV600EK in Combination with dabrafenib for the treatment of unresectable or metastatic melanomawith BRAF V600EK in Combination with dabrafenib for the treatment of metastatic NSCLC with BRAF V600E in Combination with dabrafenib for the adjuvant treatment of melanoma with BRAF V600EK in Combination with dabrafenib for the treatment of ATC that cannot be removed by surgeryor has spread to other parts of the body with BRAF V600E in In combination with vemurafenib to treat advanced melanoma with BRAF V600EK in Doseescalation of combination of RMC4630 SHP2 inhibitor and cobimetinibCombination with encorafenib for the treatment of patients with unresectable or metastaticmelanoma with BRAF V600EK in Selumetinib was approved for neurofibromatosis type with symptomatic inoperable plexiformneurofibromas according to NCT01362803Evaluation of mirdametinib in the treatment of symptomatic inoperableneurofibromatosis type1 NF1associated plexiform neurofibromas PNsCombination of mirdametinib with palbociclib in the treatment of KRasmutant nonsmall cell lung cancer NSCLCEvaluation of safety and PK of BGB283 alone and combination with mirdametinibSHR7390Phase I NCT02968485 Evaluation of safety and PK of SHR7390 0cYuan Journal of Hematology Oncology Page of Table Summary of small molecule inhibitors approved and under clinical trials for treating RasRAFmutated cancers ContinuedTargetDescriptionEvaluation of safety and PK of CS3006CompoundCS3006Development stagesPhase I NCT03516123Phase I NCT03736850ERK12UlixertinibMK8353LY3214996ASTX029ATG017KO947Phase IIINCT01781429Phase I NCT04145297Phase IINCT03698994Phase I NCT03454035Phase I NCT01358331Phase I NCT03745989Phase I NCT02972034Phase I NCT04081259Phase I NCT04391595Phase I NCT02857270Phase IINCT04386057Phase IIINCT03520075Responses to ulixertinib in NRas BRAF V600 and nonV600 BRAF mutant cancersEvaluation of ulixertinib alone or combined with hydroxychloroquine palbociclibCDK46 inhibitor in MAPK mutated cancersMK8353 was optimized from SCH772984 for better pharmacokinetics and exhibitedinhibition of BRAF V600 mutant cancersEvaluation of combination of MK8353 with selumetinib or pembrolizumab PD1 Abin advanced malignanciesEvaluation of treatment of MK8353 alone or combined with abemaciclibCDK46 inhibitorHydroxychloroquine in advanced malignanciesEvaluation of safety and PK of ASTX029Phase I NCT04305249 Evaluation of safety and PK of ATG017Phase I NCT03051035 Evaluation of safety and PK of KO947dual inhibitor RO5126766 has been developed and exhibited a strong potential against both Ras and RAFmutated cancers in phase I clinical trials [“] Thisallosteric inhibitor docks on MEK and prevents the release of MEK from RAF as well as the subsequent phosphorylation of MEK by RAF [] which gives it muchmore advantages than all other known RAF inhibitorsaccording to the regulatory mechanism of the RAFMEKERK kinase cascade [] As to small molecule inhibitors that target the terminal kinase ERK although anumber of them have been developed and are undergoing clinical trials [ ] their therapeutic values fortreating RasRAFmutated cancers remain unknownLike MEK inhibitorsthese ERK inhibitors may notachieve a good therapeutic index as single agents byvirtue of their inhibitory role in both malignant and normal tissues However they may contribute to antiRasRAF cancer therapy as synergetic agents combined withRasRAF inhibitorsOveralltargeting hyperactive MAPK signaling hasachieved exciting outcomes for treating RasRAFmutated cancers However although some effective smallmolecule inhibitors have been developed and applied toclinical treatment drug resistance and side effects remain remarkable challenges and there is still a long wayto develop a longeffective approach with manageableside effects for treating RasRAFmutated cancersAlthough hyperactive MAPK signaling has a dominantrole in cancer biology it is finetuned by other signalingssuch as PI3KAKTmTORC and AMPK during diseaseprogression [] These signaling interplays have important impacts on both cancer progression and clinicaltreatment based on MAPK inhibition In this review wewill focus on the crosstalk between MAPK and AMPKsignalingsAMPK signaling and its roles in cancer biologyAMPK signaling and cellular metabolismAMPK AMPactivated protein kinase is an energy sensorthat monitors the AMPADPATP ratio in eukaryotic cellsThis atypical protein kinase was firstly discovered as acontaminant during the purification of acetylCoA carboxylase ACC a wellstudied substrate of AMPK for fattyacid FA synthesis nowadays [“] Fig Howeverthe phosphorylation of ACC by AMPK in response to thehigh AMPATP ratio had not been revealed until a decadelater [] and the enzyme was thus named as AMPKthereafter [] Fig Biochemical studies have shownthat AMPK consists of three subunits including the catalytic α subunit and the regulatory β and γ subunits [“] Fig In mammals AMPK subunits are encoded asseveral isoforms α1 α2 β1 β2 γ1 γ2 γ3 which arepreferentially expressed in specific tissues or anisms[ ] For instance the α2 subunit associatesonly with β1 in type I muscle fibers while it binds to bothβ1 and β2 in type II muscle fibers [ ] Also theliver formulation of AMPK subunits differs among speciesas that α1β2γ1 is dominant in human whereas α1β1γ1and α2β1γ1 in dog and rat respectively [] Althoughan isoform replacement of AMPK subunits may not extensively affect the basal activity of AMPK as adaptive reit alters AMPK™ssponses such as exercise do []subcellular locations and sensitivity as well as interactionswith other signaling pathways [] The anismtissue 0cYuan Journal of Hematology Oncology Page of Fig Target hyperactive RasRAFMEKERK MAPK signaling for cancer therapy The RasRAFMEKERK MAPK signaling functions downstream ofreceptor tyrosine kinases RTKs Upon engagement by their ligands RTKs activates guanine exchange factors Sos proteins which load GTP to RasGTPases Then GTPbound Ras GTPases recruit RAFMEK heterodimers in cytosol to plasma membrane where they form transient tetramers throughthe sidetoside dimerization of RAFs The RAF dimerization not only turns on RAFs but also loosens RAFMEK heterodimerization and facilitates MEKhomodimerization on RAF dimer surface which leads to the activation of MEKs by RAFs Once MEKs are activated they phosphorylate ERKs and thenactive ERKs phosphorylate a number of downstream effectors In cancer cells hyperactive RasRAFMEKERK MAPK signaling arising from geneticmutations of Ras GTPases and BRAF can be targeted by small molecular inhibitors of Ras G12C BRAFV600E MEK and ERKstagespecific selectivity of subunit isoforms complicatesAMPK™s regulationAs a key sensor of cellular energy stress the activity ofAMPK is predominantly regulated by cellular AMPADPATP that competitively binds to the γ subunit of AMPKand thus promotes or inhibits the phosphorylation ofThr172 on α subunit by the tumor suppressor liver kinaseB1 LKB1 or the dephosphorylation of this site by phosphatases [ ] Fig Besides adenine nucleotidesintracellular calcium ions activate AMPK through calciumcalmodulindependent protein kinase kinase CAMKK2 also called CAMKKβFig which acts downstream of the hormoneactivated receptors such as muscarinic receptors and ghrelin receptor onendothelial cells or neuron cells [“] On the otherhand AMPK can be inhibited by a metabolite of glucosefructose 16bisphosphate FBP which binds to the aldolase and prevents the interaction of AMPK with LKB1 inglucoserich environments [] Fig Active AMPK[“]has more than downstream substrates that regulatethe metabolism of lipids cholesterol carbohydrates andamino acidsActive AMPK promotes the oxidation of fatty acidsand inhibits the synthesis of fatty acids and cholesterolwhich involves largely in acetylCoA AMPK phosphorylates and inhibits HMGCoA reductase HMGR that requires acetylCoA in its reduction reaction [ ] Fig Also AMPK phosphorylates ACC that converts acetylCoA to malonylCoA and therefore slowsdown the de novo fatty acid FA synthesis and increasesthe FA oxidation [] Fig Alternatively AMPK regulates the lipid metabolism through altering the mitochondria structure and function In the mitochondriaAMPK phosphorylates Akinase anchoring protein AKAP1 a key scaffold protein for protein kinase APKA and facilitates the phosphorylation of a mitochondriafusion factor dynaminrelated protein DRP1 by PKA which promotes mitochondrial fusion 0cYuan Journal of Hematology Oncology Page of Fig AMPK signaling and its downstream effectors AMPK is activated by liver kinase B1 LKB1 or calciumcalmodulindependent protein kinasekinase CAMKK2β through phosphorylation on Thr172 of α subunit and is inactivated through dephosphorylation of this site by proteinphosphatases in response to changes of cellular AMPADPATP ratio Downstream effectors activated by AMPK are indicated as arrows and thoseinhibited by AMPK are shown as barheaded linesand oxidative phosphorylation [] Moreover AMPKaccelerates the mitochondria biogenesis likely throughphosphorylating and activating the transcriptional activator proliferatoractivated receptor gamma coactivator1alpha PGC1α [ ] Fig However upon energy stress AMPK plays an opposite role in mitochondria biology Under this condition AMPK is essential forthe fragmentation of mitochondria AMPK phosphorylates mitochondrial fission factor MFF on Ser129 andthereby facilitates the translocation of DRP1 from cytosol to mitochondria membrane in energy stressdrivenmitochondria fission [ ] Then AMPK promotesthe clearance of damaged mitochondria through autophagy In this process AMPK binds directly to and phosphorylates the unc51like autophagy activating kinase ULK1 Autophagyrelated gene ATG9 and Beclin which triggers the autophagosome formation [“] Fig Active AMPK directly regulates the carbohydrate metabolism or indirectly through altering the fatty acid metabolism as described above Activation of AMPKstimulates the expression and plasma membrane translocation of solute carrier family member GLUT proteins and thereby facilitates glucose import [ “] Fig Intracellularly AMPK phosphorylates andactivatesis6phosphofructo2kinasePFK2thatfructose 26bisphoresponsible for the synthesis ofsphate a potent stimulator of glycolysis and thus accelerates glycolysis []Fig Furthermore AMPKappears to phosphorylate and inhibit glycogen synthasein the liver which dampens glycogen synthesis and thusindirectly enhances glycolysis []Active AMPK maintains cellular amino acid homeostasis mainly by controlling the activity of mammaliantarget ofrapamycin complex mTORC1 ThemTORC1 is a central sensor of cellular amino acids thatsamples amino acids in both cytosol and lysosome [] Upon activation by amino acids mTORC1 stimulates protein synthesis by phosphorylating ribosomalprotein S6 kinase B1 S6K and eukaryotic translationinitiation factor 4E binding protein 4EBP1 whichenhances the consumption of cellular amino acidsMoreover active mTORC1 blocks cellular autophagy byphosphorylating ULK1 and impairs the recycling ofamino acids [] Both effects of mTORC1 lead to a remarkable drop of cellular amino acid reservoir ActiveAMPK has been shown to inhibitthe activity ofmTORC1 direct and indirectly upon energy stresswhich limits the expenditure of amino acids Alternatively active AMPK can restrict protein synthesis byphosphorylating and thereby inhibiting eukaryotic translation elongation factor eEF2 kinase a key regulator 0cYuan Journal of Hematology Oncology Page of of protein synthesis [] To restore cellular amino acidreservoir active AMPK stimulates cellular autophagy asdiscussed above which degrades surplus or dysfunctional proteins into amino acids [] In addition it isworth noted that cellular amino acids can affect theactivity of AMPK reversely Dependent on conditionscontexts either amino acids may inhibit or stimulate theactivity of AMPK though underlying molecular mechanisms remain ambiguous [“]YAPactivity which impairsAMPK signaling in cancer biologyIt is well known that AMPK is a putative substrate oftumor suppressor LKB1 [ ] Fig Therefore AMPK has been generally considered as a key effector that mediates the tumorsuppressive function ofLKB1 Indeed a genetic ablation of the AMPK α subunitin mice accelerates Mycdriven lymphomagenesis throughfacilitating a metabolic shift to aerobic glycolysis []Simultaneously AMPK inhibitorsAMPKi promoteepithelialtomesenchymal transition EMT in breast andprostate cancers [] These studies validate AMPK as atumor suppressor under certain circumstances Furthermechanistic studies have demonstrated that AMPK prevents cancers through phosphorylating multiple targetsthat play indispensable roles on different layers of diseaseprogression AMPK phosphorylates angiomotin like AMOTL1 an adaptor protein in the HippoYap pathway and thus blocks Yes1 associated transcriptional regucells™latorproliferation and survival [] AMPK also phosphorylates TSC complex subunit TSC2 and regulatory associated protein of MTOR complex Raptor and therebyinactivates mTORC1 [ ] which in turn elevatescellular autophagy activity and inhibits cancer initiationTo bypass this inhibitory effect cancer cells can activatethe MAGE family member A MAGEA36tripartitemotif containing TRIM28 ubiquitin ligase complexthat targets the AMPK α subunit for degradation and thusreactivates mTORC1 to restrict cellular autophagy []Moreover AMPK is able to phosphorylate enhancer ofzeste polycomb repressive complex subunit EZH2and thereby disrupts the polycomb repressive complex PRC2 which relieves the epigenetic silence of tumorsuppressors in cancers [] Alternatively AMPK phosphorylates and stabilizes another epigenetic master regulator Tet methylcytosine dioxygenase TET2 whichfunctions as a putative tumor suppressor to preventtumorigenesis [] Altogether these findings indicatethat AMPK has a pronounced antitumor activity as itsupstream kinase LKB1 doescancerkillsandLICsstressleukemia TALL oncogenic Notch signaling induces ahigh level of aerobic glycolysis which needs to be restrained by AMPK and loss of AMPK results in energystressdriven apoptosis of leukemic cells and slows downdisease progression [] Similarlyin acute myeloidleukemia AML metabolic stress elevates the ROS leveland induces DNA damage in leukemiainitiating cellsLICs and AMPK confers metabolic stress resistance toLICs [] AMPK knockout or pharmaceutical inhibitionunder metabolicinhibitsleukemogenesis Moreover AMPK plays a determinantrole in maintaining the NADPH homeostasis in cancercells upon energy stress which is critic

Thyroid_Cancer

FAM83H‘AS1 is a potential modulator of cancer driver genes across different tumors and a prognostic marker for ERPR BRCA patientsMagdalena R­os‘Romero13 Alberto Cedro‘Tanda Mnica Pe±a‘Luna1 Marco Antonio Mancera‘Rodr­guez1 Lizbett Hidalgo‘Prez1 Mireya Cisneros‘Villanueva1 Fredy Omar Beltr¡n‘Anaya Roc­o Arellano‘Llamas1 Silvia Jimnez‘Morales1 Luis Alberto Alfaro‘Ru­z1 Alberto Tenorio‘Torres2 Carlos Dom­nguez‘Reyes2 Felipe Villegas‘Carlos2 Elsa Ochoa‘Mendoza1 Alfredo Hidalgo‘Miranda Breast cancer BRCA is a serious public health problem as it is the most frequent malignant tumor in women worldwide BRCA is a molecularly heterogenic disease particularly at gene expression mRNAs level Recent evidence shows that coding RNAs represent only of the total transcriptome in a human cell The rest of the of RNAs are non‘coding so we might be missing relevant biological clinical or regulatory information In this report we identified nine novel tumor types from TCGA with FAM83H‘AS1 deregulation We used survival analysis to demonstrate that FAM83H‘AS1 expression is a marker for poor survival in IHC‘detected ER and PR positive BRCA patients and found a significant correlation between FAM83H‘AS1 overexpression and tamoxifen resistance Estrogen and Progesterone receptor expression levels interact with FAM83H‘AS1 to potentiate its effect in OS prediction FAM83H‘AS1 silencing impairs two important breast cancer related pathways cell migration and cell death Among the most relevant potential FAM83H‘AS1 gene targets we found p63 and claudin CLDN1 to be deregulated after FAM83H‘AS1 knockdown Using correlation analysis we show that FAM83H‘AS1 can regulate a plethora of cancer‘related genes across multiple tumor types including BRCA This evidence suggests that FAM83H‘AS1 is a master regulator in different cancer types and BRCA in particularBreast cancer BRCA is a serious public health problem as it is the most frequent malignant tumor in women worldwide According to GLOBOCAN1 at least million new cases and a total of deaths are reported globallyBRCA is a phenotypically heterogenic disease with welldefined histological types and protein markers such as Estrogen receptor ER Progesterone Receptor PR and membrane receptor HER2 The physical and phenotypical BRCA heterogeneity is also reflected at the molecular particularly at gene expression mRNAs level This heterogeneity has been extensively studied and evidence shows that breast cancer comprises four intrinsic groups Luminal A Luminal B HER2 enriched and Basallike tumors23Molecular classification has been an important milestone in BRCA biology as it has been used to differentiate aggressive and nonaggressive tumors metastatic potential clinical prognosis and survival among other relevant cancerrelated features4 Additionally therapy responseassociated expression profiles are now available This expression profiles are able to predict if a patient can benefit from chemotherapy or antihormonal therapy5 1Laboratorio de Genmica del C¡ncer Instituto Nacional de Medicina Genmica Periferico Sur Tlalpan Arenal Tepepan Ciudad de Mxico CDMX Mexico 2Fundacin de C¡ncer de Mama FUCAM Ciudad de Mxico Mexico 3Programa de Doctorado de Ciencias Biolgicas Universidad Nacional Autnoma de Mxico Ciudad de Mxico Mexico email ahidalgoinmegengobmxScientific RepoRtS 101038s41598020710622Vol0123456789wwwnaturecomscientificreports 0cThese useful clinical advances are focused on coding RNAs profiles only however recent evidence show that coding messenger RNAs represent only of the total transcriptome in a human cell6 The rest of the of RNAs are noncoding so we might be missing relevant biological clinical or regulatory information if we only focus on messenger RNAIn this regard recent papers have focused on the role of long non coding RNAs lncRNAs in cancer biology7“ and in the role of specific lncRNAs in breast cancerFAM83HAS1 is a lncRNA whose expression impairs important cancerrelated pathways such as cell proliferation migration invasion and cell death in lung colorectal glial bladder ovarian and cervical cancer cells11“ At the molecular level one report showed that METEGFR signaling is regulated by FAM83HAS1 and16 showed that FAM83HAS1 epigenetically silenced CDKN1A by binding to EZH2 in glioma cellsIn addition two reports showed that FAM83H antisense RNA FAM83HAS1 a0is deregulated BRCA samples High expression of FAM83HAS1 indicated an unfavorable prognosis in luminal type BRCA and earlystage BRCA Altogether this evidence shows that FAM83HAS1 is an important actor in cancer biology In this paper we identified nine novel tumor types from TCGA with FAM83HAS deregulation and used a multivariate Cox regression analysis to demonstrate that FAM83HAS1 expression is a marker for poor survival in Progesterone receptor PR positive BRCA We found a significant correlation between FAM83HAS1 overexpression and tamoxifen resistance in luminal BRCA patients Using Kaplan“Meier and Cox regression analysis we found that estrogen and progesterone receptor expression levels interact with FAM83HAS1 to potentiate its effect in OS prediction Using FAM83HAS1 short hairpin knockdown coupled with microarray analysis we demonstrate that FAM83HAS1 silencing impairs two important breast cancer related pathways cell migration and cell death We further validate this phenotypic effect with in a0vitro migration and caspase assays Among the most relevant potential FAM83HAS1 gene targets we found p63 and claudin CLDN1 to be deregulated after FAM83HAS1 knockdown Using correlation analysis we show that FAM83HAS1 can regulate a plethora of cancerrelated genes across multiple tumor types including BRCAResultsFAM83H‘AS1 is deregulated in multiple tumor types Multiple studies have related FAM83HAS1 high expression levels with different tumors including luminal breast cancer11“ These findings suggest an important role for FAM83HAS1 in cancer tumor biology We therefore screened FAM83HAS1 expression levels in the TCGA database which comprises data from different tumor types and the correspondent normal tissues As expected we found significant FAM83HAS1 expression deregulation in different tumor types Fig a01A Log2FC p Some of these FAM83HAS1 expression deregulation data has been reported previously111216 but we have also found significant deregulation of FAM83HAS1 in nine additional tumor types Fig a01B Log2FC p Interestingly FAM83HAS1 was upregulated in different tumor types but downregulated in acute myeloid leukemia LAML suggesting a different mechanism for this particular malignancy Fig a01BFAM83H‘AS1 expression level is enriched in BRCA locally‘advanced tumors It was reported that FAM83HAS1 expression is a prognostic marker for luminal breast cancers12 We were interested to see if FAM83HAS1 expression was more widely associated with BRCA tumors since we and others found alterations for this lncRNA in a large number of malignancies As shown in Fig a01B FAM83HAS1 is significantly upregulated in all BRCA patients not only in the luminal subtype BRCA FAM83HAS1 overexpression is also marginally associated with BRCA locally advanced II and III clinical stages oneway ANOVA p Fig a01CFAM83H‘AS1 is a prognostic marker for ER and PR positive BRCA and its expression is related with tamoxifen resistance Altogether these widespread alterations in FAM83HAS1 expression suggested that its expression could be a prognostic biomarker for all BRCA subtypes but as mentioned above FAM83HAS1 was previously reported to have particular prognostic association with the BRCA luminal subtype1216 To test if FAM83HAS1 is a widespread or a luminal specific prognostic marker in BRCA we first screened FAM83HAS1 expression as a prognostic marker for all BRCA tumors We did not find significant association with poor OS in the Cox regression model n CI [“] Cox p value however we observed a clear tendency in poor survival prognosis in the FAM83HAS1 high expression group see Fig a02A We further validated these results in an independent Mexican patient cohort Fig a02B with all the BRCA subtypes The general clinical features of this cohort are listed in Table a0 We then tested if this effect was due to FAM83HAS1 interacting with other significant clinical and survivalrelated variables in particular luminal typerelated FAM83HAS1 predictive value was significant when interacting with Immunohistochemistry IHCdetected Progesterone receptor n HR CI [“] Cox p value supplementary Table a0 Marginal but not significant association was observed with ER status supplementary Table a0 Kaplan“Meier analysis of PR logrank p or ER positive patients logrank p showed significant association poor OS when FAM83HAS1 was overexpressed Fig a02C D No effect in survival rate was seen when FAM83HAS1 was overexpressed in PR and ER negative patients supplementary Figs a0 and This data strongly suggest that FAM83HAS1 is an independent prognostic marker for OS in PR positive BRCA subtype and confirms with further statistical analyses previous findings made by12We found a significant association with IHCdetected PR and ER status in the survival context Fig a0 C D supplementary Table a0 We then analyzed tamoxifen treatment resistance or sensitivity in our independent cohort Fig a02E and we found that FAM83HAS1 overexpression was significantly related with poor tamoxifen initial response n OR onetailed Fexact test p Scientific RepoRtS 101038s41598020710622Vol1234567890wwwnaturecomscientificreports 0cFigure a0 FAM83HAS1 expression is altered in multiple human tumors A FAM83HAS1 expression levels TPM in tumors from the TCGA database In green are shown normal tissue samples in blue tumor samples B FAM83HAS1 is aberrantly expressed in nine not previously reported tumors Color code as in A C FAM83HAS1 is enriched in locally advanced BRCA clinical stages II and IIIWe did not find a significant prognosis association for unreported tumors shown above in the Gene expression Profiling Interactive Analysis GPIA database see methods We could determine however a strong correlation between high FAM83HAS1 expression and poor OS in skin cutaneous melanoma SKCM patients n HR logrank test p Supplementary Fig a0ER and PR expression levels potentiate FAM83H‘AS1 prediction of survival in BRCA patients In order to further characterize our previous finding regarding ER and PR status and its association with FAM83HAS1 in BRCA prognosis we built a risk model taking into account ER PR and FAM83HAS1 expression levels in the same analysis This model fundamentally displays ER PR and FAM83HAS1 interaction and potentiation of the poor OS prediction in BRCAWe first calculated the Allred score17 to identify IHC ER and PR level of positivity ieER and PR expression levels from TCGA data We then obtained FAM83HAS1 expression levels from the TCGA cohort and divided it in four strata quartiles We then multiplied these two values Allred score values and FAM83HAS1 quartile values and the product was a new risk score We obtained four risk groups with the above described method shown in Fig a0 As shown here combination of very high FAM83HAS1 and ERPR expression levels potentiates Scientific RepoRtS 101038s41598020710622Vol0123456789wwwnaturecomscientificreports 0cFigure a0 FAM83HAS1 overexpression is a marker for poor prognosis in ERPR BRCA patients A Overall survival analysis Cox Regression for the BRCA TCGA cohort B Kaplan“Meier analysis for our independent BRCA cohort C Kaplan“Meier analysis for PR and D ER positive BRCA patients from the TCGA cohort E FAM83HAS1 expression is associated with tamoxifen resistance in BRCA patientsCharacteristicAgeTumor gradeClin stageERPRHER2Lymph NodesRecurrenceMetastasisClass““IIIIIISV IA IIA IIBIIIA IIIB IIICPositiveNegativePositiveNegativePositiveNegativePositiveNegativeNAPositiveNegativePositiveNegativeFrequencyPercentTable Clinicalpathological characteristics of population n In this Mexican cohort none of these clinical variables were significantly correlated with FAM83HAS1 expression levelScientific RepoRtS 101038s41598020710622Vol1234567890wwwnaturecomscientificreports 0cFigure a0 ERPR expression potentiates FAM83HAS1 death risk prediction Expression levels from ER and PR were calculated using Allread scores FAM83HAS1 expression levels were calculated from the quartile distribution of the log2 values TANRIC We then multiplied Allread scores and FAM83HAS1 expression levels in order to define risk groups see methods and main texthigh risk of decease in the TCGA cohort Kaplan“Meier model one tailed logrank p Cox hazard proportional risks for decease in these four groups were for low risk for moderate risk for high and for very high risk This data confirms a strong interaction between ER PR and FAM83HAS1 expression levels in BRCA Interaction between these three variables potentiates poor OS prediction in BRCA patients and possibly other hormonerelated human tumorsFAM83H‘AS1 potentially regulates a plethora of cancer‘related genes In order to better understand FAM83HAS1 role in BRCA we performed a differential expression analysis using the TCGA BRCA cohort We compared high versus low FAM83HAS1 RNA expression level samples see methods We found differentially expressed genes between these two groups Log2FC and ˆ’ padjvalue The vast majority of these transcripts RNAs were found to be downregulated in this analysis These results might suggest candidate target genes for FAM83HAS1 Fig a04AAmong the downregulated RNAs we identified several cancerrelated transcripts such as fibroblast growth factor FGF4 fibroblast growth factor FGF21 leptin LEP Claudin CLDN17 cadherin CDH9 Tumor Necrosis Factor receptor TNFRSF11B BCL2associated X BAX Tumor protein p53 TP53 and Phosphatase and tensin homolog PTEN see Table a0After pathway enrichment analysis we found a significant downregulation of cellular migration synthesis of steroids and lipid metabolism Fig a04B Gene set enrichment analysis GSEA also showed alterations in apoptosis and p53signalling pathways Fig a04C Taken together this evidence suggests an important regulatory role for FAM83HAS1 in cancerrelated pathwaysFAM83H‘AS1 is present both in nucleus and cytoplasm in ERPR BRCA cells To further characterize FAM83HAS1 functional role in BRCA we measured its expression in nine breast cancer cell lines including MDAMB231 HCC1187 MCF7 SKBR3 BT20 Hs578 ZR75 and one nontransformed cell line MCF10 Fig a05A As expected FAM83HAS1 was upregulated in transformed cell lines We then performed cellular fractionation assays in MCF7 cells and detected its enrichment in the cytoplasmic fraction of total input RNA Fig a05B LncATLAS screening further confirmed this observation as MCF7 cells display both FAM83HAS1 cytoplasmic and nuclear localization Supplementary Fig a0FAM83H‘AS1 knockdown deregulates transcripts expression in MCF7 cells In order to gain further insight into the potential FAM83HAS1 targets we performed shmediated FAM83HAS1 silencing experiments in MCF7 cells As shown in Fig a05C we obtained of silencing efficiency after a0h of plasmid transfection After knockdown we performed microarray experiments in MCF7 cells We identified differentiallyexpressed genes in the FAM83HAS1silenced cells FC and ˆ’ p value Fig a05D Key cancerrelated transcripts were identified such as CLDN1 TP63 FGF14 DDX60 and DRAM see Table a0 transcripts were found to be downregulated whereas were upregulated Fig a05D Table a0 Among the most upregulated RNAs we found TP63 and CLDN1 These genes can also be potential candidate target genes for FAM83HAS1 activityFAM83H‘AS1 silencing impairs cellular migration and apoptosis Pathway enrichment analysis showed that the most activated cellular processes in the shFAM83HAS1 condition are migration and cellular Scientific RepoRtS 101038s41598020710622Vol0123456789wwwnaturecomscientificreports 0cDescriptionMyosin Light chain Myosin heavy chain Actin alpha LeptinMicroRNA Nuclear receptor H4Fibroblast Growth Factor Fibroblast Growth Factor Claudin Insulin like growth factor binding protein Cadherin Hydroxydelta5steroid dehydrogenaseUDP glucuronosyltransferase family member A1Alcohol dehydrogenase 1A class I alpha polypeptideCytochrome P450 family subfamily A member Deathassociated protein kinase Tumor protein p53TNF receptor superfamily member 11bBCL2 associated X apoptosis regulatorBH3like motif containing cell death inducerPhosphate and tensin homologPathwayTight junction signalingWNT signaling pathwayWNT signaling pathwayCell migrationCell migrationCell migrationMAPK signalingFocal adhesionMAPK signalingFocal adhesionEpithelial to mesenchymal transitionPI3K pathwayCadherin signalingSteroid synthesisSteroid synthesisDrug metabolismDrug metabolismCell death regulationCell death regulationCell death regulationApoptosis regulationCell death regulationCell survival signalingFold change log2ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ padjvalue338Eˆ’951Eˆ’106Eˆ’110Eˆ’807Eˆ’147Eˆ’978Eˆ’374Eˆ’175Eˆ’244Eˆ’157Eˆ’405Eˆ’313Eˆ’115Eˆ’500Eˆ’422E ˆ’ 664Eˆ’107Eˆ’146Eˆ’Table Examples of differentially expressed genes in the FAM83HAS1 high versus low BRCA samplemotility altered molecules p value range and cellular death pathways molecules p value range Fig a05E These data are in accordance with our previous differential expression results in BRCA samples Fig a0 further supporting a master regulatory role for FAM83HAS1 in breast cancer cellsWe then aimed to functionally validate that both cellular processes are altered after FAM83HAS1 silencing We thus performed Transwell migration assays and caspase activity assays in MCF7 cells As shown in Fig a06A MCF7 cells migration significantly increases after shFAM83HAS1 transfection one tailed Ttest p We then performed matrigelinvasion assays and observed an increase a0h after transfection but this observation did not reach statistical significance Supplementary Fig a0 Furthermore we did not find significantly altered invasion related genes or pathways in the microarray assays nor the differential expression analysis in the TCGA cohort further suggesting that FAM83HAS1 is not involved in invasion in this modelCaspase assays identified a significantly increase in enzymatic activity Ttest p after a0h of FAM83HAS1 silencing which corroborates its role in apoptosis mediated cell death Caspase is the primary activator of apoptotic DNA fragmentation18 Significant increase in caspase activity in the shFAM83HAS1 condition suggests FAM83HAS1 regulation of late stage apoptosis Fig a06BMigration and cell death alterations are enriched in the FAM83H‘AS1 low expression group in BRCA samples To further validate our previous in a0vitro results we performed single sample GSEA ssGSEA in a BRCA independent cohort Gene Expression Omnibus dataset GSE115577 see methods In this approach gene sets are ranked according to absolute expression values in every sample rather than by a comparison with another sample We first stratified the GSE115577 cohort onto two groups samples with high levels of FAM83HAS1 RNA and samples with low FAM83HAS1 levels using the quartile approach described above We then aimed to know if the gene sets corresponding to migration apoptosis and other cell death processes like necrosis were significantly enriched in any of these two FAM83HAS1 expression groups As expected we found a significant enrichment of the migration Normalized Mutual Index [NMI] score AUC p value 918eˆ’ and apoptosis NMI score AUC p value processes in the FAM83HAS1low expression group Fig a0 B Interestingly we also found a strong enrichment of the necrosis pathway in this BRCA cohort NMI score AUC p value 13eˆ’FAM83H‘AS1 and its potential target genes are co‘deregulated across multiple tumor types We found that FAM83HAS1 is upregulated not only in BRCA but also in other tumors see Fig a0 We reasoned that if upregulated FAM83HAS1 may be exerting similar regulatory roles in other tumors as well In order to show this we correlated FAM83HAS1 expression levels with its potential target genes BAX CLDN1 CLDN17 DRAM DDX60 FGF4 FGF14 LEP PTEN TNFRSF11B TP53 and TP63 As shown in Fig a0 7A FAM83HAS1 is strongly correlated with these coding genes across multiple tumor types namely BLCA BRCA CESC COAD LAML LUAD LUSC OV PAAD PRAD READ SKCM STAD TGCT UCEC and UCS We then calculated the hazard ratio HR of decease event related to FAM83HAS1 and its potential targets expression in different tumor types from TCGA Fig a07B We found a significant association logrank Scientific RepoRtS 101038s41598020710622Vol1234567890wwwnaturecomscientificreports 0cFigure a0 High FAM83HAS1 levels in BRCA samples are correlated with downregulation of cancerrelated inhibitors A Volcano plot depicting differentially expressed transcripts in high versus low FAM83HAS1 levels Points in grey nonsignificant blue p value significant red fold change and p value significant B Migrationrelated and steroid metabolism genes are significantly downregulated when FAM83HAS1 is highly expressed in BRCA C GSEA analysis showed enrichment for apoptosis cell death and p53signalling pathways in the FAM83HAS1high samplestest p of FAM83HAS1 overexpression and a high decease HR in BRCA PAAD and SKCM Fig a07B The expression of FAM83HAS1 potential targets is also associated with high or low HR in these tumors Some of the coding genes that we either found in the differential expression analysis Fig a0 or after FAM83HAS1 knockdown in MCF7 cells Fig a05D are also coderegulated in other tumor types Fig a07C Altogether this evidence suggests a wide master regulatory role for FAM83HAS1 not only in ER PRBRCA but in other tumor types such as PAAD and SKCMDiscussionLong noncoding RNAs are molecules that exert numerous roles in human cancers as their biological activities involve regulation of cell proliferation cell death differentiation migration and invasion Deregulation in lncRNAs expression has also been associated with clinical outcome LncRNAs can affect expression of thousands of genes so they are regarded as key master regulators7“In this work our aim was to investigate a wider deregulation for FAM83HAS1 expression in tumors focusing on its functional and clinical role in breast cancer and the identification of potential FAM83HAS1 targets We found that FAM83HAS1 was overexpressed in nine different tumor types in the TCGA database In particular FAM83HAS1 is overexpressed and significantly correlated with a worse clinical outcome in PR positive detected by immunohistochemistry BRCA subtypes in the TCGA breast cancer cohort One previous report12 shows that FAM83HAS1 high expression indicated unfavorable prognosis in luminal breast cancer and was an independent prognostic indicator To the best of our knowledge this is the first report that suggests variable interaction between FAM83HAS1 and IHCdetected PR and ER in the clinical outcome context Furthermore we demonstrate that ER and PR expression levels can act as potentiators of FAM83HAS1 poor OS prediction In particular ER and PR high expression levels together with FAM83HAS1 overexpression confers a very high risk HR of decease in BRCA patients This data suggest an important clinical role for FAM83HAS1 in ERPR positive breast cancer It is currently unknown however if these statistical and clinical interactions are reflected at the biological or molecular level and future studies must address this issueScientific RepoRtS 101038s41598020710622Vol0123456789wwwnaturecomscientificreports 0cFigure a0 FAM83HAS1 knockdown in MCF7 cells is associated with deregulation of multiple cancerrelated genes A FAM83HAS1 expression profile in breast cancer cell lines B FAM83HAS1 is localized both in nucleus and cytoplasm but is enriched in cytoplasm in MCF7 cells C short hairpin RNA silencing of FAM83HAS1 in MCF7 cells D shmediated silencing of FAM83HAS1 induces differential expression in genes in MCF7 cells Grey nonsignificant blue p value significant green fold change significant red p value and fold change significant E Cellular migration and cell death are two significantly enriched pathways after FAM83HAS1 silencing in MCF7 cellsGene symbolDescriptionCLDN1TP63H3F3CFGF18DDX60IFI6DRAM1GPER1PARP9GSTM3Claudin Tumor protein p63H3 histone family 3CFibroblast growth factor DEAD Asp“Glu“Ala“Asp box polypeptide Interferon alphainducible protein DNAdamage regulated autophagy modulator Gprotein coupled estrogen receptor PolyADPribose polymerase family member Glutathione S S transferase mu brainPathwayEpithelial to mesenchymal transitionApoptosis signalingDNA replicationMAPK signalingFocal adhesionCell death regulationInterferon signalingCell death regulationEndocrine resistanceDNA repairDrug metabolismFold change shFAM83HAS1 vs shRANDOMˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ p valueTable Examples of differentially expressed genes after FAM83HAS1 knockdown in MCF7 cellsScientific RepoRtS 101038s41598020710622Vol1234567890wwwnaturecomscientificreports 0cFigure a0 FAM83HAS1 knockdown impairs cellular migration and induces cell death in breast cancer cells A Transwell in a0vitro migration assays in MCF7 cells B Caspase activity assays in MCF7 cells C Heatmap of the gene sets enriched in the high FAM83HAS1 expression group blue compared with that of FAM83HAS1 low expression samples green D Constellation Map of the gene sets Three connected clusters of gene sets migration apoptosis and cell death and necrosis pathways are detected in the lowFAM83HAS1 groupWe were also able to find a significant correlation between FAM83HAS1 high expression and poor tamoxifen response in BRCA patients This association could partially explain the reduced clinical response in FAM83HAS1 high expression groupWe also report that FAM83HAS1 overexpression in TCGA breast cancer samples is associated with downregulation of migration and cell deathrelated transcripts like FGF4 FGF21 LEP CLDN17 TP53 BAX and TNFRSF11B Accordingly we also found that FAM83HAS1 knockdown significantly deregulates migration and apoptosisrelated genes such as TP63 and CLND1 Transwell migration assays showed that indeed cellular migration increases after FAM83HAS1 silencing LEP and CLDN1 had been both shown to induce cellular migration and epithelial to mesenchymal transition EMT in breast cancer cells19“ further suggesting a FAM83HAS1 role in the early steps of migration Taken together this data might explain the underlying mechanisms related to FAM83HAS1 cell migration impairment in breast cancer cellsFAM83HAS1 might play a dual role probably due to cellular context FAM83HAS1 was involved in regulation of cell proliferation migration and invasion processes that were decreased after FAM83HAS1 knockdown in lung cancer cells Further analysis indicated the cell cycle was arrested at the G2 phase after FAM83HAS1 knockdown11 In the same report they found that METEGFR signaling was regulated by a0FAM83HAS1 These conflicting results may be due to cellular context or specific regulation mechanisms and henceforth specific molecular targetsWe also identified that FAM83HAS1 overexpression is associated with downregulation of cellular deathrelated transcripts like BAX TNFRSF11B and P53 In a0vitro assays also show that FAM83HAS1 silencing increases cellular death possibly by up regulating genes like p63 One previous report14 showed that cell death was markedly increased after with a0FAM83HAS1 a0knockdown in colorectal cell lines FAM83HAS1 Notch1 and Hes1 were significantly increased in colorectal cancer samples and cell lines Cell proliferation was inhibited with FAM83HAS1 knockdown and this effect mediated by a0FAM83HAS1 a0could be reversed by Notch1 regulators14It is currently not clear however if FAM83HAS1 has a direct or an indirect effect in gene regulation In this regard it has been shown that FAM83HAS1 epigenetically silenced CDKN1A by binding to EZH2 in glioma cells24 In our differential expression analysis we demonstrate that FAM83HAS1 is mostly downregulating gene expression This data might suggest an inhibitory regulation role for FAM83HAS1 Future studies must address Scientific RepoRtS 101038s41598020710622Vol0123456789wwwnaturecomscientificreports 0cFigure a0 The expression of master regulators of cancer such as p53 and p63 are dependent of FAM83HAS1 A FAM83HAS1 expression levels are strongly correlated with potential target genes TP53 TP63 BAX CLDN1 CLDN17 CDH9 TNFRSF11B PTEN FGF4 FGF14 DRAM DDX60 across different tumors BLCA BRCA CESC COAD LUAD LUSC PAAD PRAD READ OV STAD UCEC UCS SKCM B Overall survival heatmap depicting that overexpression of FAM83HAS1 confers high risk of death in BRCA PAAD and SKCM patients C FAM83HAS1 and its potential target genes are deregulated in different tumorsthese mechanisms as we cannot discard that FAM83HAS1 may regulate master gene expression via recruiting epigenetic complexes eg EZH2 In addition the exact role for FAM83HAS1 in upregulated genes remains obscure We cannot discard a subtle alternative role for this lncRNA in gene activation and future studies must address this issueOur results also show that FAM83HAS1 is present both in nucleus and cytoplasm of breast cancer cells It is possible that this lncRNA is playing a different role in cytoplasm and future studies must focus on this questionIn conclusion FAM83HAS1 is a lncRNA that is deregulated in multiple cancers and is a promising molecule that can perform as an independent prognostic factor in ER PR positive breast cancer FAM83HAS1 deregulation is associated with migration and cell death impairment in BRCA samples and breast cancer cells and may regulate a plethora of cancerrelated gene targets such as p63 BAX LEP and CLDN1MethodsThe Cancer Genome Atlas TCGA and Gene expression omnibus GEO datasets FAM83HAS1 expression levels were screened in the tumor datasets see supplementary Table a0 for details from TCGA and correspondent normal tissues using the Gene expression Profiling Interactive Analysis GPIA platform gepia cance rpkucn The tumors included are enlisted as follows Acute myeloid leukemia LAML Adrenocortical carcinoma ACC Bladder Urothelial Carcinoma BLCA Brain Lower Grade Glioma LGG Breast invasive carcinoma BRCA Cervical squamous cell carcinoma and endocervical adenocarcinoma CESC Cholangiocarcinoma CHOL Chronic Myelogenous Leukemia LMCL Colon adenocarcinoma COAD Esophageal carcinoma ESCA Glioblastoma multiforme GBM Head and Neck squamous cell carcinoma HNSC Kidney Chromophobe KICH Kidney renal clear cell carcinoma KIRC Kidney renal papillary cell carcinoma KIRP Liver hepatocellular carcinoma LIHC Lung adenocarcinomaLUADLung squamous cell carcinoma LUSC Mesothelioma MESO Ovarian serous cystadenocarcinoma OV Pancreatic adenocarcinoma PAAD Prostate adenocarcinoma PRAD Rectum adenocarcinoma READ Sarcoma SARC Skin Cutaneous Melanoma SKCM Stomach adenocarcinoma STAD Testicular Germ Cell Tumors TGCT Thyroid carcinoma THCAUterine Carcinosarcoma UCUterine Corpus Endometrial Carcinoma UCEC Uveal Melanoma UVMScientific RepoRtS 101038s41598020710622Vol1234567890wwwnaturecomscientificreports 0cPotential target genes expression correlation Hazard ratio HR map coexpression map and BRCA stage plots were also generated in the GPIA platform FAM83HAS1 expression levels were considered significantly correlated with tumors when log2FoldChange and p value Microarray generated expression data was downloaded from the GEO dataset GSE115577 This dataset includes RNA levels from BRCA samples analyzed with the Affymetrix HTA platform Downstream analysis is described belowIHC‘detected hormonal receptors and FAM83H‘AS1 risk model Clinical information of the BRCA patients was downloaded from the TCGA database porta lgdccance rgov FAM83HAS1 expression levels were downloaded from the TANRIC tool iblmdand erson tanri c_desig nbasic mainhtml We first searched for the ER and PR status and the numerical value for percent stained cells also available in the clinical data We then calculated the Allred score17 which measures the stain intensity and stain pattern for ER and PR positivity

Thyroid_Cancer

Growing evidence has demonstrated that glutathione peroxidases GPXs family genes play critical roles in onset and progression of human cancer However a systematic study regarding expression diagnostic and prognostic values and function of GPXs family genes in breast cancer remains absentMaterials and methods Several databases were employed to perform in silico analyses for GPXs family genes qRTPCR western blot and immunohistochemistry staining were introduced to validate GPX3 expression in breast cancer The functions of GPX3 in breast cancer cells were successively determinedResults By combination of receiver operating characteristic ROC curve analysis survival analysis and expression analysis GPX3 was considered as a potential tumor suppressor and a promising diagnosticprognostic biomarker in breast cancer Next low expression of GPX3 was confirmed in breast cancer cells and tissues when compared with corresponding normal controls Overexpression of GPX3 markedly suppressed proliferation colony formation migration and invasion of breast cancer in vitro Moreover two potential mechanisms responsible for GPX3 downregulation in breast cancer including hypermethylation of GPX3 promoter and release of hsamiR3245p inhibitionConclusions Collectively we demonstrate that GPX3 is markedly downregulated in breast cancer possesses significant diagnostic and prognostic values and attenuated in vitro growth and metastasis of breast cancerKeywords Glutathione peroxidase GPX3 Breast cancer Diagnosis Prognosis BiomarkerBackgroundBreast cancer is the most common diagnosed women™s malignant tumor and also the second leading cause of cancerrelated deaths in women worldwide [ ] Despite a variety of advancements have been achieved in diagnosis and therapy the total outcome of patients with breast cancer remains unsatisfactory Thus developing effective therapeutic targets and promising biomarkers for Correspondence 11718264zjueducn Peifen_Fu163comDepartment of Breast Surgery First Affiliated Hospital College of Medicine Zhejiang University QingChun Road Hangzhou Zhejiang Chinadiagnosis and prognosis prediction is very meaningful to improve prognosis of breast cancerGlutathione peroxidases GPXs consisting of eight members GPX18 are ubiquitously expressed proteins that catalyze the reduction of hydrogen peroxides and anic hydroperoxides by glutathione [] GPX family members have been well demonstrated to be frequently aberrantly expressed and are also closely linked to progression of diverse types of human cancer including kidney cancer [] pancreatic cancer [] hepatocellular carcinoma [] cervical cancer [] and gastric cancer [] However a comprehensive study about expression 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 0cLou a0et a0al Cancer Cell Int Page of function diagnostic and prognostic values of GPXs family in breast cancer remain absentIn this study we first assessed the roles of GPXs family genes in predicting diagnosis and prognosis of breast cancer and then determined the mRNA and protein expression of GPXs family genes in breast cancer using bioinformatic analysis Next the low expression of GPX3 was detected in breast cancer cells and tissues Subsequently the function of GPX3 in breast cancer cell growth and metastasis was also investigated Finally we explored the potential detailed mechanisms responsible for GPX3 downregulation in breast cancerMaterials and a0methodsROC curve analysisUsing TCGA breast cancer and normal breast expression data the diagnostic values of GPXs family genes were evaluated by ROC curve as we previously described [] Pvalue was considered as statistically significantKaplan“Meier‘plotter database analysisKaplan“Meierplotter database httpkmplo tcomanaly sis which is capable to access the effect of genes on survival in cancer types including breast cancer was employed to perform survival analysis for GPXs family genes and miRNAs in breast cancer [] Logrank Pvalue was considered as significantGEPIA database analysisGEPIA database httpgepia cance rpkucnindex html a newly developed interactive web server for analyzing the RNA sequencing expression data of tumors and normal samples from the TCGA and GTEx projects was used to determine mRNA expression profile of GPXs family genes in breast cancer [] Pvalue was considered as statistical significanceOncomine database analysisOncomine database wwwoncom ine which is a cancer microarray database and integrated datamining platform was also utilized to analyze mRNA expression of GPXs family genes in breast cancer [ ] Fold change FC Pvalue and a gene rank in top were set as the thresholds for selecting the included datasetsUALCAN database analysisThe protein expression levels of GPXs family genes in breast cancer were assessed using UALCAN database httpualca npathuabeduindex html which is a comprehensive userfriendly and interactive web resource for analyzing cancer OMICS data [] UALCAN database was also introduced to determine the promoter methylation level of GPX3 in breast cancer Pvalue of statistical analysis was considered to have significant differencesstarBase database analysisstarBase database tarb asesysueducnindex php an source platform for investigating miRNAassociated studies was used to predict the upstream binding miRNAs of GPX3 [ ] The correlation of GPX3 with miRNA in breast cancer and miRNA expression level in breast cancer were also assessed by starBase database Pvalue was considered as statistical significanceCell lines and a0clinical tissuesThe human breast cancer cell lines MCF7 and MDAMB231 and normal breast cell line MCF10A were purchased from Shanghai Institute of Biological Science Chinese Academy of Sciences Shanghai China breast cancer tissues and matched normal tissues were obtained from patients with breast cancer who received surgical resection in the First Affiliated Hospital of Zhejiang University College of Medicine Hangzhou China This study was approved by the ethics committee Table Correlation of a0 GPX3 expression with a0 various clinicopathological features in a0breast cancerFeaturesCasesBreast cancerLow expressionHigh expressionP‘valueAge ‰ Tumor size ‰ Lymph node metastasis Present AbsentHistopathological grade I“II IIIER status Positive NegativePR status Positive NegativeHER2 status Positive Negative 0cLou a0et a0al Cancer Cell Int Page of of the First Affiliated Hospital of Zhejiang University College of MedicineRNA isolation and a0qRT‘PCRTotal RNA was isolated from breast cancer cells and tissues by Trizol reagent Invitrogen USA qRTPCR was employed to detect GPX3 mRNA expression in breast cancer as we previously described [] GPX3 expression was normalized to GAPDH by the method of ˆ’ddCt The sequences of primers used in this study GPX3 forward primer ²GAG CTT GCA CCA TTC GGT CT3² GPX3 reverse primer ²GGG TAG GAA GGA TCT CTG AGTTC3² GAPDH forward primer ²AAT GGA CAA CTG GTC GTG GAC3² GAPDH reverse primer ²CCC TCC AGG GGA TCT GTT TG3²Protein extraction and a0western blotProtein of breast cancer cells was extracted using RIPA buffer Beyotime China supplemented with protease and phosphatase inhibitors Thermo Scientific USA Western blot was performed as previously described [] The primary antibodies of GPX3 and GAPDH were purchased from Abcam and antirabbit peroxidase conjugated secondary antibody was purchased from Sigma GPX3 band density was normalized to GAPDH and quantified by ImageJ softwareImmunohistochemistry IHC analysisIHC was utilized to analyze the protein expression of GPX3 in breast cancer tissues and matched normal breast tissues as we previously reported []Establishment of a0stably‘overexpressed cellFull length of GPX3 was first amplified after which the PCR product was cloned into pcDNA31PURO vector digested with BamH1 and XhoI GPX3overexpressed Lipofectamine„¢ Invitrogen USA according to the plasmid was transfected into breast cancer cells using manufactures™ instruction Then stablyoverexpressed cell was screened using puromycin a0μgmLCCK‘ assay stablyoverexpressed cells were seeded into 96well plates and cultured for varied period and a0h At the culture end of each time point a0μl CCK8 solution was added into each well and incubated for another a0 h at a0 °C Finally the optical density OD value at a0nm of each well was determined by a microplate readerColony formation assay stablyoverexpressed cells were seeded into sixwell plates and cultured for a0weeks At the end of culture the plates were washed using phosphate buffered saline PBS for two times Next the plates were fixed in methanol for a0min and stained with crystal violet solution for another a0 min Finally the visible colonies of each well were countedWound healing assayWound healing assay was introduced to detect the migrated ability of breast cancer cells × stablyoverexpressed cells were seeded into sixwell plates When the cells were grown to confluence a wound cross was made using a micropipette tip Photographs were then taken through a microscopy immediately or a0h after woundingTranswell invasion assayCell invasion was determined by Transwell invasion assay Briefly transwell inserts were firstly coated with Matrigel BD USA Then × stablyoverexpressed cells suspended in a0 mL serumfree medium were added into inserts And a0mL medium containing FBS was added to the lower compartment as a chemoattractant After culturing for a0h the cells on the upper membrane were carefully removed using a cotton bud and cells on the lower surface were fixed with methanol for a0 min and successively stained with crystal violet solution for a0min Photographs were then taken through a microscopyStatistical analysisStatistical analysis of bioinformatic analysis was performed by online databases as mentioned above The results of experimental data were shown as mean ± SD Student™s ttest was used to assess differences between two groups The diagnostic value was determined by ROC curve analysis A twotailed value of P was considered as statistically significantResultsThe diagnostic and a0prognostic values of a0GPXs family genes in a0breast cancerTo explore if the expression of GPXs family genes possesses significant diagnostic values in patients with breast cancer receiver operating characteristic ROC curve analysis was employed based on breast cancer data from TCGA database Fig a0 As shown in Fig a0 four GPXs family genes had the significant ability to distinguish breast cancer tissues from normal breast tissues including GPX2 GPX3 GPX4 and GPX8 However the other four GPXs family genes GPX1 GPX5 GPX6 and GPX7 showed no statistical diagnostic values in breast cancer Notably these findings suggested that GPX3 was the most potential diagnostic biomarker for patients 0cLou a0et a0al Cancer Cell Int Page of Fig The diagnostic values of GPXs family genes in breast cancer using ROC curve analysis a GPX1 b GPX2 c GPX3 d GPX4 e GPX5 f GPX6 g GPX7 h GPX8with breast cancer with the Area Under Curve AUC value being equal to Next we investigated the prognostic values of GPXs family genes in breast cancer using Kaplan“Meierplotter database Fig a0 Increased expression of GPX1 Fig a02a indicated poor prognosis of breast cancer Breast cancer patients with higher expression of GPX2 Fig a02b GPX3 Fig a02c or GPX5 Fig a02e had better prognosis GPX4 GPX6 and GPX7 had no significant predictive values for prognosis of breast cancer All these findings together indicated that only GPX2 and Fig The prognostic values of GPXs family genes in breast cancer determined by Kaplan“Meier plotter database a GPX1 b GPX2 c GPX3 d GPX4 e GPX5 f GPX6 g GPX7 h GPX8 0cLou a0et a0al Cancer Cell Int Page of GPX3 possessed significant diagnostic and prognostic values for breast cancerThe expression levels of a0GPXs family genes in a0breast cancerNext we further studied the expression levels of GPXs family genes in breast cancer First of all TCGA and GTEx databases were introduced to mine the mRNA expression of GPXs family genes in breast cancer The mRNA expression profile of GPXs family was shown in Fig a03a TCGA tumor tissues compared with TCGA normal tissues and Fig a03b TCGA tumor tissues compared with TCGA normal tissues and GTEx normal tissues We found that GPX2 and GPX3 were significantly downregulated in breast cancer Fig a0 3c“f Next Oncomine database was used to further analyze mRNA expression of GPXs family genes in breast cancer Fig a04a We performed metaanalysis for included studies about GPX3 and found that GPX3 mRNA expression was markedly decreased in breast cancer Fig a04b The downregulation of GPX3 mRNA expression in breast cancer of the GPX3associated studies was presented in Fig a04c“q However we found that GPX2 was not significantly downregulated in breast cancer Subsequently CPTAC database was utilized to assess the protein expression of GPXs family genes in breast cancer Fig a0 The results revealed that GPX1 GPX2 GPX3 and GPX4 protein levels were markedly decreased in breast cancer when compared with normal controls GPX7 protein expression in breast cancer was significantly increased GPX8 showed no statistical difference between breast cancer tissues and normal tissues And GPX5 and GPX6 were not found in CPTAC Taken together GPX3 was the most potential one among all GPXs family genes in breast cancer and was selected for following research Fig a0The expression level of a0GPX3 was a0confirmed in a0breast cancer and a0negatively correlated with a0tumor progressionTo further validate the results from in silico analysis we detected the mRNA and protein expression levels of GPX3 in breast cancer cells and tissues As presented in Fig a0 7a b GPX3 mRNA and protein were significantly downregulated in two breast cancer cells MCF7 and MDAMB231 when compared with normal cell MCF10A We also found that GPX3 mRNA expression in breast cancer tissues was much lower than that in adjacent matched normal tissues Fig a07c The protein expression of GPX3 was also detected using immunohistochemistry IHC analysis The results showed that GPX3 protein expression was significantly decreased in breast cancer tissues Fig a07d Collectively GPX3 mRNA and protein expression levels were significantly downregulated in breast cancer which was identical with the bioinformatic analytic results Furthermore Chi square test revealed that low expression of GPX3 was significantly negatively correlated with ERPR expression and positively linked to tumor size histopathological grade and lymph node metastasis Table a0 All these findings showed that GPX3 was negatively correlated with progression of breast cancer and might function as a tumor suppressor in breast cancerGPX3 overexpression suppressed proliferation and a0colony formation of a0breast cancer cellsGiven the low expression of GPX3 in breast cancer overexpression technology was used to study GPX3²s functions We then constructed the overexpressed plasmid of GPX3 After transfection of GPX3overexpressed plasmid GPX3 mRNA and protein expression levels were significantly upregulated in breast cancer cells Fig a0 8a b Firstly we explored the effect of GPX3 on growth of breast cancer cells CCK8 assay demonstrated that overexpression of GPX3 markedly suppressed in a0vitro proliferation of breast cancer cells MCF7 and MDAMB231 Fig a0 8c d Furthermore colony formation assay also revealed that GPX3 upregulation led to the inhibition of clonogenic capacity of breast cancer cells Fig a08e f These findings indicated that GPX3 overexpression significantly suppressed in a0 vitro proliferation and colony formation of breast cancer cellsGPX3 overexpression inhibited migration and a0invasion of a0breast cancer cellsMetastasis is another hallmark of malignant tumors including breast cancer We intended to ascertain if GPX3 affects metastasis of breast cancer Wound healing assay was first employed to investigate GPX3²s function in controlling migration of breast cancer cells and the result demonstrated that overexpression of GPX3 obviously attenuated the migrated ability of breast cancer cells Fig a09a b Moreover increased expression of GPX3 could also suppressed invasion of breast cancer cells which was detected by transwell invasion assay Fig a09c“f Taken together overexpression of GPX3 suppressed in a0vitro migration and invasion of breast cancer cellsThe potential mechanisms responsible for a0GPX3 downregulation in a0breast cancerFinally we preliminarily probed the possible molecular mechanisms that accounted for GPX3 downregulation in breast cancer Promoter hypermethylation may be responsible for expression suppression of tumor suppressors Intriguingly we found that the promoter methylation level of GPX3 was significantly upregulated in breast cancer tissues compared with normal controls Fig a010a Gene expression was also frequently negatively regulated by miRNAs at posttranscriptional 0cLou a0et a0al Cancer Cell Int Page of Fig The mRNA expression of GPXs family genes in breast cancer determined by GEPIA database a The mRNA expression profile of GPXs family genes in breast cancer tissues compared with TCGA normal breast tissues b The mRNA expression profile of GPXs family genes in breast cancer tissues compared with TCGA and GTEx normal breast tissues c d GPX2 was significantly downregulated in breast cancer e f GPX3 was significantly downregulated in breast cancer P level The miRNAs that potentially bind to GPX3 were predicted by starBase database and miRNAs were finally found For better visualization miRNAGPX3 network was established Fig a0 10b Based on the action mechanism of miRNA there should be negative correlation between miRNA and target gene We performed expression correlation analysis for miRNAGPX3 pairs As listed in Table a0 four potential miRNAs hsamiR3245p hsamiR3283p hsalet7a5p and hsamiR449b5p which were inversely associated 0cLou a0et a0al Cancer Cell Int Page of Fig The mRNA expression of GPXs family genes in breast cancer determined by Oncomine database a The mRNA expression of GPXs family genes in breast cancer b Metaanalysis for the included GPX3associated datasets in breast cancer c“q The mRNA expression of GPX3 was markedly downregulated in breast cancer in included GPX3assocaited datasets 0cLou a0et a0al Cancer Cell Int Page of Fig The protein expression of GPXs family genes in breast cancer detected by UALCAN database a GPX1 b GPX2 c GPX3 d GPX4 e GPX7 f GPX8 P 0cLou a0et a0al Cancer Cell Int Page of Fig The visual flowprocess diagram of this studywith GPX3 expression in breast cancer were identified The prognostic values of the four miRNAs in breast cancer were also evaluated by Kaplan“Meierplotter database Fig a0 10c d Survival analysis revealed that among the four miRNAs only high expression of hsamiR3245p indicated poor prognosis for patients with breast cancer Fig a0 10c The expression levels of four miRNAs in breast cancer was subsequently determined by starBase Fig a010g“j and showed that miR3245p and hsamiR449b5p were significantly upregulated whereas hsamiR3283p and hsalet7a5p were markedly downregulated in breast cancer compared with normal controls By combination of survival and expression analysis miR3245p was considered as the most potential upstream miRNA of GPX3 in breast cancer The above results implied that promoter hypermethylation and miR3245pmediated suppression were two potential mechanisms that may be responsible for GPX3 downregulation in breast cancer Fig a010lDiscussionBreast cancer is the most common cancer type in women The molecular mechanism of carcinogenesis of breast cancer is still unclear and need to be further investigated Increasing findings have showed that GPXs are critical regulators in onset and progression of human cancer However the knowledge of GPXs in breast cancer is still limitedROC curve and survival analysis for GPXs family revealed that some of them might serve as promising diagnostic and prognostic biomarkers for breast cancer especially GPX2 and GPX3 Expression analysis demonstrated the significant low expression of GPX3 in breast cancer GPX3 was reported to act as a tumor suppressor 0cLou a0et a0al Cancer Cell Int Page of Fig The expression levels of GPX3 in breast cancer cells and tissues The mRNA a and protein b expression of GPX3 in breast cancer cells was significantly lower than that in normal breast cell c The mRNA expression of GPX3 was markedly decreased in breast cancer tissues compared with matched normal breast tissues d IHC analysis of GPX3 expression levels in normal breast tissues and breast cancer tissues Bar scale um P in human cancer For example Cai et a0al indicated that GPX3 prevented migration and invasion of gastric cancer by targeting NFkBWnt5aJNK signaling [] Lee et a0al suggested that GPX3 arrested cell cycle and functioned as a tumor suppressor in lung cancer [] Hua et a0 al showed that silencing GPX3 expression promoted tumor metastasis in human thyroid cancer [] Caitlyn et a0 al revealed that plasma GPX3 limited the development of colitis associated carcinoma [] However the function and mechanism of GPX3 in breast cancer have not been reported and need to be further elucidatedNext we confirmed the low expression of GPX3 in breast cancer cells and tissues using qRTPCR western blot and IHC which supported the results of bioinformatic analysis Functional experiments revealed that overexpression of GPX3 significantly inhibited in a0 vitro proliferation colony formation migration and invasion of breast cancer cellsPrevious studies have showed the effect of promoter methylation level in regulating gene expression [] Thus we preliminarily evaluated the promoter methylation level of GPX3 in breast cancer and found that it was significantly upregulated in breast cancer compared with normal breast tissues Moreover Mohamed et a0 al also demonstrated the link between promoter hypermethylation of GPX3 and inflammatory breast carcinogenesis [] The report together with our finding revealed that hypermethylation of GPX3 promoter might be a potential mechanism responsible for GPX3 downregulation in breast cancermiRNAs are involved in multiple biological processes by suppressing gene expression [ “] We also explored the upstream regulatory miRNAs of GPX3 By combination of correlation analysis survival analysis and expression analysis for these miRNAs miR3245p was regarded as the most potential miRNA which was overexpressed negatively correlated with GPX3 expression and possessed poor prognosis in breast cancer Numerous studies have demonstrated that miR3245p served as an oncogenic miRNA in human cancer For example miR3245p promoted progression of papillary thyroid carcinoma via microenvironment alteration [] miR3245p facilitated progression of colon cancer by activating Wntbetacatenin pathway [] Moreover the 0cLou a0et a0al Cancer Cell Int Page of Fig Overexpression of GPX3 inhibited proliferation and colony formation of breast cancer cells in vitro a“b The overexpression effect of GPX3overexpressed plasmid in breast cancer cells c“d Overexpression of GPX3 inhibited proliferation of MCF7 and MDAMB231 cells e“f Overexpression of GPX3 inhibited colony formation of MCF7 and MDAMB231 cells P relationship between GPX3 and miR3245p has already been reported in lung cancer [] Thus overexpressed miR3244p might be another mechanism that accounted for GPX3 downregulation in breast cancer In the future the oncogenic roles of miR3245p need to be further investigated by in a0vitro and in a0vivo assaysConclusionsIn summary our current findings indicate that GPX3 is markedly downregulated in breast cancer promotes in a0 vitro growth and metastasis of breast cancer cells and servers as a promising diagnostic or prognostic biomarker for patients with breast cancer Moreover we also elucidate that promoter hypermethylation and miR3245pmediated suppression may be two 0cLou a0et a0al Cancer Cell Int Page of Fig Overexpression of GPX3 suppressed migration and invasion of breast cancer cells in vitro a b Increased expression of GPX3 attenuated migration of MCF7 and MDAMB231 cells c d Increased expression of GPX3 attenuated invasion of MCF7 cell e f Increased expression of GPX3 attenuated invasion of MDAMB231 cell Bar scale um P See figure on next pageFig The potential mechanisms responsible for GPX3 downregulation in breast cancer a The promoter methylation level of GPX3 was increased in breast cancer compared with normal controls b The miRNAGPX3 network c“f The prognostic values of four miRNAs in breast cancer g“j The expression levels of four miRNAs in breast cancer k The intersection analysis of survival analysis and expression analysis l The model of GPX3²s function and dysregulated mechanism in breast cancer P was considered as statistically significant 0cLou a0et a0al Cancer Cell Int Page of 0cLou a0et a0al Cancer Cell Int Page of Table The expression correlation of a0GPX3 with a0predicted miRNAs using TCGA breast cancer datamiRNAhsamiR3245phsamiR3283phsalet7a5phsamiR449b5phsamiR6295phsamiR47565phsamiR642a5phsalet7d5phsamiR449ahsamiR5895phsamiR181d5phsamiR21145phsamiR34a5phsamiR449c5phsamiR23a3phsamiR3150a3phsamiR47315phsamiR23b3phsamiR4915phsamiR4739hsamiR181c5phsamiR3612hsamiR5823phsamiR650hsalet7b5phsamiR3383phsamiR2278hsamiR1225phsamiR181a5phsamiR181b5phsamiR3139hsamiR4644hsamiR5013phsamiR26825phsamiR4306hsamiR95phsamiR620hsamiR1321hsamiR985phsalet7e5phsamiR1855phsamiR1270hsalet7 g5phsamiR2055phsamiR371a5phsamiR8735phsamiR34b5phsamiR5325phsamiR2963pRˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ P‘valueTable continuedmiRNAhsamiR7085phsamiR35295phsamiR5745phsamiR8765phsamiR2965phsamiR5023phsamiR1365phsamiR285phsamiR3615phsamiR520 hhsamiR6683phsamiR520 g3phsamiR376b3phsamiR6753phsalet7f5phsamiR34c5phsamiR665hsamiR1385phsamiR146a5phsamiR376a3phsamiR223phsamiR2995phsalet7i5phsamiR4953phsamiR1433phsamiR8893phsamiR146b5phsamiR3795phsamiR2233phsalet7c5pRP‘valuepotential mechanisms responsible for GPX3 downregulation in breast cancer These results provide key clues for developing effective therapeutic targets and biomarkers for breast cancerAcknowledgementsNot applicableAuthors™ contributionsWL and PF designed this work performed experiments analyzed data and draft the manuscript BD performed some experiments SW revised the manuscript All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsThe data in this work are available from the corresponding author on reasonable request 0cLou a0et a0al Cancer Cell Int Page of Lou W Ding B Fan W High expression of pseudogene PTTG3P indicates a poor prognosis in human breast cancer Mol Therapy Oncolytics “ Lou W Liu J Ding B Jin L Xu L Li X Chen J Fan W Five miRNAsmediated PIEZO2 downregulation accompanied with activation of Hedgehog signaling pathway predicts poor prognosis of breast cancer Aging “ Chen D Si W Shen J Du C Lou W Bao C Zheng H Pan J Zhong G Xu L et al miR27b3p inhibits proliferation and potentially reverses multichemoresistance by targeting CBLBGRB2 in breast cancer cells Cell Death Dis Cai M Sikong Y Wang Q Zhu S Pang F Cui X Gpx3 prevents migration and invasion in gastric cancer by targeting NFsmall ka CyrillicBWnt5aJNK signaling Int J Clin Exp Pathol “ An BC Choi YD Oh IJ GPx3mediated redox signaling arrests the cell cycle and acts as a tumor suppressor in lung cancer cell lines Plos One 2018139e0204170 Zhao H Li J Li X Han C Zhang Y Zheng L Guo M Silencing GPX3 expression promotes tumor metastasis in human thyroid cancer Curr Prot Peptide Sci “ Barrett CW Ning W Chen X Smith JJ Washington MK Hill KE Coburn LA Peek RM Chaturvedi R Wilson KT et al Tumor suppressor function of the plasma glutathione peroxidase gpx3 in colitisassociated carcinoma Cancer Res “ Kulis M Esteller M DNA methylation and cancer Adv Genet “ Mohamed MM Sabet S Peng DF Nouh MA ElShinawi M Promoter hypermethylation and suppression of glutathione peroxidase are associated with inflammatory breast carcinogenesis Oxid Med Cell Lou W Liu J Ding B Chen D Xu L Ding J Jiang D Zhou L Zheng S Fan W Identification of potential miRNAmRNA regulatory network contributing to pathogenesis of HBVrelated HCC J Transl Med Lou W Liu J Gao Y Zhong G Chen D Shen J Bao C Xu L Pan J Cheng J et al MicroRNAs in cancer metastasis and angiogenesis Oncotarget “ Lou W Liu J Gao Y Zhong G Ding B Xu L Fan W MicroRNA regulation of liver cancer stem cells Am J Cancer Res “ Yang Y Xia S Zhang L Wang W Chen L Zhan W MiR3245pPTPRDCEBPD axis promotes papillary thyroid carcinoma progression via microenvironment alteration Cancer Biol Therapy “ Yan D Liu W Liu Y Luo M LINC00261 suppresses human colon cancer progression via sponging miR3243p and inactivating the Wntbetacatenin pathway J Cell Physiol “ Lin MH Chen YZ Lee MY Weng KP Chang HT Yu SY Dong BJ Kuo FR Hung LT Liu LF et al Comprehensive identification of microRNA arm selection preference in lung cancer miR3245p and3p serve oncogenic functions in lung cancer Oncol Lett “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsEthics approval and consent to participateThis study was approved by the ethics committee of the First Affiliated Hospital of Zhejiang University College of MedicineConsent for publicationNot applicableCompeting interestsThe authors state that they have no conflicts of interestReceived June Accepted July References Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “Lou W Liu J Ding B Xu L Fan W Identification of chemoresistanceassociated miRNAs in breast cancer Cancer Manag Res “ Matouskova P Hanouskova B Skalova L MicroRNAs as potential regulators of glutathione peroxidases expression and their role in obesity and related pathologie

Thyroid_Cancer

distribution and reproduction in any medium provided the original work isproperly citedLacrimal gland neoplasms comprise up to of all orbital masses clinically and histologically Much of our current coreknowledge regarding lacrimal gland tumors stems from prior study of their more common counterparts the salivary glandsThe prognosis for each lacrimal gland tumor is contingent upon proper clinical evaluation and ultimately the histopathologicdiagnosis We describe a case of an invasive carcinoma expleomorphic adenoma CaexPA with a cystadenocarcinomacomponent arising from the lacrimal gland in the absence of any previously diagnosed pleomorphic adenoma benign mixedtumor or prior incisional surgery This case illustrates the importance ofincludingimmunohistochemistry and genetic testing to narrow a diï¬erential diagnosis and potentially aid or guide therapy in the futureOur finding suggests that carcinoma of the lacrimal gland may be derived from previously undiagnosed and perhaps evensubclinical pleomorphic adenomathe histopathologic assessment IntroductionMalignant mixed tumor of the lacrimal gland also knownas carcinoma expleomorphic adenoma CaexPA is thethird most frequent epithelial lacrimal gland tumor afterpleomorphic adenoma PA and adenoid cystic carcinomaACA [] Clinically aï¬ected patients tend to be to years older than those with PA and often present with aninsidiously progressive mass of a lacrimal fossa that suddenly becomes symptomatic Other reported clinical settings are patients without any previously known lacrimalgland tumor who develop a rapidly growing mass associated with pain and patients with one or more recurrencesof PA who undergo malignant transformation [] Diagnostic imaging is critical for the clinical diagnosis of a lacrimalgland neoplasm Computed tomography CT and magnetic resonance imaging MRI are ideal but MRI remainsthe preferred method to visualize surrounding tissue anddetect radiographic features concerning aggressive malignancy [] The incidence of a malignant transformation ofPA increases with the duration of the tumor Prior studiesreport that approximately less than of PA undergoesmalignant change within years after the first treatmentand by the end of years [] CaexPA can arisein patients without a known preexisting lacrimal glandtumor The malignant component is most often adenocarcinoma not otherwise specified however other histologicsubtypes have been described such as adenoid cystic carcinoma ductal carcinoma mucoepidermoid carcinoma andsquamous cell carcinoma 0cCase Reports in PathologyHerein we report a case of an invasive CaexPA with anepithelial component consistent with a cystadenocarcinomain the absence of any previously diagnosed PA and supportedby immunohistochemical and molecular studies Case PresentationA 64yearold male patient presented to the emergencydepartment due to left lateral canthal pain tearing and ipsilateral hearing loss over four months The initial examinationrevealed a visual acuity of bilaterally The pupils werereactive without an aï¬erent pupillary defect Intraocularpressures were and mmHg respectively Confrontational visual fields and color plates were unremarkable Therewas a complete abduction restriction of the left eye Theexternal examination revealed edematous upper and lowereyelids predominantly overlying the lateral orbital rim associated with temporal sloping and a nontender palpable andfixed mass of the temporal fossa a There wasptosis of the left upper eyelid with a reduced marginal reflexdistance of two and a half millimeters mm Exophthalmometry measured mm and mm with a base measurement of mm The anterior segment was otherwisenormal The fundus exam revealed symmetrically sharp andpink disc margins without pallor or edemaMaxillofacial CT scan with contrast showed a lytic lesion ofthe left orbital wall with associated heterogeneous soft tissuemass measuring — cm medially displacing the left lateralrectus muscle b Magnetic resonance imaging of thebrain and orbits revealed an enhancing ltrating mass of theleft lateral orbital wall extending into the left supra zygomaticmasticator space c A core guided needle biopsywas performed and the hematoxylineosin HE stainedslide revealed a moderately diï¬erentiated adenocarcinomainvolving fibrous connective tissue and demonstrating a cribriform architectural pattern with moderate cytologic atypia andindividual cell necrosis Figures 2a and 2bPositron emission tomography and CT of the chest abdomen and pelvis did not reveal any underlying malignancy orevidence of metastases Subsequently the patient underwentleft orbital exenteration with eyelid sparing Grossly the specimen included orbital contents frontal bone portions of thefrontal sinus and zygomatic bone Serial sectioning revealeda — cm multilocular cystic mass involving the lacrimalgland fossa abutting the globe superotemporallyHistopathologically the HEstained sections disclosedpredominantly neoplastic cystic structures in the proximityof the lacrimal gland acini measuring to mm in diameterltrating fibrous connective tissue and bone cA small focus of pleomorphic adenoma was identified associated with a lowgrade ductal carcinoma in situ The invasivecystic component revealed intraluminal papillary architecture and cribriform arch formations of the lining epitheliumd The neoplastic epithelium was composed ofmedium to largesized cuboidal cells with intercalated ductcell appearance eosinophilic cytoplasm and apocrine features Small foci of invasive solid components were observeddemonstrating cribriform architecture moderate to severenuclear pleomorphism and up to mitotic figures per highpower field Columnar cells with pseudostratified nucleiwere also present with moderate nuclear atypia Foci of ruptured cysts with hemorrhage granulation tissue lymphocyticltrate and dystrophic calcification were also seen No lymphovascular or perineural invasion was identifiedImmunohistochemical studies using monoclonal antibodies were performed with appropriate positive controls encompassing Gata3 mouse monoclonal antibody “ predilute Cell Marque Rocklin CA gross cystic disease fluidprotein GCDFP15 mouse monoclonal antibody dilute Thermo Fisher Scientific Fremont CA androgen receptor AR rabbit monoclonal antibody dilute Cell Marque Rocklin CA SOX10 rabbit polyclonalantibody dilute Cell Marque Rocklin CAprostatic specific antigen PSA mouse monoclonal antibody predilute Cell Marque Rocklin CAthyroid transcription factor TTF1 mouse monoclonalantibody predilute at micrograms Ventana Medical Services Tucson AZ p63 mouse monoclonal antibody predilute “ Biocare Medical Concord CA p53mouse monoclonal antibody predilute at microgramsVentana Medical Services Tucson AZ high molecularweight cytokeratin HMWK903 mouse monoclonalantibody predilute Cell Marque RocklinCA CAM mouse monoclonal antibody predilute Ventana Medical Services Tucson AZ estrogen receptor ERmouse monoclonal antibody diluted VectorLaboratories Burlingame CA progesterone receptor PRmouse monoclonal antibody prediluted Leica BiosystemsNewcastle UK Her2neu rabbit monoclonal antibody predilute Ventana Medical Services Tucson AZ and Ki67rabbit monoclonal antibody predilute Ventana MedicalServices Tucson AZ Immunohistochemical studies wereperformed on an automated stainer Leica BiosystemsInc BOND III System Buï¬alo Grove IL All antibodiesand testing were performed in a CLIAcertified laboratoryThe invasive component of the tumor was positive fata3 AR HMWK903 and CAM52 and focally positivefor GCDFP15 while negative for p63 ER PR SOX10 PSAand TTF1 stains Figures 2e“2h p53 was positive in lessthan of the tumor cells The Ki67 proliferative index ofthe tumor cells was low p63 and SOX10 stainshighlighted the PA and the in situ component of the tumorDetection of HER2 status by immunohistochemistry wasequivocal and reflex testing using fluorescence in situhybridization FISH was negative for HER2neu geneamplification based on the updated guidelines of theAmerican Society of Clinical OncologyCollege of AmericanPathologists criteria for HER2neu testing in breast cancerMolecular profiling using a nextgeneration sequencingNGS based assay Foundation One was performed onthe lacrimal gland tumor paraffinembedded tissue to identify genomic alterations within cancerrelated genes Thefollowing genomic alterations were detected HRAS G13RPIK3CA E542K and BCORL1 H215fsˆ— Microsatellitestatus MDstable and tumor mutation burden TMB low1MutsMbThe combined histopathologic immunohistochemicaland molecular findings supported the diagnosis of invasive 0cCase Reports in PathologyaabcbcFigure a Clinical photo of the patient at time of presentation direct view b Computed tomography maxillofacial axial cut Illustrating a cm mass of the lateral orbit eroding the lateral orbital wall with a soft tissue component extending into the orbit causing proptosis andnonaxial displacement of the globe c MRI brain and orbit T1 fatsuppressed image with gadolinium showing a lobulated mass withmixed cystic and solid components and inhomogeneous enhancement involving the left lateral orbital wall and suprazygomatic leftmasticator space Additionally the mass demonstrates edema and enhancement suggesting ltration by the massCaexPA disclosing a carcinomatous component with apredominant cystic growth pattern and focal solid areasreminiscent of a cystadenocarcinoma The surgical marginswere negative In addition the microscopic examination ofthe left eye globe revealed pseudoexfoliation syndrome andFuchs™ endothelial dystrophyThe patient underwent postoperative adjuvant chemoradiation followed by excision of the eyelids with no residualtumor Followup examination showed no evidence of recurrence or metastatic disease nine months after completingadjuvant therapy DiscussionMuch remains unknown about both the natural histologicprogression and malignant transformation of CaexPAWe know that lacrimal gland neoplasms comprise oforbital masses clinically [ ] and of orbital masseshistologically [ “] and nearly half of epithelial tumorsare malignant [] Although uncommon malignant transformation of PA can occur with incomplete excision InvasiveCaexPA can represent malignant transformation of PAmany of which develop recurrence or distant metastasis tothe lung bone abdomen and CNS [] This casehighlights the importance of an immunohistochemical andgenetic evaluation in complex lacrimal tumorsThe malignant epithelial component of CaexPA hasmorphological varieties including adenocarcinoma adenoidcystic carcinoma squamous cell carcinoma mucoepidermoid carcinoma and ductal carcinoma It could be likelya mixture of subtypes [] Rarely the only evidence ofpleomorphic adenoma is the presence of large areas of hyalinized stroma composed of myoepithelial cells and a fewductal structuresIn the current case the carcinomatous component of thetumor discloses predominantly an ltrative cystic growthpattern reminiscent of a cystadenocarcinoma previouslydescribed in neoplasms of the salivary and lacrimal glandsThe term cystadenocarcinoma of the salivary gland hasevolved It encompasses a variety of tumors depicting a cysticpattern of growth to a subset of papillary and cystic malignant neoplasms that have indolent behavior This is alsoobserved in other lowgrade salivary gland carcinomashowever it is important to note that they can demonstrateltrative growthlocal recurrence and metastasize toregional lymph nodes at the time of diagnosisPreviously Foss [] in a review of cases ofcystadenocarcinoma of the salivary gland used the followingdiagnostic criteria occurrence within a salivary gland invasive growth a predominantly cystic pattern ofgrowth with or without a papillary component and theabsence of acinar or mucoepidermoid diï¬erentiation or evidence of origin in a PA In the same review the predominantcell type varies among tumors and includes small cuboidalintercalated ductlike cellslarge cuboidal cells and tallcolumnar cells The subgroups of the large cuboidal cells 0cCase Reports in PathologyababcHMWCK903Androgen receptordedp53ep63fcfghghFigure a Moderately diï¬erentiated adenocarcinoma involving cores of fibrous connective tissue HE 2x b High power view of thetumor revealed a cribriform pattern moderate nuclear pleomorphism and scattered mitoses HE 40x c Neoplastic cystic structures inclose proximity to lacrimal gland tissue star in a hyalinized stroma and lymphoid proliferation HE 2x d Tumor that arose from afocus of pleomorphic adenoma star depicting cystic structures with small papillae HE 4x Inset papillary projections lined by largecuboidal cells with moderate nuclear atypia and apocrine features HE 20x e Tumor cells were strongly positive for HMWK903 4xf Nuclear expression of androgen receptor was present in the tumor 20x g p53 positive in less than of tumor cells 4x h p63was negative within the invasive component of the tumor 4xhave central nuclei abundant eosinophilic cytoplasm largenucleoli and apocrine features which are similar to thoseobserved in this case The fourth group has a combinationof cell types Furthermore Foss suggest that tumorswith predominantly columnar cells are associated withincreased metastatic potentialThe cystic growth pattern characteristic of this tumor isoften associated with an ‚ammatory response due toruptures of the dilated structures as noted in this case Cystformation in neoplasias of the salivary and lacrimal glandscan behave as a mimicker of an ‚ammatory process Pakdel report a case of a spontaneous rupture of a PAmasquerading as orbital cellulitis [] Histologically aruptured cystic space of a PA surrounded by a monocyticltrate and foreign bodytype granulomas is described Inthis paper the authors consider the spontaneous rupture ofthe cystic space as an underlying mechanism for the acutepresentation of this tumorCystadenocarcinoma can have a broad diï¬erentialdiagnosis The principal considerations include papillary cystic acinic cell adenocarcinoma secretory carcinoma mucoepidermoid carcinoma MEC and ductal carcinoma Aciniccell carcinoma and secretory carcinoma are typically indolent monotypic tumors that can disclose a papillary cysticarchitecture Histologicallysecretory carcinoma sharesnearly identical growth patterns to acinic cell carcinoma 0cCase Reports in Pathologybut instead shows a multivacuolated eosinophilic cytoplasmoften with luminal and intracytoplasmic mucin and no truezymogen granules Immunohistochemically secretory carcinoma is S100 and mammaglobinpositive and typicallynegative for DOG1 while acinic cell carcinoma shows theopposite staining profile Additionally secretory carcinomaharbors t1215p13q25 resulting in an ETV6NTRK3 genefusion [] MEC is usually composed of a mixture ofpredominantly epidermoid squamoid cells abundant intermediate cells ranging from small basal cells with basophiliccytoplasm to larger cells with eosinophilic cytoplasm andmucous cells Welldiï¬erentiated MEC is a circumscribedtumor that can disclose glandular and cystic structures linedby a single layer of mucussecreting cells however the intermediate and highgrade tumors show solid nests or sheets ofcells composed of primarily epidermoid cells with a scant cystic component and obvious invasion severe pleomorphismnecrosis and increased mitosesOn the other hand primary ductal adenocarcinoma ofthe salivary gland SDC originates from the excretoryportion of the salivary duct is a rare aggressive malignantepithelial tumor and accounts for only of epithelial lacrimal gland tumors Histologically the tumor is highly ltrative and usually solid with occasional cystic areas disclosingbreastlike ductal carcinoma features with central necrosisOccasionally cystadenocarcinoma with large cuboidal cellseosinophilic cytoplasm and highgrade nuclear atypia bearssome similaritiesto ductal adenocarcinoma Howeverpapillarycystic invasive growth is not usually seen in ductaladenocarcinoma [] The invasive component features trabeculae ducts and sheets of neoplastic cells in a desmoplasticstroma with perineural and vascular invasion The latter isnot commonly observed in cystadenocarcinoma Immunohistochemically the tumor cells are positive for a low molecular weight cytokeratin CAM52 CK7 CEA EMA andGCDFP15 Other than epithelial markersthis tumorexpresses AR in up to of invasive cases HER2 positivityin of cases and p53 overexpression in all reportedcases The Ki67 proliferative index is over The tumoris ER and PR negativeFurthermore in this case the tumor also demonstratescytomorphologicalfeatures similar to that of a low tointermediategrade ductal carcinoma in situ of the breast Thelatter findings might correlate with the previously describedlowgrade cribriform cystadenocarcinoma of the salivary glandalso known as lowgrade salivary ductal carcinoma and salivaryductal carcinoma in situ currently categorized as intraductalcarcinoma low grade and high grade respectively These typesof tumors show a variety of growth patterns both solid and cystic ranging from cribriform to solid to micropapillary and arereminiscent of lowgrade ductal carcinoma in situ of the breastFocal ltration may be noted []The exact pathogenesis of CaexPA remains controversial Prior studies have demonstrated that PA and CaexPA of the salivary and lacrimal glands share similar genomicprofiles and frequently overexpress the PLAG1 oncoprotein[ ] Harrison [] in their review indicate that thedevelopment of CaexPA follows a multistep model of carcinogenesis with the progressive loss of heterozygosity at 8qthen 12q and finally 17p Alterations including amplificationgene fusion and translocations in 12q genes such as HMGICHMGA2 and MDM2 may play a role in the malignant transformation In the same study the authors also mention thatloss of 17p is usually common in CaexPA indicating tumorsuppressor gene p53 loss as this tumor evolves Additionallyit appears that CaexPA and other malignant epithelialtumors other than ACC do not harbor MYB gene rearrangements or fusions It is important to note that there does notappear to be a correlation between rearrangement statusand clinical outcome []Additional mutational analysis of the lacrimal gland carcinomas has been also evaluated [] demonstrating that RASKRAS NRAS PIK3CA and MET mutations are frequent indiverse epithelial neoplasms of the lacrimal gland with thehighest proportion of mutations found in adenoid cystic carcinoma PIK3CA and MET mutations can coexist with RASmutationsPIK3CA and HRAS mutations are detected in this casewhich correlates with alterations already described in lacrimal gland carcinomas however alterations in MDM2HMGA2 NTRK3 p53 PLAG1 and ETV6 among others werenot observedIn summary this case demonstrates an invasive CaexPA with a malignant epithelial component that resemblesthe cystadenocarcinoma mixed cell type described by Foss [] Immunohistochemically the tumor was ARpositive while negative for ER PR and Her2 with expression of p53 in less than of the tumor cells Theprevalence of AR varies between the diï¬erent subtypes ofsalivary gland carcinomas SGC In recent medical literature data AR expression has been detected in as many as of SDC [] Dalin [] in the same study alsoindicate that adenocarcinoma and acinic cell carcinoma ofthe salivary gland express AR in and of the casesrespectivelyOur findings emphasize the importance ofThe current classification of the salivary gland tumors[] and the combined histopathologic and immunohistochemical features suggest that this tumor could representthe results of the natural course of a lowgrade cystadenocarcinoma with focal transformation into an intermediatehighgrade invasive ductal carcinoma expleomorphic adenomafurtherexploration of CaexPA pathogenesis especially the extentof disease and the histologic subtype In additiontheimmunohistochemical and genetic testing provides important support for the diagnosis as well as potentially guidesfuture therapy and prognostic evaluation Correctly identifying the type of malignant epithelial component is a significant factor in the survival of aï¬ected patients To ourknowledge this patient represents the first case in whichCaexPA of the lacrimal gland reveals an indolent epithelial malignancy with a low proliferative index arising froman undiagnosed PA after many likely consecutive molecular alterationsLastly due to the rarity of these tumors arising from thelacrimal gland further studies are necessary to evaluate theirbiologic behavior and determine any correlation betweenCaexPA and pseudoexfoliation syndrome 0cCase Reports in PathologyEnd Results  International Journal of Ophthalmology vol no pp “ [] F A Jakobiec J R Bily and R L Font œOrbit in OphthalmicPathology An Atlas and Textbook W H Spender Edpp “ WB Saunders Co Philadelphia 4th edition[] M H Devoto and J O Croxatto œPrimary cystadenocarcinoma of the lacrimal gland Ophthalmology vol no pp “ [] R H Simpson œSalivary duct carcinoma new developmentsmorphological variants including pure in situ high gradelesions proposed molecular classification Head and NeckPathology vol Supplement pp “ [] R D Foss G L Ellis and P L Auclair œSalivary gland cystadenocarcinomas a clinicopathologic study of cases TheAmerican Journal of Surgical Pathology vol no pp “ [] L Barnes J W Eveson P Reichart and D Sidransky Pathology and Genetics of Head and Neck Tumours IARC [] J Antony V Gopalan R A Smith and A K Y Lam œCarcinoma ex pleomorphic adenoma a comprehensive review ofclinical pathological and molecular data Head and NeckPathology vol no pp “ [] F Pakdel N Pirmarzdashti S Soltani Z Nozarian F AAmoli and A Kassaee œSpontaneous rupture of lacrimalgland pleomorphic adenoma Ophthalmic Plastic and Reconstructive Surgery vol no pp e41“e43 [] R R Seethala œHead and neck pathology Surgical PathologyClinics vol no [] S L von Holstein A Fehr M Persson œLacrimal GlandPleomorphic Adenoma and Carcinoma ex PleomorphicAdenoma Genomic Profiles Gene Fusions and Clinical Characteristics Ophthalmology vol no pp “[] T Y Chen M G Keeney A V Chintakuntlawar et alœAdenoid cystic carcinoma of the lacrimal gland is frequentlycharacterized by MYB rearrangement Eye vol no pp “ [] M Dalin P Watson A Ho and L Morris œAndrogen receptor signaling in salivary gland cancer Cancers vol no p Conflicts of InterestNone of the authors declare any competing interests Currently Dr Del Valle Estopinal is the Director of OphthalmicPathology and Assistant Clinical Professor of the Departments of Ophthalmology and Pathology at University ofCalifornia Irvine The City Dr S Orange CA Authors™ ContributionsDr Maria Del Valle Estopinal was the ocular pathologistthat performed the histopathologic immunohistochemicaland genetic evaluation Dr Bryant Carruth wastheoculoplasticstrained ophthalmologist who performed thecore biopsy The other authors participated in the clinicalcare and preparation of this manuscript All authors readand approved the final paperAcknowledgmentsThe authors acknowledge the cooperation of the patient Theyacknowledge the help of the SUNY Upstate Departmentof Pathology and Eye Plastic and Reconstructive Surgeonsof Central New York in the contribution to evaluationand treatment of this patientReferences[] C L Shields J A Shields R C Eagle and J P Rathmelllacrimal glandœClinicopathologic review of cases oflesions Ophthalmology vol no pp “ [] R L Font J O Croxatto and N A Rao Tumors of the Eye andOcular Adnexa American Registry of Pathology WashingtonDC [] W Harrison P Pittman and T Cummings œPleomorphicadenoma of the lacrimal gland a review with updates ontransformation and molecular genetics SaudimalignantJournal of Ophthalmology vol no pp “ [] SO Baek YJ Lee SH Moon YJ Kim and YJ JunœPrimary adenocarcinoma of the lacrimal gland Archives ofPlastic Surgery vol no pp “ [] D Bell M C Sniegowski K Wani V Prieto and B EsmaeliœMutationallacrimal gland carcinomas andimplications for treatment Head Neck vol Supplement pp E724“E729 landscape of[] J A Shields C L Shields J A Epstein R Scartozzi and R CEagle Jr œPrimary epithelial malignancies of the lacrimalgland the Ramon L Font lecture Ophthalmic Plastic Reconstructive Surgery vol no pp “ [] Lacrimal Gland Tumor Study Group œAn epidemiologicalsurvey of lacrimal fossa lesions in Japan number of patientsand their sex ratio by pathological diagnosis Japanese Journalof Ophthalmology vol no pp “ [] S L von Holstein M H TherkildsenJ U PrauseG Stenman V D Siersma and S Heegaard œLacrimal glandlesions in Denmark between and  Acta Ophthalmologica vol no pp “ [] W M Hassan M S Bakry H M Hassan and A S AlfaarœIncidence of orbital conjunctival and lacrimal gland malignant tumors in USA from Surveillance Epidemiology and 0c'

Thyroid_Cancer

RANK are expressed in different cell types and tissues throughout thebody They were originally described for their essential roles in bone remodeling and theimmune system but have subsequently been shown to provide essential signals fromregulating mammary gland homeostasis during pregnancy to modulating tumorigenesisThe success of RANKLRANK research serves as a paragon for translational researchfrom the laboratory to the bedside The case in point has been the development ofDenosumab a RANKLblocking monoclonal antibody which has already helped millionsof patients suffering from postmenopausal osteoporosis and skeletal related events incancer Here we will provide an overview of the pathway from its origins to its clinicalrelevance in disease with a special focus on emerging evidence demonstrating thetherapeutic value of targeting the RANKLRANKOPG axis not only in breast cancer butalso as an addition to the cancer immunotherapy arsenalKeywords osteoimmunology RANKLRANKOPG malignant tumor targeted therapy DenosumabEdited byLinda ConnellyCalifornia University of Science andMedicine United StatesReviewed byDhivya R SudhanINTRODUCTIONUniversity of Texas SouthwesternMedical Center United StatesSophia HL GeeUniversity of Miami United StatesCorrespondenceJosef M PenningerjosefpenningerubccaSpecialty sectionThis was submitted toWomen™s Cancera section of the journalFrontiers in OncologyReceived February Accepted June Published August CitationMing J Cronin SJF and Penninger JM Targeting theRANKLRANKOPG Axis for CancerTherapy Front Oncol 103389fonc202001283In the œseed and soil theory was first proposed by Stephen Paget for tumor metastasesto distant ans When tumor cells œseeds leave their primary site of origin and spreador metastasize the microenvironment œsoil of the target an is usually favorable for tumorcell anchoring and expansion of metastatic cells Bone is not only the site for primary bonetumors such as giant cell tumors and osteosarcoma but is also one of the most common distantmetastatic sites for solid tumors such as multiple myeloma MM breast cancer prostate cancerand nonsmall cell lung cancer NSCLC suggesting that the bone environment can serve asœsoil for tumor development and might also serve as a œseed for further metastatic spread Recentresearch on the bone microenvironment and its involvement in cancer biology has focused on thefield of osteoimmunology which includes the crosstalk between bone stromal cells osteoblastsand osteoclasts and immune cells Identifying key players regulating bone homeostasis couldpave the way for potential therapeutic cancer targets in particular to break the vicious circle ofmetastasis to the bonesThe receptor activator of the nuclear factor kappaB ligand RANKL also known as TNFSF11together with its receptor RANK TNFRSF11A the decoy receptor osteoprotegerin OPGTNFRSF11B and the recently identified receptor Leucinerich repeatcontaining Gproteincoupled receptor LGR4 has been shown to play critical bottleneck functions not only inregulating bone metabolism but also in immunity and tumorigenesis In this review we will brieflyintroduce the key functions of the RANKLRANKOPG axis in maintaining bone homeostasisand regulating immunity Furthermore we will discuss the role of this pathway from primaryFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertumorigenesis to cancer metastasis with particular attention tobreast cancer and the hormonal control of this pathway We willalso discuss recent data pointing to the RANKLRANK axis as anovel therapeutic target in BRCAmutated breast cancers and asa novel promising cancer immunotherapy agentRANKLRANKOPG AND BONEHOMEOSTASISBone provides strength and structure protects vital ans storesminerals such as calcium and is essential in the productionof hematopoietic cells Bone homeostasis is maintained by thebalance between mainly two types of cells osteoblasts derivedfrom mesenchymal cells which build bone and osteoclastsderived from bone marrow hematopoietic precursor cells whichresorb bone Figure Osteoblasts act as both mechanicalsensors together with osteocytes and coordinators for the boneremodeling process which is controlled by local growth factorsand systemic factors for example calcitonin or sex hormonessuch as estrogen The pathological imbalance between boneformation and resorption leads to the development of local orsystemic bone diseases such as osteopetrosis and osteoporosis The interaction and communication between osteoclasts andosteoblasts is intricately regulated in feedback loops to maintainbone homeostasis and this constant remodeling process of thebone matrix is critical for healthy bone strength and efficienthematopoiesis RANK TNFRSF11A OFE ODFR TRANCER ODARCD265 and RANKL TNFSF11 TRANCE ODF andOPGL “ are a receptorligand pair of the TNF receptorsuperfamily discovered at the end of the last millennium and wereidentified as key regulators of osteoclast development and bonemetabolism Figure Factors that can induce boneresorption such as the sex hormone progesterone vitamin D3PTHrP IL1 IL11 IL17 or TNFα “ act on osteoblaststo induce RANKL expression which then binds to its receptorRANK on the surface of osteoclast progenitor cells inducing preosteoclast diï¬erentiation into multinucleated fullyfunctionalosteoclasts RANKL also plays an important role in the continuedsurvival and function of osteoclasts “ Figure RANKLis produced as a membranebound protein which can alsobe shed as a soluble trimeric protein SheddaseresistantRANKL mice have been generated in which soluble RANKLis undetectable in the circulation bone mass or boneFIGURE Role of RANKLRANKOPG axis on bone homeostasis and immune system RANKL is secreted by osteoblasts and osteocytes when stimulated byparathyroid hormone PTH vitamin D andor prostaglandin PGE2 RANKL binds to RANK on the membrane of osteoclast progenitors preosteoclasts whichresults in bone resorption by mature osteoclasts Osteoprotegerin OPG binds to RANKL thus inhibiting RANK signaling and bone resorption RANKRANKL alsoplays a role in immune cell regulation and the crosstalk between both systems termed osteoimmunology T cells can also express RANKL which can both act onpreosteoclasts but can also act on dendritic cells DCs to promote their survival and to prolong T“DC interactions DCs can exhibit modulating effects onRANKmediated osteoclastogenesis through the secretion of OPG HSC hematopoietic stem cell MSC mesenchymal stem cellFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerstructure was not aï¬ected during development in these mice butadult mice displayed reduced osteoclast numbers and increasedcancellous bone mass Importantly the bone loss caused byestrogen deficiency was unaï¬ected by the lack of soluble RANKLThus these data show that it is the membranebound formof RANKL which is largely responsible for the physiologicalfunctions of RANKL although the soluble form can contributeto bone remodeling in adult mice Osteoprotegerin OPG TNFRSF11B acts as a decoy receptorfor RANKL and is induced by estrogen IL4 or transforminggrowth factor beta TGF OPG competitively binds toRANKL thereby interfering with RANKL“RANK interactionsand blocking bone resorption “ The relative levels ofOPG and RANKL are precisely controlled to ensure healthybone During pathological conditions such as menopauserelatedosteoporosis decreased estrogen levels result in decreased OPGand subsequently increased RANKL resulting in enhancedosteoclast activation and bone loss Recently leucinerichrepeat G proteincoupled receptor LGR4 was identifiedas an additional receptor for RANKL Similar to RANKLGR4 is expressed on osteoclasts but unlike RANK LGR4 is anegative regulator for osteoclast diï¬erentiation Therefore bothOPG and LGR4 are endogenous inhibitors of RANKLRANKsignaling A recent study has shown that RANKL reversesignaling from osteoclasts to osteoblasts couples bone resorptionto bone formation processes This is achieved through thesecretion of small extracellular vesicles from osteoclasts thatcontain RANK The authors showed that these RANK vesiclesbind membranebound RANKL on the osteoblasts and therebypromote bone formation by triggering RANKL reverse signalingvia activation of Runtrelated transcription factor Runx2Targeting RANKL reverse signaling represents a novel strategyto avoid the reduced bone production associated with inhibitionof osteoclastogenesis As RANKL is an important regulator of bone loss in bonemetastases associated with cancers such as multiple myelomaand in postmenopausal osteoporosis a specific fully human IgG2monoclonal RANKL antibody mAb has been developed whichneutralizes the activity of RANKL which has been designated asDenosumab The efficacy of Denosumab has been confirmed inmultiple clinical trials and Denosumab therapy is now approvedand widely used for the treatment of various boneassociateddiseases “RANKLRANKOPG IN THE IMMUNESYSTEMApart from bone homeostasis the RANKLRANKOPG axis isalso involved in various physiological immune processes RANKwas originally discovered on dendritic cells DCs and RANKLmediates the survival of DCs The interaction betweenactivated T cellderived RANKL and RANK expressed on DCsincreases the antigenpresenting capabilities of the latter thusaugmenting the number and cell cycle of antigenspecific Tcells as well as enhancing the immune response of memory Tcells Interestingly phenotyping of rankl and rankdeficient micerevealed a complete absence of peripheral lymph nodes but intactspleen and Peyer™s plaque structures “ Subsequent studieshave found that during embryogenesis RANKL is expressedby hematopoietic lymphoid tissue inducing LTi cells andmesenchymallymphoid tissue anizer LTo cells “RANKL has been demonstrated to stimulate lymphotoxin LTexpression and regulate LTi cell accumulation FurthermoreRANKL also triggers the proliferation of adult lymph nodestroma indicating that RANKL may directly activate LTo cells“ In the thymus the RANKLRANK pathway is criticalfor CD80 AIRE medullary thymic epithelial cell mTECmaturation involved in central immune tolerance RANKdeficient mice display mild autoimmunity at an advancedage RANKLRANK activation in lymphatic endothelialcells LECs is important for the tissueresident macrophagesnamely sinusoidal macrophage maturation not only duringembryogenesis but also after inflammationinduced loss of thesecells Moreover group innate lymphoid cells ILC3s inthe intestine use RANKLRANK interactions to control theirown abundance and intestinal homeostasis Genetic ablation ofRANKL specifically in IL3C cells leads to an increased number ofthese cells with enhanced levels of proinflammatory cytokinessuch as interleukin17A IL17A and IL22 during intestinalinfection Human patients carry RANK mutations and mice lackingRANKL or RANK exhibit a defect in B cell developmentresulting in a significant reduction in B cell numbers however these eï¬ects might be indirect because in themousetissuespecific deletion of Rank in B cells showedno diï¬erence in function nor development of B cells andblocking RANKRANKL with Denosumab does not apparentlyaï¬ect B cell physiology in osteoporosis patients In addition reports using Bcellspecific rankldeficient micehave shown that B cellderived RANKL increases osteoclastnumbers and bone loss brought on by estrogen deficiency Overexpression of RANKL in keratinocytes results in functionalalterations of epidermal dendritic cells and systemic increases inregulatory CD4CD25 T cells Tregs numbers Thereforeenvironmental stimuli can rewire the local and systemic immunesystems via RANKL The RANKLRANK system is alsoinvolved in M microfoldcell development a specific antigensampling cellular subtype found in the intestine as mesenchymalcells produce RANKL that can directly interact with intestinalepithelial cells to regulate M cell diï¬erentiation “Inhibition of mesenchymal RANKL impairs M celldependentantigen sampling and B celldendritic cell interaction in thesubepithelial dome SED resulting in decreased IgA productionand microbial diversity In addition B cells are absentin cryptopatches CPs and isolated lymphoid follicle ILFsformation was abrogated in rankl null mice Whether B cells or T cells are essential for bone loss isstill controversial Ovariectomy has been shown to enhance Tcelldependent TNFalpha production in a bone loss mousemodel because of the enhanced macrophage colonystimulatingfactor MCSF and RANKL In contrast anotherstudy suggested T cells are not involved in ovariectomyinducedFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertrabecular bone loss Nevertheless it has been reportedin postmenopausal women that increased T cell activity andincreased RANKL production by T cells are associated withosteoporosis Furthermore studies in conditionalknockout mice to specifically eliminate RANKL in B cells or Tcells have shown that RANKL produced by B cells but not T cellsleads to bone loss by the induction of osteoclastogenesis Thelack of mature B cells does not prevent bone loss suggestingthat RANKL is derived from immature B cells Moreover it hasbeen reported that deletion of rankl in T cells does not change thenumber of T cells but results in impaired mature B cell numbersin the bone marrow suggesting that RANKL might promote Bcell maturation via paracrine signaling RANKLRANKLOPG IN MAMMARYGLAND PHYSIOLOGY AND BREASTCANCERBreast cancer is the most prevalent female malignancy Studies based on large populations have shown that womenwho receive estrogen plus progesterone hormone replacementtherapy called combined HRT are more vulnerable to breastcancer compared to women who receive estrogen only “furthermore progesterone levels have been demonstrated to bean independent risk factor for increased breast cancer incidence In rankl knockout mice our group was the first to reportthat during pregnancy RANKL deficiency results in a totalblock in the development oflobuloalveolar milksecretingstructures Whereas estrogen triggers the expansion ofthe mammary epithelium in puberty progesterone drives theproliferation of mammary epithelial cells in the estrous cycleand in pregnancy induces the growth and diï¬erentiation of themammary epithelium into ultimately milksecreting acini Figure Mechanistically progesterone induces progesteronereceptor PRpositive mammary epithelial cells to expressRANKL resulting in the proliferation of neighboring RANKmammary epithelial progenitor cells in an autocrine and alsoparacrine fashion “ Moreover RANKL can induce theproliferation of RANKpositive ductal epithelial cells through theFIGURE RANKRANKL pathway in mammary gland physiology and breast cancer A RANK is constitutively expressed on the membrane of luminal and basalepithelial cells including mammary stem cells MaSCs Stimulation with progesterone induces RANKL expression and secretion in progesterone receptor PRpositiveluminal epithelial cells RANKL binds in an autocrine fashion to RANK on luminal epithelial cells which stimulates further RANKL expression and in a paracrine fashionto RANK on basal epithelial cells resulting in enhanced RANK expression on basal mammary epithelial cells and the activation of the IKKαNFκB“cyclin D1 signalingaxis to induce a variety of physiological responses necessary for mammary gland development B Heterozygous BRCA1 mutationcarrying women canspontaneously lose the remaining wildtype BRCA1 gene from somatic mutation or epigenetic silencing Subsequently loss of BRCA1 protein can result in increasedgenomic instability DNA damage and genetic mutations eg TP53 Progesterone as well as synthetic progestins upregulate RANKL expression in PR luminalbreast epithelial cells which stimulates RANKmediated cell proliferation of adjacent progenitor cells as discussed in A Altogether the genotoxic stress and amplifiedproliferation cues culminate in uncontrolled proliferation and the development of breast cancerFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerinduction of Rspondin Therefore RANK and RANKL linksex hormones to mammary progenitor cell proliferation duringthe estrous cycle and in pregnancy Figure Clinically an increase in serum progesterone and RANKLlevels is associated with an increase in breast cancer risk inpostmenopausal women Higher concentrations of solubleRANKL are positively correlated with an increased risk ofestrogen receptorpositive but not estrogen receptornegativebreast cancer indicating that the RANKRANKLOPG axis maybe involved in the tumorigenesis of ER breast cancer Indeed in a hormoneinduced spontaneous mouse breast cancermodel RANKL is critical for the development of sex hormonedriven breast cancer Deletion of RANK and Ikkα akey downstream regulator of the RANK signaling pathway inmammary epithelial cells also significantly delayed progestinMPA and DNAmutation DMBAinduced mammary tumorformation further indicating that the RANKRANKL pathwaydrives breast cancer Furthermore the selective inhibitionof RANKL by RANKFc not only attenuated breast tumorprogression in a hormone and carcinogendriven mouse breastcancer model but also decreased the progression of breast cancerin a transgenic spontaneous tumor model BRCA1 and BRCA2 mutations are the most prevalent geneticdrivers for hereditary breast cancer in humans Interestinglywomen with germline BRCA12 mutations usually exhibithigher progesterone and estrogen levels during the gestationalphase ofthe estrous cycle compared to women withoutthese mutations Inversely decreased serum OPG levelsare associated with increased breast cancer incidence Moreover high levels of RANK expression were observed inbreast cancer samples from premalignant lesions and patientswith BRCA1 mutations SNP data analysis from theCooperative Tumor GeneEnvironmental Research iCOGSincluding approximately BRCA1 and BRCA2mutation carriers identified SNPs which were significantlyassociated with breast cancer risk at the TNFRSF11A locusencoding RANK Altogether these human data stronglysupport the idea that the RANKLRANKOPG axis is intimatelyinvolved in the tumorigenesis of BRCA12 mutationdrivenbreast cancerSubsequent animal studies provided direct evidence thatRANKL and RANK are critically involved in the oncogenesis ofBRCA1 mutationdriven hereditary breast cancer Figure Genetically engineered mice carrying Brca1 and Tp53 mutationsshowed hyperproliferation and malignancy in their mammaryglands at months of age the inactivation of the RANKLRANKpathway in these mice largely prevented the occurrence ofmalignanttumors and resulted in significantly prolongedsurvival Additionally the pharmacological blockade of RANKLusing RANKFc completely abolished the development ofprecancerousin the Brca1Tp53 doublemutatedbreast cancer model Amplification of RANKexpressingmammary duct progenitor cells can be found in the nontumorbreast tissue of BRCA1 mutant carriers and these cells havesimilar molecular characteristics as basallike breast cancercells RANKL inhibition also significantly suppressedthe proliferation oftumor anoids derived from BRCA1mutant human breast biopsy specimens and RANKLRANKlesionspathway blockade strongly reduced tumorigenesis in patientderived xenograft PDX breasttumor mouse model Thus independent work among diï¬erent laboratories usingdiï¬erent mouse models as well as studies using humanbreast epithelial progenitor assays hasled to the sameconclusion RANKLRANK aï¬ect mammaryprogenitorcells and are critically involved in the BRCA1mutation drivenmammary tumorigenesisTherefore we and others have proposed that the monoclonalantibody Denosumab which specifically inhibits RANKRANKLinteractions could potentially be used for the prophylactictreatment of breast cancer in BRCA12 carriers Indeed weposit that healthy women with BRCA1 mutation will benefit notexcluding an eï¬ect on other TNBCs In a pilot clinical studytermed BRCAD the proliferation marker Ki67 was significantlydownregulated in the breast biopsy of BRCA1 mutation carrierswho received shortterm treatment with Denosumab suggestingthat RANKL inhibition may be a feasible method for the chemoprevention of breast cancer in women with BRCA1 mutationsThis study requires additional patient data which is currentlyongoing Another clinical study DBEYOND which aimedto investigate whether neoadjuvant RANKL inhibition therapycan reduce tumor proliferation in premenopausal early breastcancer patients found no significant change in Ki67positivetumor cells in the breast cancer tissues treated with Denosumabbut the density of tumorinfiltrating lymphocytes TILs wasincreased in the stroma and tumor tissues upon Denosumabtreatment In addition to the now experimentally well validated role ofRANKLRANKOPG in the sex hormone and BRCA1 mutationdriven mammary cancer tumorigenesis it has also been reportedthat this pathway can induce epithelialmesenchymal transitionEMT in breast cancer cells as well as in prostate andendometrial cancers “ suggesting that RANKLRANKsupports tumorigenesis in various epithelial cancers Moreoverour group has recently reported on the role of RANKL andRANK in lung cancer We demonstrated that the inactivation ofrank in lung epithelial cells disrupts mitochondrial bioenergeticsand significantly reduceslung cancer development bothculminating in increased survival This genetic modeling inthe mouse supports findings in human clinical trials in whichRANK inhibition with the monoclonal antibody Denosumabresulted in prolonged survival especially in patients withnonsmall celllung cancer NSCLC adenocarcinomas andsquamous tumors Notably this Denosumabdependent survivaladvantage occurred in lung cancer patients irrespective of visceralmetastasis hinting that the underlying eï¬ects of RANKLRANKblockade in addition to those targeting the bone are involved Epidemiological reports have also uncovered genderdiï¬erences particularly in lung cancer with respect to etiologyprogression and treatment response believed to be due tosexrelated hormonal factors “ though the underlyingmolecular mechanisms are poorly understood We have recentlyshown in our experimental lung cancer model that by ablatingthe sex hormones in female mice we could eï¬ectively eliminatethe survival advantages brought about by loss of rank in the lungtumors Furthermore synthetic progesterone MPAdependentenhanced lung cancer initiation required RANK expressionFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in CancerTogether these data suggest that the sex hormone regulation ofRANKLRANK could also explain the gender diï¬erences seen inhuman lung cancerRANKRANKL AS REGULATORS OFMETASTASISStudies have now shown that the RANKLRANKOPG axis playsa role in the progression of malignant tumors by promotingtumor cell migration stimulating tumor neovascularizationand promoting distant metastasis of tumor cells Disseminated tumor cells are responsible for the earlymetastasis of tumors which frequently can be detected inthe bone marrow of patients with malignant tumors Thisœmicrometastases niche forms a favorable microenvironmentfor the development of metastatic spread protecting cancer cellsfrom various antitumor treatments and modulating anticancerimmune responses thereby allowing the tumor cells to escapeimmune surveillance The tumor microenvironment is acomplex milieu composed of distinct factors such as cytokinesextracellular matrix components and various cell types suchas fibroblasts endothelial cells and immune cells all ofwhich participate in cancer development progression andmetastasis In bone tissue the tumor microenvironmentincludes immune and tumor cells as well as osteoblasts andosteoclasts all of which participate in a œvicious cycle thataccelerates osteolysis and cancer cell proliferation through inpart the RANKRANKLOPG axis For instance cancercells can increase the expression of RANKL in osteoclastsby secreting parathyroid hormonerelated peptide PTHrP Tumor cells can also directly express RANKLand secrete cytokines such as interleukin IL1α TNFα macrophage colonystimulating factor MCSF orprostaglandin E2 PGE2 all of which promote osteoclastdiï¬erentiation and survival resulting in local osteolysis whichsupports metastatic growth “ Subsequently growthfactors released by the bone matrix such as insulinlike growthfactors IGFs fibroblast growth factor FGFs plateletderivedgrowth factor PDGF or bone morphogenetic proteins BMPspromote cancer cell proliferation “ In addition tocytotoxic drugs and endocrine disruptive drugstherapiestargeting the RANKRANKLOPG axis exhibit direct andorindirect antitumor eï¬ects by blocking the vicious cycle betweenbone and cancer cells “In a murine model of melanoma metastasis it was foundthat for malignant tumors with RANK expression RANKLproduced by osteoblasts and bone marrow stromal cells couldact as a chemical attractant and promote the migration andmetastasis of malignant tumors to these sites Similareï¬ects were also found in malignant tumors such as breastcancer “ prostate cancer “ and lung cancer The activation of phospholipase C PLC proteinkinase C PKC ERK and phosphatidylinositol3OH kinasePI3K pathways were involved in RANKinduced tumor cellmigration “ RANK engagement by RANKL inducestrimerization of the RANK receptor which then stimulates therecruitment and activation of the adapter protein TRAF6 viaTRAF6binding sites in the Cterminus of RANK™s cytoplasmictail TRAF6 in turn complexes with many other downstreamadapters and kinases to activate the aforementioned pathwaysMoreoverthe RANKLRANK pathway was also shown topromote the formation of new blood vessels and regulate thetumor microenvironment at the primary tumor site to promotethe migration of tumor cells into the bloodstream and formetastasis to distant ans “In breast cancer RANKL is also produced by Foxp3expressing Tregs and tumorassociated macrophages TAMsthat can aï¬ect tumor growth tumor cell dissemination andmetastasis RANKL expression on tumorinfiltratingregulatory T cells may also be involved in cancer metastasis TAMs are either M1 or M2 macrophages with M1 being antitumor and M2 TAMs promoting tumorigenesis ImportantlyM2 macrophages express RANK and are attracted by RANKLproduced by the tumor microenvironment The RANKLRANKpathway in M2 macrophages can regulate the production ofchemokines and promote the proliferation of Treg lymphocyteswhich supports the immunosuppressive milieu within the tumormicroenvironment Recently it has been reported that estrogenrelatedreceptoralpha ERRα an important factor of cancer cell invasivenesspromotes breast cancer cell dissemination from primarymammary tumors to the bone Intriguingly RANK hasbeen shown to be a target for ERRα Furthermore the metaexpression analysis of breast cancer patients has uncovereda positive association between metastases and ERRαRANKexpression as well as a positive correlation between ERRαand BRCA1 mutation carriers revealing a novel pathwaywhereby ERRα in primary breast cancer could promote earlydissemination of cancer cells to bone Moreover it wasrecently shown that RANKL serum levels are significantlyincreased in breast cancer patients who developed bonemetastases p and patients within the highest quartileof RANKL had a significantly increased risk of developing bonemetastases compared to those in the lowest HR 95CI“ p This study further suggests a role ofRANKL in breast cancer metastasis TARGETING RANKLRANK IN HUMANCANCERIn light of the diï¬erent roles of the RANKLRANK pathwayin bone metabolism and immune system functions therapytargeting this axis may not only control primary tumordevelopment such as in the case of breast cancer and reducebone metastasis which has been demonstrated in clinical trials but also exert a direct antitumor eï¬ect via regulatinglocal tumorassociated immune responses as observed in studiesusing the monoclonal RANKL antibody inhibitor Denosumab In randomized clinical trials Denosumab has shown rapideï¬ectiveness by directly impairing osteoclast activity andinducing osteoclast apoptosis Moreover Denosumabwassignificantly more eï¬ective in reducing urinary Nterminal peptides a biochemical marker for bone turnoverFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerand more eï¬ective in delaying skeletalrelated events SREssuch as pathologic fractures spinal cord compression andhypercalcemia which greatly aï¬ect quality of life in patients withbreast cancer and castrationresistant prostate cancer CRPCbone metastases However the eï¬ect of Denosumab to delaySREs in patients with NSCLC and multiple myeloma MMpatients with bone metastases is comparable to bisphosphonatedrugs “ Moreover the benefit of Denosumab andbisphosphonates is not only restricted to osteolytic cancers suchas breast myeloma and NSCLC but also evident in osteoblasticcancers Recently it was demonstrated in osteoblastic cancerssuch as prostate cancer that Denosumab or bisphosphonate canaï¬ect the osteoclastosteoblast balance in the œvicious cycle ofbone destruction induced by metastasized cancer cells which highlights the potential rationale in treating osteoblasticcancer patients with Denosumab or bisphosphonatesIn a randomized phase III clinical trial comparing Denosumaband bisphosphonate zoledronic acid ZA in patients with solidtumors breast cancer prostate cancer multiple myeloma andbone metastases the results showed that Denosumab was similarto ZA in preventing or delaying the onset of primary SREs However in nonsmallcell lung carcinoma NSCLCn treatment with Denosumab showed a significantimprovement in overall survival In these patients nostatistically significant SRE delay was observed in Denosumabtreated patients suggesting that this survival advantage maybe independent of the bone system The result of arandomized phase III trial of multiple myeloma MM patientsn also demonstrated the eï¬ectivity of Denosumab toreduce the occurrence of primary SRE events moreover the useof Denosumab significantly improved progressionfree survivalPFS Whether this survival benefit is due to the decreasein the incidence of bone metastasis or whether Denosumab hasother antitumor eï¬ects requires further researchIn the randomized placebocontrolled phase III ABCSG18trial which enrolled postmenopausal female patients withearly hormone receptorpositive breast cancer the first clinicalfracture of the Denosumabtreated group was compared with theplacebo group and a significant protection of bone breaks wasdemonstrated hazard ratio [HR] · [ CI ·“·] p A median followup of months showeda significant improvement in the diseasefree survival DFS inthe Denosumabtreated group HR CI “Cox p These data suggest that adjuvant Denosumabcan significantly improve the DFS rate of HR postmenopausalbreast cancer patients However in another randomizedphase III clinical trial of breast cancer DCARE recent reportshave shown that adjuvant Denosumab does not reduce therisk of breast cancer recurrence or death in earlystage breastcancer patients receiving standard adjuvant therapy Theseinconsistencies which could be explained by diï¬erent cohortsfor patient stratifications eg more advanced early cases ofbreast cancer were included in the DCARE trials as comparedto the ABCSG18 study need to be further evaluated with largercohorts of patients and multiplecenter analysis Importantlya recent followup study of the ABCSG18 trial confirmed thethat blocking RANKL in an adjuvant breast cancer therapysetting not only markedly reduces the risk of breaking bones butalso significantly reduces the reoccurrence of the breast tumors It should be also noted that although there was nodiï¬erence in bonemetastasesfreesurvival in the DCARE trialDenosumab treatment significantly reduced the time to bonemetastasis at the site of first occurrence DENOSUMAB AS A NOVEL CANCERIMMUNOTHERAPYThe field of cancer immunotherapy has paved the way for a newparadigm to combat cancer by coaxing the body™s own immunesystem to seek out specifically target and destroy cancer cellsAmong the various approaches immunecheckpoint inhibitorsthattarget CTLA4 as

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

"fluoroscopicallyguided interventional FGI procedures by orthopedic surgeons has beenincreasing This study aimed to investigate the occupational radiation exposure among orthopedic surgeons inSouth KoreaMethods A nationwide survey of orthopedic surgeons was conducted in South Korea in October Thedosimetry data of the participants were obtained from the National Dosimetry Registry The orthopedic surgeonswere categorized by job specialty [spine or trauma specialists other orthopedic specialists and residents] anddescriptive statistics for the demographics and workrelated characteristics were presented Multivariable logisticregression analysis was used to evaluate the risk factors for the orthopedic surgeons who were not linked with thedosimetry dataResults Among the total participants n of the orthopedic surgeons spent more than of theirtime working with the FGI procedures when compared with their overall work The average frequency of the FGIprocedures among the orthopedic surgeons was days per month Less than of the participants wereregularly provided with radiation monitoring badges The proportion of subjects who always wore lead aprons andthyroid shields were and respectively The residents group experienced more unfavorable workingconditions of radiation exposure than the other specialists The dosimetry data were not significantly linked amongthe residents odds ratio [OR] confidence interval [CI] “ and orthopedic surgeons working atsmall hospitals OR CI “Conclusions Although orthopedic surgeons often performed FGI procedures they wore protective gear lessfrequently and a large proportion of orthopedic surgeons were not monitored by the national radiation dosimetrysystem As the number of radiation procedures performed by the orthopedic surgeons increases more intensiveapproaches are needed to reduce radiation exposure especially for spine and trauma surgeonsKeywords Fluoroscopy Health professional Occupational exposure Orthopedics Radiation Correspondence leewjkoreaackr2Department of Epidemiology and Health Informatics Graduate School ofPublic Health Korea University Seoul South Korea3Department of Preventive Medicine Korea University College of Medicine Goryeodaero Seongbukgu Seoul South KoreaFull 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 0cKang Journal of Occupational Medicine and Toxicology Page of BackgroundWith the markedly increased use offluoroscopicallyguided interventional FGI procedures during surgerythe risk of exposure to the ionizing radiation has increased for the orthopedic surgeons [ ] Fluoroscopicprocedures have been beneficial for patients undergoingorthopedic surgery because these minimally invasiveprocedures provide better direct visualization for soft tissue dissection spare blood supply and result in fewercomplications than surgeries [ ] However during the fluoroscopic procedures the surgeons may beexposed to the primary beam and scattered radiation [] As the use of fluoroscopy is continuously increasingin orthopedic surgery and orthopedic surgeons are oftenclosely exposed to the radiation source during operations they are considered a highrisk group of occupational radiation exposure [“]A few epidemiologic studies have reported an increased risk of cancer from radiation exposure amongorthopedic surgeons worldwide [“] Diverse harmful effects such as cancer cataracts chromosomal abnormalities and other chronic diseases have also beenreported in physicians performing FGI procedures globally [] In addition orthopedic surgeons have concerns about the hazards of radiation but they have beenshown to demonstrate lower rates of wearing personalprotective equipment and dosimeters [ ] Howeverprevious studies on orthopedic surgeons were mainlylimited by the small sample sizes and limited information on the FGI procedurerelated work practices andoccupational radiation exposureIdentifying the occupational characteristics and radiation exposure could provide scientific evidence andserve as a fundamental step in developing strategies toprotect against occupational radiation exposure Therefore this study aimed to investigate the work practicesrelated to radiation exposure among orthopedic surgeons in South Koreasurgeonsatamong orthopedicMethodsStudy populationWe conducted a field survey using a selfadministeredquestionnairetheconference of the Korean Orthopedic Association inOctober The association includes all memberorthopedic surgeons and the conference is the representative meeting for orthopedic surgeons in SouthKorea A total of orthopedic surgeons participated in this study Written informed consent wasvoluntarily obtained from all study participants priorto enrollment The protocols ofthe study werereviewed and approved by the Institutional ReviewBoard of our university KUIRB1736A2Questionnaire and dosimetryA detailed questionnaire was developed from a previousstudy for interventional medical radiation workers []The questionnaire included demographics date of birthgender workplace address work history job title specialty years since beginning worktotal duration ofwork work practices proportion of interventional procedures performed for the recent year working days permonth working hours per week name of the main procedure performed badge wearing wearing protectiveequipment and concern for developing radiationassociated diseases 5point Likert scale The questionnaire is provided as a supplementary material Supplementary Table The Korea Center for Disease Control and PreventionKCDC has been monitoring the hospital personnel exposed to radiation since it maintains a centralizedNational Dosimetry Registry NDR and implements alifelong followup management system for radiation doseusing a personal thermoluminescent dosimeter TLD[] The NDR has collected dose measurements quarterly by five personnel monitoring centers designated bythe KCDC for all diagnostic radiation workers Thestandard protocol of wearing the NDR badge for alldiagnostic radiation workers is wearing one TLD badgebeneath the apron on the left side of the chest To evaluate the individual radiation dose the survey data werelinked with the NDR up to the second quarter of with respect to the participant™s name gender date ofbirth and workplace address This effective dose measured in Sievert was derived from the personal doseequivalent at a depth of mm Hp [] The lowestdetectable quarterly level of the NDR is mSv Incases where the dose was below the minimum detectablelevel the dose was considered as half of the detectablelevel owing to the highly skewed distribution []Data analysisStudy participants were classified by job specialty asspine or trauma specialists ST other orthopedic specialists Others and residents based on previous studiesin which spine or trauma surgeons were reported to beexposed to radiation more than the other orthopedicsurgeons [ ] Descriptive statistics for the demographics and workrelated characteristics are presented Thelevel of concern for developing radiationassociated diseases was recategorized as low very unlikely and unlikely medium and high likely and very likely Usingthe chisquare analysis the three job specialty groupswere compared according to the demographics occupational characteristics such as involvement with fluoroscopy and wearing the badges and protective devicesMultivariable logistic regression analysis was used toevaluate the risk factors for the orthopedic surgeons 0cKang Journal of Occupational Medicine and Toxicology Page of linked with the dosimetry data afterwho were notadjusting for agelocation of medical facility and jobspecialty Among the orthopedic physicians who werelinked with the TLD data the individual quarterly badgedoses recorded during the study period were summedand divided by the number of years to obtain the annualeffective doses this was compared according to the jobspecialty using oneway analysis of variance ANOVASTATA version StataCorp College Station TXUSA was used for statistical analysis and pvalues were considered statistically significantResultsA total of ST Others and residents participated in this study Table Most orthopedic surgeonswere aged years and worked at general hospitalsmany were young residents who were in training Therates of high concern for occupational radiation riskwere higher among the ST than the other groups Ourstudy participants comprised of all the members ofthe Korean Orthopedic Association but the residents inour study accounted for of all the members of theassociation Supplementary Table Approximately of the orthopedic surgeons spentmore than of their time working with the FGI procedures when compared with their overall work Table The average work duration of performing FGI procedures was years and the average frequency of interventions was every days per month The specialistsworked with fluoroscopy for longer periods than the residents however within the same period ie workloadper week or month the residents group performedfluoroscopy procedures more frequently than the specialists The proportion of subjects who always woreprotective gearsleadglasses and gloves ranged from to and the residents wore the protective gears less frequently than theother specialiststhyroid shieldslead apronsAmong the orthopedic surgeons who respondedto the survey only were linked with theTLD data Table The odds ratios OR of not beinglinked with the dosimetry data was significantly increased among the surgeons working at small hospitalsOR confidence interval [CI] “ andresidents group OR CI “ afteradjusting for potential confounding factors Among theorthopedic doctors who were linked with the dosimetrydata the annual effective dose was higher in the ST mSv than in Others mSv or residents mSv Supplementary Table DiscussionThe orthopedic surgeons in this study often performedfluoroscopy during the course of their work howeverthe rate of wearing the dosimetry badges and protectivedevices has been shown to be low In addition most participants in this study were not linked to the nationaldosimetry data The residents group experienced moreTable Characteristics of the orthopedic surgeons according to the job specialty in South KoreaCharacteristicsTotaln Na SpecialistsSTn N Age group year “‰¥ Type of the medical facilityGeneral hospitalSmall hospitalLocation of the medical facilityMetropolitanProvince Level of concern for occupational radiation exposureLowMediumHigh ST spine or trauma specialists Others other orthopedic specialistsaNumbers may not reflect the total owing to missing valuesbpvalue for the chisquare test or Fisher™s exact test Othersn N Residentsn N pvalueb 0cKang Journal of Occupational Medicine and Toxicology Page of Table Occupational characteristics of the orthopedic surgeons according to the job specialty in South KoreaResidentsOccupationaln characteristicsN Totaln Na SpecialistsSTn N Others n N Fluoroscopy workCalendar year began working with fluoroscopy ““““‰¥ Years working with fluoroscopy “‰¥ Proportion time spent working with fluoroscopy“““ Working days per month with fluoroscopy “‰¥ Number of fluoroscopic procedures per week “‰¥ Working hours per week with fluoroscopy “‰¥ Badge wearingProvided with the badges regularlyNoYes Proportion of personnel wearing the badges“““ Personal protective equipment useProportion of personnel wearing the lead aprons pvalueb 0cKang Journal of Occupational Medicine and Toxicology Page of Table Occupational characteristics of the orthopedic surgeons according to the job specialty in South Korea ContinuedOccupationalcharacteristicsTotaln Na “““ Proportion of personnel wearing the thyroid shields“““ Proportion of personnel wearing the lead glasses“““ Proportion of personnel wearing the lead gloves“““ SpecialistsSTn N Others n N Residentsn N pvalueb ST spine or trauma specialists Others other orthopedic specialistsaNumbers may not reflect the total owing to missing valuesbpvalue for the chisquare test or Fisher™s exact testunfavorable working conditions in terms of radiation exposure than the other specialists Among the orthopedicsurgeons who were linked with the dosimetry data theST had higher radiation doses than the other orthopedicsurgeons To the best of our knowledge this study is thefirst attempt to investigate the status of occupational radiation exposure among the orthopedic surgeons inSouth Korea Our findings may contribute to the increasing awareness of the radiation protection and itspotential risks among hospital workersThe rates of use of fluoroscopy and protective devicesin our study were comparable with those in a worldwidestudy on orthopedic surgeons that reported that morethan half of the procedures performed by of thesurgeons involved radiation exposure whereas the ratesof using lead aprons thyroid shields and lead glasseswere and respectively [] According to aUS survey of the subjects reported that lead apronswere not available and the remaining half reported thatthey were not appropriately sized [] The other studyreported that one out of three orthopedic surgery residents were not provided protective gowns in the US[] These rates of personal protective equipment usewere lower among the orthopedic surgeons than amongthe interventional cardiologists particularly the rates ofwearing the lead aprons thyroid protectors and leadglasses were and respectively [] Thepossible reasons for low rates of apron use was not beingproperly provided with aprons and inconvenience whilewearing aprons among the Korean Intern and ResidentsAssociation []The rate of always wearing a badge was generally comparable with those in a worldwide survey that reportedthat about onefifth of the orthopedic surgeons wear adosimeter [] The Irish orthopedic surgeons also reported that the regular use of dosimeters among theorthopedic trainees was [] and only of theorthopedic surgeons were reported to wear the TLD 0cKang Journal of Occupational Medicine and Toxicology Page of Table Odds ratios of not being linked with dosimetry data among the orthopedic surgeons in South KoreaCharacteristics CIORNot linked with the dosimetry n Na Linked with the dosimetry n N Age group year “‰¥ Type of medical facilityGeneral hospital Small hospital Location of medical facilityMetropolitanProvince Level of concernLowMediumHighJob specialtySTOthersResidents Calendar year began working with fluoroscopy ““““‰¥ Years working with fluoroscopy “‰¥ Proportion time spent working with fluoroscopy “““ Proportion of personnel wearing the badges“““ “referencereferencereferencereferencereferencereferencereferencereferencereferenceOR odds ratio AOR adjusted odds ratio ST spine or trauma specialists Others other orthopedic specialists CI confidence intervalsaNumbers may not reflect the total owing to missing valuesbAdjusted for age location of medical facility and job specialtyORb CI“referencereferencereferencereferencereferencereferencereferencereferencereference 0cKang Journal of Occupational Medicine and Toxicology Page of during surgery in Turkey [] A possible reason for theorthopedic surgeons not preferring to wear the dosimeters is that they may believe that it will affect their performance and make them uncomfortable [ ]Another reason may be that if their radiation exposurewas greater than the specified limit they would be prohibited from operating with fluoroscopy for a specifiedperiod of time therefore they do not routinely wear thepersonal dosimetry badges []Approximately threefourth of our participants werenot linked with the national dosimetry data and the riskof not being linked was increased among the residentsgroup and those who worked at smaller medical facilities A possible reason may be owing to the incompleteness by which the radiation safety managers at eachmedical facility select the radiation exposed orthopedicsurgeons In members of the Korean Internand Resident Association responded that of themhad been exposed to fluoroscopy however only ofthem wore the TLD when exposed to radiation duringfluoroscopy [] These results suggest that the monitoring system for the orthopedic surgeons with radiationexposure appears to be unsuccessful”in particular forresidents and for small hospital workers”and additionalefforts to improve dosimetry monitoring system are required in South KoreaThe doses monitored in the dosimetry data of theorthopedic surgeons in this study were lower than thosereported in Italy [] The Philippines [] India []and South Korea [ ] This finding supports the notion that the radiation exposure among the orthopedicsurgeons may be widely ranged depending on the workprocedures and experience [] The higher radiationdose among ST in this study may be attributed to longerexposure time and more fluoroscopic shots in ST [“]than those of other surgeons However the actual exposuremight be underestimated because of the orthopedic surgeons™ low rate of wearing the badges and protective devices Owing to the irregular and inconsistent use ofdosimeters estimating the radiation dose with personal dosimeters needs to be improved among orthopedic surgeonsMore than onethird of the orthopedic surgeons weregreatly concerned about health problems caused by occupational radiation exposure which is similar to thatreported in a previous worldwide study on orthopedicsurgeons [] Among all the participants the ST surgeons showed a higher rate of concern than the othergroups and this may be related to them performing ahigher proportion of the FGI procedures than other doctors Previously a lack of radiation knowledge andawareness about fluoroscopy were also associated with ahigh level of concern and low rate of wearing the protective devices [ ] therefore education about radiation exposure is warranted for orthopedic surgeonsThis study is the first attempt to investigate the statusof occupational radiation exposure in a relatively largenumber of orthopedic surgeons in South Korea However the study did not represent all members of the Korean Orthopedic Association although the residentsgroup may represent the total population of the orthopedic residents The high proportion of residents in thissurvey may be owing to their high participation in theconference and having greater interests in radiation exposure because of their harsh working conditions Ourfindings represent mainly male orthopedic surgeonsowing to a very small number of female participants Inaddition this study recruited few orthopedic surgeonswho worked at clinics which limited our findings mainlyto orthopedic surgeons at large hosptials Recall biasmay be present because of using a selfadministeredquestionnaire However the participants were a relatively young and highly educated group and the questionnaire items were related to their daily worktherefore the bias should be minimal The informationon selfreported working practices regarding radiationexposure has been reported as reliable among South Korean radiologic technologists []ConclusionsWe reported the occupational characteristics and radiation exposure among orthopedic surgeons in SouthKorea Although many orthopedic surgeons performinterventional FGI procedures unfavorable work characteristics”such as the low rate of wearing protective devices and dosimeters”may increase the radiation riskAdditionally badge monitoring was not noted for therelatively large proportion of the orthopedic surgeonswho performed FGI procedures The orthopedic surgeons are at a risk of occupational radiation exposureand more intensive approaches are needed to reduce radiation exposure and protect possible workrelatedhealth effectsSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1299502000276xAdditional file Supplementary Table Questionnaire used fororthopedic surgeons at the conference of the Korean OrthopedicAssociation in Additional file Supplementary Table Comparison of selectedcharacteristics between the target population and the study participantsAdditional file Supplementary Table Annual effective doses byoccupational characteristics and specialties among orthopedic surgeonsin South KoreaAbbreviationsNDR National Dosimetry Registry TLD Thermoluminescent dosimeterKCDC Korea Centers for Disease Control and Prevention ST Spine or trauma 0cKang Journal of Occupational Medicine and Toxicology Page of specialists Others Other orthopedic specialists OR Odds ratioCI Confidence intervalsAcknowledgmentsWe would like to thank all study participants for their voluntary participationthe Korea Centers for Disease Control and Prevention for providingdosimetry data and the Korean Orthopedic Association for providingdemographic data of their membersAuthors™ contributionsSK and WJL conceptualized the research SK performed the data analysesand wrote the first draft of the manuscript ESC YJB TWN DL and SYSinterpreted the findings and provided advice on the data analyses WJL andSK revised the final manuscript All authors contributed to the draft revisionand approved the final manuscriptFundingThis work was supported by the National Research Foundation of KoreaNRF grant funded by the Korea government MSIT No2020R1A2C1008891Availability of data and materialsThe datasets generated during the current study are not publicly availablebecause the detailed individual data is restricted for both legal and ethicalconcerns but are available from the corresponding author on reasonablerequestEthics approval and consent to participateThe protocols of the study were reviewed and approved by the InstitutionalReview Board of Korea University KUIRB1736A2 Written informedconsent was voluntarily obtained from all study participants prior toenrollmentConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Occupational and Environmental Medicine COMWEL AnsanHospital Korea Workers™ Compensation Welfare Service GyeonggidoSouth Korea 2Department of Epidemiology and Health Informatics GraduateSchool of Public Health Korea University Seoul South Korea 3Department ofPreventive Medicine Korea University College of Medicine GoryeodaeroSeongbukgu Seoul South Korea 4Department of Policy andAdministration National Radiation Emergency Medical Center Korea Instituteof Radiological and Medical Sciences Seoul South Korea 5Department ofRadiation Effects Research National Radiation Emergency Medical CenterKorea Institute of Radiological and Medical Sciences Seoul South Korea6Department of Orthopedic Surgery Gyeongsang National UniversityHospital Jinju South KoreaReceived February Accepted August Gausden EB Christ AB Zeldin R Lane JM McCarthy MM Trackingcumulative radiation exposure in orthopaedic surgeons and residents whatdose are we getting J Bone Joint Surg Am “Jung GH Jang JH Kim JD Kim CK Radiation exposure over the course ofa year from an image intensifier in the orthopaedic operating room JKorean Fract Soc “Oddy M Aldam C Ionising radiation exposure to orthopaedic trainees theeffect of subspecialty training Ann R Coll Surg Engl “Rampersaud YR Foley KT Shen AC Williams S Solomito M Radiationexposure to the spine surgeon during fluoroscopically assisted pediclescrew insertion Spine “Tunçer N Kuyucu E Sayar Ş Polat G Erdil Ä° Tuncay Ä° Orthopedic surgeons™knowledge regarding risk of radiation exposition a survey analysis SICOTJ Chou LB Chandran S Harris AH Tung J Butler LM Increased breast cancerprevalence among female orthopedic surgeons J Women's Health “ Chou LB Lerner LB Harris AH Brandon AJ Girod S Butler LM Cancerprevalence among a crosssectional survey of female orthopedic urologyand plastic surgeons in the United States Womens Health Issues “ Mastrangelo G Fedeli U Fadda E Giovanazzi A Scoizzato L Saia BIncreased cancer risk among surgeons in an orthopaedic hospital OccupMed “Ko S Kang S Ha M Kim J Jun JK Kong KA Health effects fromoccupational radiation exposure among fluoroscopyguided interventionalmedical workers a systematic review J Vasc Interv Radiol “Fan G Wang Y Guo C Lei X He S Knowledge deficiency of workrelatedradiation hazards associated with psychological distress among orthopedicsurgeons a crosssectional study Medicine 20179621e6682Joeris A Goldhahn S Kalampoki V Gebhard F Intraoperative radiationexposure of orthopaedic surgeons“mismatch between concerns andprotection Occup Med Health Aff Ko S Chung HH Cho SB Jin YW Kim KP Ha M Occupational radiationexposure and its health effects on interventional medical workers studyprotocol for a prospective cohort study BMJ 2017712e018333Korea Centers for Disease Control Prevention KCDC ReportOccupational Radiation Exposure in Diagnostic Radiology in Korea p httpwwwcdcgokr Accessed Feb Hornung RW Reed LD Estimation of average concentration in the presenceof nondetectable values Appl Occup Environ Hyg “ Hassan M Patil A Channel J Khan F Knight J Loos M Do we glowEvaluation of trauma team work habits and radiation exposure J TraumaAcute Care Surg “ Bowman JR Razi A Watson SL Pearson JM Hudson PW Patt JC Whatleads to lead results of a nationwide survey exploring attitudes andpractices of orthopaedic surgery residents regarding radiation safety J BoneJoint Surg Am 20181003e16Jacob S Boveda S Bar O Brézin A Maccia C Laurier D Interventionalcardiologists and risk of radiationinduced cataract results of a Frenchmulticenter observational study Int J Cardiol “The Korean Intern and Resident Association Summary of ResidentsRadiation Exposure Experience Survey httpyoungmd Accessed Feb ReferencesRehani M CirajBjelac O Vañó E Miller D Walsh S Giordano B et alRadiological protection in fluoroscopically guided procedures performedoutside the imaging department Ann ICRP “Singer G Occupational radiation exposure to the surgeon J Am AcadOrthop Surg “ Matityahu A Duffy RK Goldhahn S Joeris A Richter PH Gebhard F Thegreat unknown”a systematic literature review about risk associated withintraoperative imaging during orthopaedic surgeries Injury “ Miller DL Overview of contemporary interventional fluoroscopy proceduresHealth Phys “Richter P Dehner C Scheiderer B Gebhard F Kraus M Emission of radiationin the orthopaedic operation room a comprehensive review OAMusculoskeletal Med Nugent M Carmody O Dudeney S Radiation safety knowledge andpractices among Irish orthopaedic trainees Ir J Med Sci “Kesavachandran CN Haamann F Nienhaus A Radiation exposure of eyesthyroid gland and hands in orthopaedic staff a systematic review Eur JMed Res Klein LW Tra Y Garratt KN Powell W LopezCruz G Chambers C et alOccupational health hazards of interventional cardiologists in the currentdecade results of the SCAI membership survey Catheter CardiovascInterv “ Rosario M Garduce V Geronilla M Radiation exposure of Filipino orthopedicresidents in tertiary level hospitals Bone Rep Recommendations Mahajan A Samuel S Saran AK Mahajan MK Mam MK Occupationalradiation exposure from C arm fluoroscopy during common orthopaedicsurgical procedures and its prevention J Clin Diagn Res 201593RC01“ Bae SY Kim JO Yoo JD Yoon SY Jang JW Radiation exposure fromfluoroscopy during orthopaedic surgical procedures J Korean Frac Soc“ 0cKang Journal of Occupational Medicine and Toxicology Page of Kim JW Kim JJ Radiation exposure to the orthopaedic surgeon duringfracture surgery J Korean Fract Soc “Kim MJ Cha ES Ko Y Chun BC Lee WJ Reliability of selfreportedquestionnaire on occupational radiation practices among diagnosticradiologic technologists Am J Ind Med “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

"evaluate the clinicopathologic characteristics of Lymph Node metastasisbetween investing layer of Cervical fascia and deep fascia of infrahyoid strap Muscles LNCM in papillary thyroidcarcinoma PTCMethods Retrospective review of patients with PTC who underwent thyroidectomy and central compartment neckdissection CND from January to January was performed in two tertiary referral academic medicalcenters A total of consecutive patients with PTC who underwent thyroidectomy and CND were included inthe retrospective review The LNCM was resected as a separate specimen by the surgeon and the clinicopathologiccharacteristics of the patients were recorded Multivariate logistic regression analysis was performed to identify riskfactors for LNCM metastasisResults Of PTC patients patients had lymph nodes in the LNCM Among them caseswere confirmed to be positive in the LNCM In total the metastasis rate of LNCM in PTC patients was Univariate analysis revealed that the metastasis of LNCM were more likely to have a primary site in theinferior pole extrathyroidal extension ETE central cervical metastasis level III and level IV metastasis Multivariateanalysis further showed tumor location in the inferior pole ETE level III and level IV metastasis conferred asignificantly increased odds ratio for LNCM metastasisConclusion Attention should be paid to the lymph tissue in the LNCM for PTC patients especially in presence of aprimary site in the inferior pole ETE level III and level IV metastasisKeywords Thyroid carcinoma Surgery Central compartment neck dissection Recurrence Suprasternal spaceIntroductionPatients with papillary thyroid carcinoma PTC have afavorable prognosis with central neck locoregional recurrence varying from to [] The goal of a prophylacticor therapeutic central compartment neck dissection pCNDor tCND is to decrease the incidence of local recurrenceby removing all lymphatic tissue within the levels VI and Correspondence wugaosongwhueducn1Department of Thyroid and Breast Surgery Zhongnan Hospital of WuhanUniversity Donghu Road Wuhan Hubei People™s Republic of ChinaFull list of author information is available at the end of the VII compartments which are generally the first and themost commonly involved with metastasis [] For patientswithout evidence of lymph node metastasis on preoperativeevaluation the additive value of a pCND at the time ofthyroidectomy is controversial Some authors advocatepCND considering high rate “ of occult metastaticnodal disease in cN0 PTC [] while other authors considerthat there is no highlevel evidence in favor of pCND []The performance of pCND is dependent on the weightgiven to the risks and benefits of pCND [] Consideringthe oncologic benefits of CND and the risks of a repeat 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 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of neck operation performing pCND is recommended toevery patient in China [ ]Although American Thyroid Association ATA guideline has defined the boundary of central neck compartment there is also significant variability in terms of theextent of CND In routine clinical practice CND canrange from sampling a few nodes in the paratrachealregion to a complete clearance from left carotid artery toright carotid artery and down to and including the uppermediastinum [] Owing to the variant extent of CNDsome central compartments are easily to be neglectedFor thyroid carcinoma patients with specific clinicopathologic characteristics incomplete lymph node dissectionmay result in increased recurrence reoperation andreoperationassociated complications [] Lymph Nodebetween investing layer of Cervical fascia and deep fasciainfrahyoid strap Muscles LNCM has not beenofreported The LNCM compartment is defined as followssuperiorly by the hyoid bonelaterally by the carotidarteries anteriorly by the investing layer of cervicalfascia and posteriorly by the deep fascia of infrahyoidstrap muscles LNCM space includes suprasternal spaceand intrainfrahyoid strap muscle spaceAnatomically LNCM is located anterior to the strapmuscles We consider that what is special about the concept of the LNCM is that it is belong to level VI but is aneasily overlooked anatomical area by a strap musculatureinvolving the sternohyoid and sternothyroid musclesduring selective or modified neck dissection Although themetastasis in LNCM was seldom it did occur in somePTC patients with regional recurrence As part of LCNMsuprasternal space metastasis for thyroid cancer wereinvestigated in three studies [“] Thus we routinelydetected the suprasternal space and intrainfrahyoid strapmuscle space Fig This study was performed to identify the clinicopathologic characteristics and indication forlymph node metastasis dissection in the LNCMMaterials and methodsPatientsA retrospective review from the clinical and histopathologydatabase of two tertiary referral academic medical centersTongji Hospital of Huazhong University of Science andTechnology and Zhongnan Hospital of Wuhan Universityfrom January to January were conducted In theinstitutions preoperative examinations consisted of athorough physical examination neck ultrasound a clinicalevaluation of thyroid nodules and neck lymph nodes Fineneedle aspiration cytology FNAC were performed in patients who were suspected thyroid nodules or lymph nodeWith a pathological confirmation of PTC all the patientsreceived a thyroidectomy with CND Accordingly a pCNDwas performed for cN0 patients and a therapeutic CNDwas performed for cN1 patients The inclusion criteria forthe patients were as follows the clinical history completely recorded the LNCM was resected as a separatespecimen by the surgeon PTC patients who underwentthyroidectomy plus CND with or without lateral neckdissection A total of consecutive PTC patients wereenrolled The medical ethic committee of ZhongnanHospital of Wuhan University approved the procedure andinformed written consent was obtained from all patientsSurgical approachAll the operations were performed by the same seniorsurgeon Gaosong Wu with the patients under generalFig Four subdivisions Level VI 1st Level VI 2nd Level VI 3rd and Level VI 4th of central neck compartment are divided by deep fascia ofinfrahyoid muscles pretracheal visceral fascial and right recurrent laryngeal nerve 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of anesthesia Thyroidectomy was performed with a standard technique of fine capsular en bloc dissection andresection from inferior pole to superior pole [“]Intraoperative neuromonitoring was employed for all ofthe thyroidectomies [] Superior parathyroid glandswere identified and preserved in situ inferior parathyroidglands were protected in situ or autotransplanted in thesternocleidomastoid muscle according to three certaintypes based on their blood supply and location [ ]After the incision of the investing layer of cervicalfascia the interval between sternohyoid and sternothyroid muscles and the space anterior to the sternohyoidmuscle above the clavicle and the sternum weredetected If there was fibrofatty tissue Instead of the enbloc removal of the entire centrallymph nodes theLNCM was resected as a separate specimen if occurredThe presence or absence of lymph node metastasis wasdefined according to postoperative pathological reportsWhile dissecting paratracheal lymph nodes intraoperative neuromonitoring was employed to detect RLN fromdistally to proximally minimizing morbidity from injuryto RLN during compartment nodal dissection LNCMand other compartment lymphatic tissue were processedfor routine hematoxylin and eosine HE separatelyThe pathologic results were independently determinedby two qualified pathologists without any prior knowledge of the patients™ clinical dataData collection and statistics analysisTo determine the relation between LNCM metastasis andclinicopathologic factors such as age sex primary tumorsite lateral cervical lymph node metastasis level VI metastasis the chisquare test and Fisher™s exact test were usedas appropriate Multivariate logistic regression analysiswas performed to identify risk factors for LNCM metastasis of PTC P was considered statistically significantAll calculations were performed using SPSS for windows Postthyroidectomy hypocalcemia lasting for morethan months was considered as permanent VCP All patients were followed up every “ months postoperativelyResultsPatients detected with LNCMAfter reviewing patients who underwent thyroidectomy plus CND with or without lateral neck dissectionfrom January to January patients were detected with LNCM and patients wereabsent of LNCM The average tumor size of LNCM was cm and the mean number of lymph nodes sampledfrom LNCM was ranging from to Table showsthe comparison of clinicopathologic characteristics between the present LNCM group and the absent groupIn univariate analysis Hashimoto™s disease p multifocality leisions p the tumor located ininferior portion p extrathyroidal extensionETE p central cervical metastasis p level III and level IV metastasis p were significantly associated with high prevalence of LNCMPatients with metastatic LNCMAmong a total of patients with LNCM metastaticLNCM was found in patients Table compares the clinicopathologic characteristics between themetastatic LNCM group and the nonmetastatic LNCMgroup Three hundred eightythree patients were confirmed free of LNCM metastasis of themwith clinically negative node performed pCND and of them with clinically positive performed tCND All thepatients in the metastatic LNCM group performedtCND Lateral neck dissection was performed in cases in the metastatic LNCM group and cases in the nonmetastatic group all lateral neckdissection wastherapeutically performed Univariateanalysis was performed for the patients with and patients without metastatic LNCM Age at diagnosisgender and tumor size coexistent thyroid disease tumorfocality and level II metastasis were not correlated withLNCM metastasis Univariate analysis identified tumorlocated in the inferior pole central cervical metastasisETE level III and level IV metastasis as significant predictors of LNCM metastasis in our study population Multivariate analysis further showed that tumor location ETElevel III and level IV metastasis conferred a significantlyincreased odds ratio for LNCM metastasis Table ComplicationsThe median followup time was months range “ of patients had voice changes all of whomrecovered within “ months Temporary vocal cord paralysis was confirmed in patients by laryngoscope andthirteen permanent hypocalcemia was observed aftersurgeryDiscussionIn order to achieve the best chance of cure and effectivedisease control thoroughness of dissection has to betaken into account We prospectively performed comprehensive CND for PTC patients who underwent thyroidectomy and CND In addition data were analyzed for PTC patients to investigate the clinicopathologic characteristics for LNCM metastasis The occurrence rate ofLNCM was and of the patients harbored metastatic LNCM In total the positiverate of the LNCM was In this studymultivariate analysis revealed that a primary site in the inferior pole ETE level III and level IV metastasis were ofhigher LNCM metastasis rate which was consistent withthe findings by the previous report oflymph node 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Table Univariate analysis of demographic and clinicopathologic factors for patients who had lymph nodes in LNCM compared tothose who did notVariablesAge mean ± SDAbsent n Present n P value‰¤ GenderFemale MaleTumor size cm ‰¥ cmCoexistent thyroidNodular goiterYes NoToxic goiterYes NoHashimoto™s diseaseYes NoTumor focalityMultifocality UnifocalityTumor locationInferior portionUpperMiddle portionExtrathyroidal extensionYes NoCentral cervical metastasisYes NoLateral cervical metastasisLevel IIYes NoLevel IIIYes NoLevel IVYes No LNCM Lymph Node between superficial layer of deep Cervical fascia and deep fascia of infrahyoid Musclesmetastasis between sternocleidomastoid and sternohyoid muscle []Several studies have emphasized the importance of similar compartment in neck dissection for thyroid carcinomaSun pioneered the confirmation of the significantinvolvement of lymph node metastasis between sternocleidomastoid and sternohyoid muscle LNSS in lateralneck dissection [] which anatomically classified as part ofthe space of Burns They concluded that the positive rate ofLNSS was in clinically nodepositive cN PTCwhich was correlated with a primary site in the inferiorpole the lateral nodal metastasis level III and level IV nodalmetastasis [] Then Homma [] reported two casesof PTC patients with level III and IV lymph node metastases as well as metastasis in the suprasternal space Yu et al[] investigated the clinical significance of the suprasternalspace lymph node SSLN in pathological nodepositivepN PTC patients They concluded that metastasis rate ofSSLN was and the high SSLN metastasis of PTC wascorrelated with primary cancer site in the inferior thyroidpole strap muscle invasion level IV metastasis and LNSSmetastasis In our experience LNCM was rarely occurredin the central neck compartment and the positiveLNCM in PTC patients was infrequent as well Notably among the patients with pN PTC themetastasis rate of LNCM was which was muchlower than the metastasis incidence of SSLN described by Yu [] According to their results onefifth patients with pN PTC were performed incompleteCND and remained metastatic lymph nodes 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Table Univariate analysis of demographic and clinicopathologic factors for positive LNCMVariablesAge mean ± SDMetastasis n Nonmetastasis n ‰¤ GenderFemale MaleTumor size cm ‰¥ cmCoexistent thyroidNodular goiterYes NoToxic goiterYes NoHashimoto™s diseaseYes NoTumor focalityMultifocality UnifocalityTumor locationInferior portionUpperMiddle portionExtrathyroidal extensionYes NoCentral cervical metastasisYes NoLateral cervical metastasisLevel IIYes NoLevel IIIYes NoLevel IVYes NoLNCM Lymph Node between superficial layer of deep Cervical fascia and deep fascia of infrahyoid MusclesP value The total number of lymph nodes in the central neckcan range from to [] There is no consensus on thenumber of nodes removed or examined that would constitute an adequate dissection Aimed to allow surgeons tomore accurately report the extent of lymphadenectomyTable Multivariate analysis of predictors for LNCM positivityVariablesTumor locationp valueCentral cervical metastasisExtrathyroidal extensionLevel III metastasisLevel IV metastasisOR CI “ “ “ “ “LNCM Lymph Node between superficial layer of deep Cervical fascia and deepfascia of infrahyoid Musclesvisceralwe divide the central neck compartment into four subdivisions by deep fascia of infrahyoid strap muscles pretrachealfascial and right RLN Fig Theproposed LNCM compartment is bounded superiorly bythe hyoid bone laterally by the carotid arteries anteriorlyby the investing layer of the cervical fascia and posteriorlyby the deep fascia of infrahyoid strap muscles which is defined as Level VI 1st In the current study suprasternalspace composed part of the LNCM Fig Compared toSSLN reported by Yu [] LNCM encompasseslymph nodes in the suprasternal space and lymph nodesbetween sternohyoid and sternothyroid muscles Figs and LNCM can fall under the normal subdivisions ofthe central compartment Subdivisions can actually recordthe extent of the CND which is able to provide detailedinformation for the possible second operation 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Fig LNCM coverage area in vivo Lymph nodes between sternohyoid and sternothyroid muscles a and lymph nodes in the suprasternal space bIncluding LNCM as an anatomical part of the centralneck allows for removal of previously unrecognized micrometastatic disease in of PTC patients with the inferiorportion lesions ETElevel III and level IV metastasisDissection of the LNCM space is less invasive and easy toachieve and is not timeconsuming It is at the entrance ofcentral neck compartment which is easy to expose and haslow risk of damaging RLN or parathyroid With the application ofintraoperative neuromonitoring and in situpreservation or autotransplantation of parathyroid theoccurrence of vocal cord paralysis and permanenthypoparathyroidism in the current study were lowerin this study [ ] Therefore in cases where LNCMspace metastasis is suspected or preoperative ultrasoundand CT suggests LNCM metastasis greater attentionshould be paid to the nodal tissue in the LNCM space inthyroid carcinoma patients These patients might benefitfrom a reduced risk of regional recurrence central neckreoperative morbidity and improved decision making inrelation to the use of radioiodine ablationThere are severallimitations in the present studyThe retrospective design is a limitation of the studyAnd this was two tertiary referral centers retrospective review and routine prophylactic nodal surgerywas offered in China however it is not standard elsewhere in the world which is a major limitation Aprospective randomized trial with a long time followup period may help to further evaluate the clinicalsignificance of LNCM in PTC patientsConclusionsIn summary additional dissection of nodes in theLNCM were accessible and might not increase morbidity Therefore attention should be paid to the lymphtissue between investing layer of cervical fascia and deepfascia ofinfrahyoid strap muscles for PTC patientsespecially in presence of inferior portion lesions ETElevel III and level IV metastasisAcknowledgementsThe authors thank the studied patients for their willingness to cooperatewith our studyAuthors™ contributionsGaosong Wu Study concepts and design Qianqian Yuan Study designmanuscript preparation and editing Jinxuan Hou Data analysis andmanuscript editing Yiqin Liao Data acquisition Lewei Zheng Manuscriptpreparation Fang Lu Data acquisition Kun Wang Quality control of dataand algorithms The authors read and approved the final manuscriptFundingThe authors have no support or funding to reportAvailability of data and materialsNot applicableEthics approval and consent to participateThis research was comprised of human participants and was approved byMedical Ethics Committee of Wuhan University Zhongnan Hospital Informedconsent was obtained from all individual participants included in the studyConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Thyroid and Breast Surgery Zhongnan Hospital of WuhanUniversity Donghu Road Wuhan Hubei People™s Republic of China 2Department of Thyroid and Breast Surgery Tongji Hospital of TongjiMedical College of Huazhong University of Science and Technology Jiefang Avenue Wuhan Hubei People™s Republic of China Received April Accepted August ReferencesLang BH Ng SH Lau LL Cowling BJ Wong KP Wan KY A systematic reviewand metaanalysis of prophylactic central neck dissection on shorttermlocoregional recurrence in papillary thyroid carcinoma after totalthyroidectomy Thyroid “So YK Seo MY Son Y Prophylactic central lymph node dissection forclinically nodenegative papillary thyroid microcarcinoma influence onserum thyroglobulin level recurrence rate and postoperative complicationsSurgery “Hughes DT Rosen JE Evans DB Grubbs E Wang TS Solórzano CCProphylactic central compartment neck dissection in papillary thyroid 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Cancer and effect on Locoregional recurrence Ann Surg Oncol “ McHenry CR Is prophylactic central compartment neck dissection indicatedfor clinically nodenegative papillary thyroid Cancer the answer isdependent on how the data are interpreted and the weight given to therisks and benefits Ann Surg Oncol “Selberherr A Riss P Scheuba C Niederle B Prophylactic œfirststep centralneck dissection level does not increase morbidity after Totalthyroidectomy Ann Surg Oncol “Hartl DM Leboulleux S Al Ghuzlan A Baudin E Chami L Schlumberger M Optimization of staging of the neck with prophylactic central andlateral neck dissection for papillary thyroid carcinoma Ann Surg “ McAlister ED Goldstein DP Rotstein LE Redefining classification ofcentral neck dissection in differentiated thyroid cancer Head Neck“ Miller JE AlAttar NC Brown OH Shaughness GG Rosculet NP Avram AM Location and causation of residual lymph node metastasis aftersurgical treatment of regionally advanced differentiated thyroid CancerThyroid “Sun G Wang Y Zhu Y Wang Y Xu K Wei W Lymph node metastasisbetween sternocleidomastoid and sternohyoid muscle in clinically nodepositive papillary thyroid carcinoma Head Neck “ Homma A Hatakeyama H Mizumachi T Furusawa J Kano S Sakashita T Lymph node metastasis in the suprasternal space from thyroidpapillary cancer Int Cancer Conf J “ Yu S Ge J Sun B Wei Z Lei S Lymph node metastasis in suprasternal spacein pathological node“positive papillary thyroid carcinoma Eur J Surg Oncol“ Wu G Kong D Thyroidectomy with Wu Gaosong's ProcedureVideoEndocrinologr httpsdoi101089ve20150050 Wu G Wang K Intraoperative Neuromonitoring and Protection of theSuperior Laryngeal Nerve with Wu Gaosong's ProcedureVideoEndocrinology httpsdoi101089ve20160070 Wu G Cui Q Wang K Carbon Nanops for Identifying Lymph Nodesand Protecting Parathyroid Glands in Thyroid Lobectomy with IpsilateralCentral Compartment Lymph Nodes Dissection VideoEndocrinology httpsdoi101089ve20160064Kong D Cui Q Gaosong W A Novel Classification of Parathyroid Glands andTheir Preservation in Thyroidectomy VideoEndocrinology httpsdoi101089ve20170093 Wang K Wu G Intraoperative Neuromonitoring in Selective Neck Dissectionfor Thyroid Cancer SND IIa Vb with Wu Gaosong's ProcedureVideoEndocrinology httpsdoi101089ve20160082 Yuan Q Wu G Hou J Liao X Liao Y Chiang F Correlation betweenelectrophysiological changes and outcomes of vocal cord function in recurrent laryngeal nerves with visual integrity during thyroidectomyThyroid “ Cui Q Li Z Kong D Wang K Wu G A prospective cohort study of novelfunctional types of parathyroid glands in thyroidectomy Medicine 9552e5810Tavares MR Cruz JA Waisberg DR Toledo SP Takeda FR Cernea CR et alLymph node distribution in the central compartment of the neck ananatomic study Head Neck “ Wang K Cai H Kong D Cui Q Zhang D Wu G The identificationpreservation and classification of the external branch of the superiorlaryngeal nerve in thyroidectomy World J Surg “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

"Nippleareola complex NAC reconstruction is a technique used in breast reconstructive surgerywhich is performed during the final stage of breast reconstruction after total mastectomy of primary breast cancerComposite nipple grafts utilizing the contralateral NAC are common however to our knowledge there are noreports of new primary invasive ductal carcinoma development within the graft Here we describe one such casefor the first timeCase presentation A 54yearold woman was referred to us by the Department of Plastic and ReconstructiveSurgery in our medical center for further evaluation of right nipple erosion She had undergone total mastectomyof the right breast following a breast cancer diagnosis years ago at which time tumor biological profilingrevealed the following estrogen receptor ER positive progesterone receptor PgR negative and humanepidermal growth factor receptor HER2 undetermined She received adjuvant chemotherapy and endocrinetherapy She defaulted endocrine therapy for a few years and years after surgery she underwent autologousbreast reconstruction with a deep inferior epigastric perforator DIEP flap In the following year NAC reconstructionwas performed using a composite graft technique Seven years after the NAC reconstruction erosion appeared onthe nipple grafted from its contralateral counterpart scrape cytology revealed malignancy The skin on the rightside of her chest around the NAC and subcutaneous fat tissue consisted of transferred tissue from the abdomen asthe DIEP flap and grafted nipple were located on the graft skin The right nipple carcinoma arose from the tissuetaken from the left nipple Magnetic resonance imaging MRI or computed tomography showed no malignantfindings in the left breast As the malignant lesion seemed limited to the area around the grafted right nipple onMRI surgical resection with sufficient lateral and deep margins was performed around the right nipple Pathologicalfindings revealed invasive ductal carcinoma with comedo ductal components infiltrating the graft skin andunderlying adipose tissue Immunohistochemistry revealed positive for ER PgR and HER2Continued on next page Correspondence mar016outlookjp1Department of Breast and Thyroid Surgery Yokohama City UniversityMedical Center Urafunecho Minamiku Yokohama Japan4Department of Gastroenterological Surgery Yokohama City UniversityGraduate School of Medicine Yokohama 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 httpcreativecommonslicensesby40 0cKimura Surgical Case Reports Page of Continued from previous pageConclusions To our knowledge this is the first case involving the development of invasive ductal carcinoma in anipple graft constructed on the skin of a DIEP flap with the origin from the contralateral breast™s nippleKeywords Breast cancer Autologous breast reconstruction Nipple graft Contralateral breast cancer Nippleareolacomplex reconstruction Deep inferior epigastric perforator flap Reconstructed nipple Donor nipple Compositenipple grafting Invasive cancer of graftBackgroundAutologous breast reconstruction ABR after mastectomy for breast cancer has become common over thepast decades [] An ABR with a perforator flap is a goodoption for a lot of patients who may be concerned abouttheir body image and nippleareola complex NAC reconstruction is performed in women who have undergone a total or skinsparing mastectomy which differsfrom a nipplesparing mastectomy It is an integral partof the breast reconstruction process as patients associatethis stage with the end of their plastic surgery treatmentand a sense of completion [] It was once a concernthat surgical trauma could activate dormant micrometastases however more recent reports have shown no increased risk of breast cancer recurrence following breastreconstruction [] New primary carcinomas arisingfrom the grafted breast tissue have rarely been reportedfollowing surgery for breast cancer Here we present arare case of a new primary carcinoma in a reconstructednipple which originated from the contralateral nipplewithout any malignancy detected through imaging in either the left nipple or left mammary glandCase presentationA 54yearold woman was referred to us by the Department of Plastic and Reconstructive Surgery in our medical center for further evaluation of prolonged rightnipple erosion She had previously received a diagnosisof right breast cancer and undergone total mastectomyand axillary dissection years ago in another hospitalHistopathology identified an invasive ductal carcinomawith a tumor diameter of cm and a nuclear grade of one of lymph nodes showed metastasis Tissue profiling revealed the following estrogen receptor ERpositive progesterone receptor PgR negative and human epidermal growth factor receptor HER2 undetermined As adjuvant therapy she received six cyclesof cyclophosphamide methotrexate and 5fluorouracilCMF followed by tamoxifen for years Then shedefaulted her endocrine therapy Seven years after thesurgery the doctors from our medical center performedABR with a deep inferior epigastric perforator DIEPflap at another facility The following year her right nipple was reconstructed by Vshaped resection of the leftnipplegraftingnipplesharingautologousandantechnique The right areola was reconstructed with apenetrating skin graft from the proximal thigh and leftareola Concurrent mastopexy was performed for the leftbreast Fig 1aAfter years right nipple erosion appeared and shevisited the Department of Plastic and ReconstructiveSurgery in our medical center At first it appeared thatshe had an eczematous nipple lesion caused by an infection and she was treated with antibiotics however theerosion progressed and enlarged over the course of afew months She was eventually referred to our department The skin on the right side of her chest around theNAC and the subcutaneous adipose tissue consisted oftransferred tissue from her abdomen as the DIEP flapand grafted nipple were constructed on the skin graft Inthe right nipple normal tissue was almost completely affected by erosion and there was no abnormality itchingor pain in the right areola Fig 1b Scrape cytology revealed malignancy of the epithelial cells and that theright nipple carcinoma originated from the tissue takenfrom the left nipple On magnetic resonance imagingMRI the malignant lesion seemed limited to the areaaround the grafted right nipple Fig 2a with no malignancy observed in the left breast on MRI and computedtomography CT Fig 2b In addition no distant metastases were observed on CT Paget™s disease was clinically suspected and we performed surgical treatmentThough the standard surgical operation for mammaryPaget™s disease is mastectomy we performed partialbreast excision including the right nipple with sufficientlateral and deep margins Fig 3a“c because there wasno mammary tissue in the right reconstructed breast except for the nipple and areola The incision was closedwith investing suturesThe specimen submitted for surgical pathology wascomposed of epithelial and adipose tissue grafted fromthe abdomen areolar tissue grafted from the base of thethigh and left areola and a nipple graft from the contralateral side Macroscopically the lesion spread aroundthe nipple and adipose tissue Fig 4a Pathologicalexamination identified invasive ductal carcinoma with afew comedo ductal components within the nipple extensive infiltration of grafted epithelial and adipose tissueFig 4b and a tumor diameter of mm The nucleargrade score was nuclear atypia score was and 0cKimura Surgical Case Reports Page of Fig a This is an image obtained about months after autologous breast reconstruction ABR with a deep inferior epigastric perforator DIEPflap followed by nippleareola complex NAC reconstruction of the right breast b Image of the right NAC shows erosion and marginal crustingwith a small amount of normal tissue at the upper right side of the grafted nipple The erosion was hemorrhagic on scraping The grafted areolahad no abnormalitymitotic count score was there was no lymphatic orvascular invasion and the lateral and deep margins werenegative Immunohistochemical staining showed strongpositive for ER weak positive for PgR positive for HER2with a score of and cells showing positive Ki67stainingAs the biological profile classified the tumor as a luminal HER2 type weekly paclitaxel trastuzumab andendocrineadjuvantadministered astherapy weretherapies No distant metastases or local recurrence wereseen year after the surgeryDiscussionNAC reconstruction is a technique used in the finalstage of breast reconstruction following total mastectomy for primary breast cancer Composite nipple grafting has been described in the literature since the early1970s and the technique is now widely performed Tilldate only one report of breast cancer originating in aFig a Magnetic resonance imaging MRI shows enhancement of the lesion of the right nipple and slight subcutaneous adipose tissue bThere is no abnormal enhancement in the left breast 0cKimura Surgical Case Reports Page of Fig Preoperative image of the patient a and markings for skin incision b Intraoperative image after excision of the lesion with sufficientlateral and deep margins cnipple reconstructed from tissue obtained from thecontralateralunaffected side has been identified via aPubMed search however that case involved Paget™s disease not an invasive carcinoma [] A de novo carcinoma developed years after the NAC reconstructionand the donor nipple had no identifiable lesion similarto the findings in this case To our knowledge that report documented the first case of Paget™s disease arisingfrom a grafted nipple in the US literatureBreast cancer is considered to develop from the epithelia of terminal duct lobular units TDLUs includingcases of in situ and invasive carcinomas TDLUs usuallyexistin distal mammary glands through a series ofbranches On the other hand major lactiferous ductsterminate in and exit from the mammary glands at thenipple although there are cases where TDLUs exist inadjacent lactiferous ducts in the nipple Kryvenko et aland occultinvestigated thefrequency of TDLUsneoplastic epithelial proliferation in grosslyclinically unremarkable nipples [] They observed TDLUs in ofnipples of tissue specimens from therapeutic or prophylactic mastectomies while occult neoplastic epithelialproliferation was seen in of grosslyclinically unremarkable therapeutic mastectomy nipples They also reported that the nipples were unremarkable in all cases ofprophylactic mastectomies Moreover occult neoplasticproliferationsin grossly unremarkable nipples werelargely correlated with underlying carcinomas but onlytwo patients had a primary malignant neoplasm in theirnipples Based on these reports it is difficult to confidently state whether grossly unremarkable donor nipplesare afflicted by malignant neoplasms or notThis case is also rare in terms of the origin of the invasive ductal carcinoma of the nipple Very little has beenpublished about breast cancers developing within thenipple especially invasive ones The first large series ofFig Histopathological findings a The boundary of the tumor is delineated with a red line on the macroscopic image The tumor is localizedwithin epithelial and subcutaneous adipose tissue The macroscopic image shows extensive infiltration and growth of tumor cells b At the lowerperipheral area tumor cells are infiltrating into the adipose tissue across the desmoplastic border black arrow The infiltrating tumor cells showtrabecular sheetlike acinar and nesting growth patterns with a few ductal carcinomas in situ with comedo necrosis red dotted circle 0cKimura Surgical Case Reports Page of carcinomas originating in the nipple was published in [] Of approximately cases of malignantdisease of the breast in patients undergoing surgery atthe Mayo Clinic in years only of cases originated in the nipple Secondly Sanders have reported common features ofinvasive primary breastcarcinomas originating in the nipple [] The frequencyof those carcinomas was in the report and only patients among more than breast carcinoma patients investigated in their study presented with symptoms related to the nipple Of these patients hadepithelial changes associated with in situ ductal carcinomas involving the skin of the nipple Paget™s diseasewith small foci of invasion into the dermis The rest ofthe patients presented with a nipple mass with or without skin changes Consistent with the case reported hereHER2 positivity was observed in tissue obtained from of patients with Paget™s disease and of patientswith a nipple mass The likelihood of lymph node metastases was not higher than that for carcinomas of similarsizes and there was no disease recurrence followingproper adjuvant therapy Thus invasive primary nipplecarcinomas are rare but conventionaltherapies forbreast cancer treatment are also useful in such cases Inour case we initially suspected Paget™s disease becauseof nipple erosion and the fact that no solid tumor wasidentified by palpation The patient exhibited pagetoidspread of an invasive ductal carcinoma with comedoductal components without the presence of a palpablesolid tumor However contrast scrape cytology suggested a ductal carcinoma because no melanin granuleswere observed in the malignant cells [] In contrastconsidering that HER2 positivity is more frequent inPaget™s disease than in invasive ductal carcinoma it cannot be denied thatthis carcinoma originated fromPaget™s disease in terms of the immunoprofile HER2 isoverexpressed in “ ER and PgR are positive in approximately and respectively in Paget™s disease[] in contrast with “ positive for HER2 in invasive ductal carcinoma However there were only a fewPaget cells within the squamous epithelium of the nipple and most malignant cells had directly infiltrated thenipple surface resulting in ulceration In addition theinfiltrating malignant cells were notlarge cells withabundant eosinophilic cytoplasm like Paget cells Therefore they had likely originated from the invasive ductalcarcinoma rather than Paget™s diseaseThere has been a lot of discussion about the oncological safety of ABR after mastectomy for invasivebreast cancer however a current literature review failedto show significant risks of either concurrent or delayeddistant or local recurrence following ABR relative to therisk in the absence of ABR In this case there were noremnant breast tissues in the right breast except for thenipple and areola and the malignant neoplasm arosefrom the flap of abdominal skin from a DIEP flap []Thus the malignancy was not defined as a local recurrence but as a new primary carcinoma considering therewere no right breast tissue components such as skinblood and lymphatic vessels around the malignant neoplasm Although total mastectomy is usually recommended in cases of local recurrence of breast cancer inthis case we performed a partial resection because thelesion was quite localized and there was no mammarygland except for localized tissue beneath the nipple ofthe right breast based on imaging and the carcinomaseemed not to be related to lymphovascular invasion ofthe initial breast cancerIt was unclear whether the de novo carcinoma arosebefore or after the NAC surgery Surgical treatmentmight trigger the occurrence of malignant changes although there are few case reports describing cancers developing in transferred tissues to support this scenarioCancer development is a multifaceted dynamic seriesof events and some new carcinogenic hypotheses havebeen described Recently the role of the microenvironment in driving tumor progression has been increasinglyrecognized [] Tumor formation begins when geneticabnormalities occur in cells that undergo rapid unchecked proliferation However a tumor is not solelycomprised of cancer cells it is a heterogeneous collection of both cancer cells and surrounding noncancerousor stromal cells that work in concert with one anotherto promote unrestricted growth infiltration and propagation of malignancy throughout the body In the microenvironment of carcinomas tumor cells recruit varioustypes of stromal cells such as fibroblasts inflammatorycells and endothelial cells and their cellular interactionsare important drivers of progressive tumor growth []Expansion of the tumor stroma is often observed in invasive carcinomas these changes result in desmoplasiaswhere tumors and stroma actively interact Pathologically our case presented scirrhous spread of a carcinomain the absence of a palpable tumor thus we assumedthat there was an expansion of a desmoplasia aroundthose tissuesA hypoxic environment is one of the key physiologicaland microenvironmental characteristics that differentiatetumors from normal tissues The transcription factorhypoxiainducible factor 1α and angiogenesis are important factors that regulate hypoxiainduced signalingcascades [] Hypoxia can drive and maintain geneticinstability resulting in a mutated phenotype Moreoverhypoxia along with acidosis increases clonal selectionresulting in aggressive cancer phenotypes As a resultstressorsincluding surgery may induce oncogenicchanges in tissue In contrast œnipple banking was formally performed to rebuild a nipple which is a 0cKimura Surgical Case Reports Page of technique in which a nipple is taken from a site on theipsilateral breast banked in the groin and then laterreturned to the chest [ ] Nevertheless the nipplewas taken in the case without apparent involvement ofthe nipple with carcinoma some cases of the development of heterotopic carcinoma in the transplanted nipple were reported This indicates that cancer cells in thetransplanted nipple could survive in a hypoxic or ischemic environment In our case surgical stressors mightalso have affected the donor tissue of the left nipplethough the timing of generation of malignant cells wasnot definitive Generally careful preoperative screeningfor contralateral breast cancer should be performed before NAC reconstruction whether malignantlesionsexist though there were no signs of malignancy in theleft breast in this case Moreover postoperative examinations are important because of the possibility of development of occult malignant tumors in the left breastthus we have continued to monitor the patient™s leftbreast carefullyConclusionsTo our knowledge this is the first report of an invasiveductal carcinoma developing in a grafted nipple following ABR This might be a rare case but clinicians mustconsider the possibility that carcinomas can develop in agraft as long as there are remnants of mammary glandtissue within the graftAbbreviationsABR Autologous breast reconstruction CMF Cyclophosphamidemethotrexate and 5fluorouracil 5FU CT Computed tomographyDIEP Deep inferior epigastric perforator ER Estrogen receptor HER2 Humanepidermal growth factor receptor MRI Magnetic resonance imagingNAC Nippleareola complex PgR Progesterone receptor TDLU Terminalduct lobular unitAcknowledgementsWe would like to thank Editage wwweditagecom for English languageeditingConsent to participateThis study was carried out in accordance with the principles of theDeclaration of HelsinkiAuthors™ contributionsMK wrote the manuscript and performed the breast cancer surgery whicharose in the grafted nipple in this case in which KN assisted and instructed TSperformed all plastic surgery operations which were mentioned in this reportfor this patient and supervised this manuscript from the point of plastic surgeryKN TI and IE supervised this manuscript from the point of oncology and breastsurgery MT and YI supervised this manuscript from the point of pathology HSSI MM SA AY KS and YI served as the attending physicians of the presentedpatient All authors read and approved the final manuscriptFundingThe authors have no financial contributions to discloseEthics approval and consent to participateNot applicableConsent for publicationThe patient described in this report provided informed consent for thepublication of the case detailsCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Breast and Thyroid Surgery Yokohama City UniversityMedical Center Urafunecho Minamiku Yokohama Japan2Plastic and Reconstructive Surgery Yokohama City University MedicalCenter Yokohama Japan 3Diagnostic Pathology Yokohama City UniversityMedical Center Yokohama Japan 4Department of GastroenterologicalSurgery Yokohama City University Graduate School of Medicine YokohamaJapan 5Department of Oncology Yokohama City University Graduate Schoolof Medicine Yokohama Japan 6Department of Breast Oncology and SurgeryTokyo Medical University Shinjuku Tokyo JapanReceived January Accepted July ReferencesNahabedian MY Patel K Autologous flap breast reconstruction surgicalalgorithm and patient selection J Surg Oncol “Sisti A Grimaldi L Tassinari J Cuomo R Fortezza L Bocchiotti MA et alNippleareola complex reconstruction techniques a literature review Eur JSurg Oncol “Geers J Wildiers H Van Calster K Laenen A Floris G Vandevoort M et alOncological safety of autologous breast reconstruction after mastectomy forinvasive breast cancer BMC Cancer Basu CB Wahba M Bullocks JM Elledge R Paget disease of a nipple graftfollowing completion of a breast reconstruction with a nipplesharingtechnique Ann Plast Surg “Kryvenko ON Yoon JY Chitale DA Lee MW Prevalence of terminal ductlobular units and frequency of neoplastic involvement of the nipple inmastectomy Arch Pathol Lab Med “Congdon GH Dockerty MB Malignant lesions of the nipple exclusive ofPaget™s disease Surg Gynecol Obstet “Sanders MA Brock JE Harrison BT Wieczorek TJ Hong X Guidi AJ et alNippleinvasive primary carcinomas clinical imaging and pathologicfeatures of breast carcinomas originating in the nipple Arch Pathol LabMed “Sakorafas GH Blanchard K Sarr MG Farley DR Paget™s disease of the breastCancer Treat Rev “Hoon Tan P Ellis I Allison K Brogi E Fox SB Lakhani S The WHOclassification of tumours of the breast Histopathology Allen RJ Treece P Deep inferior epigastric perforator flap for breastreconstruction Ann Plast Surg “Spaw M Anant S Thomas SM Stromal contributions to the carcinogenicprocess Mol Carcinog “ Yamaguchi Y Hayashi S Estrogenrelated cancer microenvironment ofbreast carcinoma Endocr J “ Bristow RG Hill RP Hypoxia DNA repair and genetic instability Nat RevCancer “ Cucin RL Gaston JP Case report implantation of breast cancer in atransplanted nipple a plea for preoperative screening CA Cancer J Clin“Snyderman RK Nipple banking CA Cancer J Clin “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

Circulating tumor cells CTCs derived from primary tumors andor metastatic tumors aremarkers for tumor prognosis and can also be used to monitor therapeutic efficacy andtumor recurrence Circulating tumor cells enrichment and screening can be automatedbut the final counting of CTCs currently requires manualintervention This not onlyrequires the participation of experienced pathologists but also easily causes artificialmisjudgment Medicalimage recognition based on machine learning can effectivelyreduce the workload and improve the level of automation So we use machinelearning to identify CTCs First we collected the CTC test results of patientsAfter immunofluorescence staining each picture presented a positive CTC cell nucleusand several negative controls The images of CTCs were then segmented by imagedenoising image filtering edge detection image expansion and contraction techniquesusing python™s CV scheme Subsequently traditional image recognition methodsand machine learning were used to identify CTCs Machine learning algorithms areimplemented using convolutional neural network deep learning networks for trainingWe took cells from patients for training and testing About cells wereused for training and the others were used for testing The sensitivity and specificity ofrecognition reached and respectively We will further revise our modelshoping to achieve a higher sensitivity and specificityKeywords circulating tumor cells CTCs imFISH machine learning image segmentation CNN networkINTRODUCTIONThe metastasis of cancers is a complex and multistage process The circulating tumor cells CTCsare the œseeds shed from the primary tumor andor metastatic lesions and rooted in a new œsoiltransferred by the circulatory system Paget Circulating tumor cell is an intermediate stageof cancer metastasis correlated with cancer aggressiveness and the likelihood of metastasis andtherefore can be used to predict disease progression and survival on a realtime basis by liquidbiopsy Lindsay Praharaj Anand and Roszik Baek Maly Marcuello Pan Riebensahm The molecular subtypesof CTCs not only the CTCs count are interrelated with the prognosis BanysPaluchowski Cristofanilli Dong Stefanovic What™s more the PDL1Edited byCheng GuoColumbia University United StatesReviewed byJuanying XieShaanxi Normal University ChinaKhanh N Q LeTaipei Medical University TaiwanCorrespondenceBinsheng Hehbscsmu163comQiliang Zhou13974942986163comYuebin LiangliangybgeneiscnGeng Tiantianggeneiscn These authors have contributedequally to this workSpecialty sectionThis was submitted toComputational Genomicsa section of the journalFrontiers in Bioengineering andBiotechnologyReceived March Accepted July Published August CitationHe B Lu Q Lang J Yu HPeng C Bing P Li S Zhou Q Liang Yand Tian G A New Methodfor CTC Images Recognition Basedon Machine LearningFront Bioeng Biotechnol 103389fbioe202000897Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine Learningexpression in CTCsis correlated with the response toimmunity inhibitors Kloten PDL1EMTCTCs were associated with significantly poorer survival aftercurative surgery showing that PDL1 expression and EpithelialMesenchymal Transition EMT of CTCs are negative survivalpredictors for Nonsmall celllung cancer NSCLC patientsJanning Manjunath Pretreatment PDL1 CTCs are usually associated with a bad prognosis in patientstreated with PD1 inhibitors in NSCLC such as nivolumabGuibert The liquid biopsies worked as an ongoing monitoring systemto assess tumor heterogeneity and make it possible to detect asingle CTC or clusters of cells Wan Merker Praharaj Asante The breakthroughfor CTCdetection is the application of immunomagnetic CTCenrichment combined with flow cytometry which is still theœgold standard of CTCdetection Racila Howeverthis method that lack of the cancer specific markers still remainslots of limitation Grover Ferreira Gabriel Keller Thusthe multimarker immunofluorescence staining is required for recognizeCTCs Antibodies against chromosome centromere duplicationCEP8chromosome centromere duplication CEP17 areused to mark the rapidly dividing tumor cells antibodies againstCD45 as typical leukocytes filaments as well as 4cid486diamidino2phenylindole DAPI for labeling nuclears Koudelakova Lu Liu Lee Although there are great advantages in enrichment technologythe automatic recognition of CTCs still remains problemsManual identification is very timeconsuming and unreliableWith the continuous deepening of the application of CTCsrecognition in various cancer diseases the demand for rapidand automatic identification and counting methods of CTCs isincreasing Several studies have reported the automated screeningprocess Nagrath Yang Kraeft performed a fluorescencebased automated microscope systemREIS for cell detection This scanning can quantify the numberof cells reliably and reproducibly and categorize positive cellsbased on the marker expression profile Ligthart redefined the CTCs by computer algorithms after the manualcounting The stricter definition with the standard deviationof the signal in the CKPE channel the peak signal value inboth the DNADAPI and CD45APC channels and the size ofthe objects used as classifier was well validated CTC by clinicaloutcome using a perfectly reproducing automated algorithmMingxing reported an automated CTC enumerationZhou Allimages with diï¬erent colors weretransferred to a grayscale image and the grayscale images wereused to identify the position and outline of cells Howeverdespite the widely accepted these classification methods stillremain subjective as the rules are set artificially The fixedconditions may not identify the morphologically heterogeneousCTCs integrally What™s more diï¬erent technologies usually usediï¬erent antibodies making comparison and standardizationacross diï¬erent platforms challenging Marcuello With the maturity of artificial intelligence AI recent yearsmachine learning become an exciting field for research TheUS Food and Drug Administration FDA has approved severalcommercial products using machinelearning algorithms in themedical diagnosis and research The cardiovascular MRI analysissoftware of Arterys was the world™s first internet platform formedical imaging AI powered and FDA cleared This software isable to analyze multiple multiperiod MR images to determineblood flow in heart and main vessels The cloud platformwill enable software to collect and analyze the vast amount ofcardiovascular data from MR scanners in real time which willspeed up doctors™ diagnosis This artificial machine is consistentand tireless and is able to identify characters beyond humanperception which provided a substantial interest in the fieldof medical research specifically medical images Dominguez Erickson Lundervold and Lundervold Maier Many algorithms are developed forselecting the best weights for features involving neural networksHornik decision trees Quinlan supportvector machines Cristianini and ShaweTaylor the na¯veBayes Lowd and Domingos knearest neighbors Zhouand Chen and deep learning McBee Wainberg Zou Deep learning as wellas deep neural network learning refers to the use of neuralnetworks with more than layers able to integrate vast datasetslearn arbitrarily complex relationships and incorporate existingknowledge Convolutional neural networks CNNs is a powerfulalgorithm for advancing biomedical image analysis as it assumesthat the input layer has a geometric relationship such as the rowsand columns of images Anthimopoulos Poplin It has been successfully applied in the cancer diagnosisand nuclei or tissue identification Le Le Xing present a novel method for automatednucleus segmentation powered by CNNs The features involvedin the images are considered as a part of the search processand there is no need to limit the features compared to thetraditional machine learning methods which will eliminate thebias created subjective Here we apply deep learning to therecognition of CTCs in order to reduce the artificial errors andimprove accuracyMATERIALS AND METHODSPatients and Samples PreparationA cohort of patients with cancers were enrolled inthis study during “ which was approved by theethics committee of Chifeng Municipal Hospital The clinicalpathological characteristics of patients including age genderCTC number and cancer type are summarized in Table Fourmilliliter of peripheral venous blood was routinely collectedfor every patient The first ml blood samples obtainedafter puncture was discarded in order to avoid the skinepithelial cells contamination Then the blood was placed inanticoagulation tubes and store at room temperature The testwas completed within hAll the patients were divided into two parts according tothe collecting date The earlier patients we collected wereused as the training data the others were used as the independentFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningTABLE Clinical pathological characteristicsClinicopathologic variableAgeGenderSamples typeCTC numberCancer typeCategoryMeanMaleFemaleUnknownPeripheral bloodMeanLung cancerLiver cancerGastrointestinal cancerBreast cancerCarcinoma of thyroidNPCOtherClinical level““testing data Thousand three hundred cells images in the earlierreceived patients were selected to build the CTC recognitionmodel which will be further tested by the cells images of thetest dataset There was no cross part between the two datasets inorder to avoiding the overfittingEnrichment and imFISH Identification ofCTCsThe Cyttel method was used to isolate and enumerate CTCsThe peripheral blood was first centrifuged at g for minto get the precipitation and then washed by CS1 buï¬er CyttelBiosciences Co Ltd Beijing China Then the red blood cellswere lysed by CS2 buï¬er Cyttel After centrifuged at gfor min the precipitate was washed by CS1 buï¬er Thenthe cells were incubated completely with antiCD45 monoclonalantibodyconjugated beads Cyttel for min Three milliliterseparation medium was used to separate the beads and the CTCsby gradient centrifugation at g for min Then the upper rarecell layer was centrifuged at g for min and resuspendedby CS1 The tube was put on a magnetic stand for min Aftersmeared fixed and dried cells were used to perform the imFISHThe slides were fixed dehydrated and then dried at roomtemperature µl CEP8CEP17 antibody was added to the cellsand the slides were placed in a hybridization and denatured for h at —¦C The probe was eluted and the slides were washedtwice in — SSC Then the CD45 fluorescent antibody was addedto the sample area and the slides were put in a wet box andincubate for h at —¦C After incubation CD45 fluorescentantibody was aspirated and µl mounting media containingDAPI was added to the sample area After mounted the cells canbe observed and counted under a fluorescence microscopeThe Manual Interpretation Standard ofCTCs CountingAfter imFISHlots ofimages were acquired with diï¬erentfluorescent colors Usually manual counting is the œgoldstandard but it™s a time consuming and exhausted processionThe Manual interpretation standard of CTCs counting is Eliminates the aggregation superposition and interference ofnucleus or impurity DAPI positive CD45 negative and Three or more than three CEP8CEP17 signal points Itwill be regarded as one signal point if the distance between twosignal points is smaller than the diameter of one pointThe Image Segmentation Method WasUsed to Segment Single Nucleus andGive Labels of Cells Instead of ManualSince the obtained microscopic image is very huge the algorithmwill be limited by the memory and cannot be executed normallyon a conventional computer We first selected part of the imagecontaining one CTC cell and several nonCTC cells around toperform the following test The chosen resolution is — The CV package of python was used to process theCTCs images including conversion of color and morphologicaltransformations The RGB image was converted to the gray image The derivatives were calculated using the CVfunction Sobel from an image Morphological transformations operations based on theimage shapeThe Morphological package of python was used to segmentthe images of CTCs by image denoising image filtering edgedetection image expansion and contractionNuclei were segmented in the blue channel DAPI and theproportion of red in the red channel was detected based onthe position of the nucleus The nucleus with proportion of redhigher than was defined as having a common leukocyteantigen The orange channel was used to detect the number ofCEP8 chromosomes and the green channel was used to detectthe number of centromere probes extracted by CEP17 Diï¬erentcell types were distinguished by diï¬erent colors Figure The CNN Deep Learning Method WasUsed for CTCs IdentificationWith the development of AI machine learning has been wildlyused in the procession of medical images Deep learning is a bigimprovement on artificial neural networks allowing higherlevelfeature extraction and better data prediction with more layersAfter segmentation CNN network were used to identify CTCcells in single nucleus Finally it enters the output layer andoutput the result ie CTCs or nonCTCsOur CNN model was built based on AlexNet which wasfirst introduced in Krizhevsky The networkconsists of eight weighted layers Figure the first five layersare convolution layers and the remaining three layers are fullconnection layers The output of the last full connection layeris the input of the dimensional softmax values which willgenerate the distribution network of two types of labelsThe fivefold cross validation was used to prevent overfittingand select hyperparameters of the model The best crossvalidation score was obtained by searching the hyperparameterspace round and round The final hyperparameters involved inFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The imFISH result and the segmentation of chromosome and nuclear A“C The imFISH result of CEP8 CD45 and DAPI D The merge of panelsA“C E The CTCs were identified by CV segmentation method and marked in red box a“c The CEP8 signal points were identified by CVsegmentation method and marked in red boxour model are activation function kernel regularizer type andregularization factor The workflow is shown belowSp The grid was defined on 3dimensions with eachofthese maps for hyperparameter sets eg hyperparameters activation function kernel regularizer typeregularization factor activation function œsoftmaxœReLU œtanh kernel regularizer type œl1 œl2regularization factor œ œ The range of possible values were defined of eachdimension Allestablishing the best onethe possibleconfigurations weresearched forEvaluation Criteria for ClassificationModelsAfter segmentation some performance evaluation criteria Xie were involved in to evaluate the performance of theclassification model such as sensitivity Se or recall specificitySp precision F1 score and area under the receiver operatingcharacteristic curve AUCSerecall TPTP FNTNTN FPTPprecision TP FPF1 — precision — recallprecision recallIn the equations TP stands for the number of positive CTCcells which are correctly recognized as positive CTC cells FPstands for the number of negative CTC cells that are incorrectlyrecognized as positive CTC cells FN stands for the numberof positive CTC cells incorrectly recognized as negative CTCcells TN stands for the number of negative CTC cells correctlyrecognized as negative CTC cells Table RESULTSPatient CharacteristicsA total of patients were enrolled in this study from January to June The average age is years old Patients withlung cancer count of all patients and the next is breastcancer and gastrointestinal cancer Table Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The layers of the CNN model The first five layers are convolution layers and the remaining layers are full connection layersThree SubImages Were Required forManual CountingWe performed imFISH for all the patients and required images of CTCs cells Every image was divided into or channels with diï¬erent color The orange channel representedthe chromosome with CEP8 Figure 1A the green channelthecentromereofthechromosomerepresentedCEP17 Supplementary Figure S1represented the whitethe blueFigure 1C The mergence wasWe then manually labeled all withred channelcell with CD45 Figure 1Brepresented the nuclei with DAPIshown in Figure 1Dthese subimages accordingchannelFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningTABLE Confusion matrix definitionsTABLE The confusion matrix of the models for test datasetConfusion MatrixPredictionMethodConfusion MatrixPredictionPositiveNegativePositiveNegativeTruePositiveNegativeTrue positive TPFalse positive FPFalse Negative FNTrue Negative TN CVALexNetTrueTruePositiveNegativePositiveNegativethetoare CTCs positivestandard AmongourresultspatientsTABLE Tuning of the hyperparameters of AlexNetActivation functionKernel regularizer typeRegularization factorThe Segmentation of Nuclear andIdentifying CTCs by CVSegmentation MethodIn order to avoid the artificial error and save costs we performedthe traditional image identification method for CTCs countingFigure The nucleus was separated in the blue channelDAPI Figure 1E and the red proportion of the red channelwas detected according to the location of the cell nucleus Theproportion higher than was defined as the number of theCEP8 chromosome detected by the common antigen orangechannel of white blood cells Figures 1A“C the number ofcentromeric probes detected by the green channel such as CEP17Supplementary Figure S1After segmentation of nuclear we used CV segmentationmethod to identify CTC cells from single nucleus regions in testing dataset by the manual interpretation standard ofCTCs counting After identification and judgment cells of negative nuclei were recognized as CTC negative About cells of positive nuclei were recognized as CTCnegative The sensitivity and specificity were and while the precision and F1 score reached and respectively Table We also applied the regionbased image segmentationalgorithm such as watershed algorithm in the segmentationprocess The watershed algorithm was implemented the bywatershed function in CV python and CV In this method optimal threshold value was used respectivelyin binaryzation process by setting THRESH_OTSU mode Thetraditional watershed algorithm was sensitive to noise and theaccuracy was lower than our segmentation method on CTCnegative data set in size of Supplementary Table S3The HyperParameters Selected forEvaluating the CNN MethodWe used GridSearchCV class in scikitlearn by providinga dictionary of hyperparameters to determine the hyperparameters of the model After the crossvalidation processactivation function was set to ReLU kernel regularizer type wasset to l2 and regularization factor was set to as shown inTable with the best performance Further the hyperparameterswe selected were used to construct the model on the wholetraining datasetsoftmaxReLUtanhl1l2l1l2l1l2The underline value shows the best result of AUC value in the tuning process of thehyperparameters of AlexNetThe Identification of CTCs by CNNMethodWe got nuclei of patients by segmentation processFigure showed the whole flowchart of the experiment About nuclei were used for training the left were usedfor testing We use the same images for testing cells of negative nuclei were recognized as CTC negative and cells of were recognized as CTC positive The sensitivityand specificity were and while the precisionand F1 score reached and respectively Table and Figure Before that we also compared the performance of AlexNetmodel with others such as ResNet and Xception All ofthem have close AUC values Figure butthe AlexNetwas less timeconsuming in the training and test processSupplementary Table S1DISCUSSIONThis study showed a method for CTC counting powered bymachine learning The use of machine learning for imageinterpretation can capture important image features reduceerrors caused by manually setting interpretation standardsand save time and labor costs Although this method showsa higher sensitivity and specificity in CTC countingisslightly worse than the first method for the data used inthis study Actually we have analyzed that the main reasonis that there are fewer positive samples for training and thealgorithm cannot extract features of more positive samplesin the group were excludedIn additiondue to quality problems Unfortunatelythe CTC imagesincluded in the group doesn™t cover the whole film but asome picturesitFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The flowchart of the whole experimentFIGURE The ROC curve of AlexNet ResNet and Xception modelpicture just focused on a certain CTCpositive cell under themicroscope which results in that the machine learning methodhas no advantage in recognition speed compared with thetraditional image recognition method Enlarging the scope ofimages and collected more samples is also that need to beimproved in the futureDeep learning has already been shown to be suitablefor detection of CTCs because of the high sensitivity andspecificity in CTC counting We had changed the filter sizeand number in all convolution layers in order to find thebest CNN parameters We found diï¬erent filter size andnumber will influence the results largely We changed filternumber from range to in our training process Wefound that the training result was not convergence when thenumber was less than It showed that the range of thefeature number of the image is about “ We tried toincrease the filter size from to but the result was notchanged a lot and the convergence speed even became slowerwhen the filter size higher than From this process wesummarized that the feature size in CTCs could not be greaterthan pixels Furthermore there are many appropriately AImodels such as VGG InceptionV14 We will apply themon the CTCs dataset to establish a more suitable model inthe later testingtumorsCirculating tumor cellis an important marker for earlyscreening and prognosis ofIn addition CTCsoriginating from the primary tumor may be more eï¬ectivefor tumor tissue tracing and molecular classification Imagerecognition can only obtain the characteristics ofthe cellsurface If strict tissue tracing is required other molecularbiological experimental data such as the isolation of CTCcells and single cell sequencing may be required Besidesin this study we also evaluated the performance of AlexNetmodel in variant types of cancers Supplementary Table S2and Figure S2 showed that our model presents a betterperformance in Lung cancer than Gastrointestinal cancer andBreast cancer One of the reasons may be that the trainingdata size of Lung cancer is much larger than those ofGastrointestinal cancer and Breast cancer Furtherpostoperative recurrence may occur in approximately of patients even after complete resection of NSCLC Yano These proteins especially epithelial proteinssuch as EpCAM PIK3CA AKT2 TWIST and ALDH1may have more activitiesHanssen whichwilllead more influence in the morphology of cells andaï¬ecting the recognition performance thereby Therefore themultiimage omicsincluding CT images HE staining andimmunohistochemical images as well as the sequencing datamay be urgently needed at this stageFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCONCLUSIONIn orderIn the present study we established a CTC cell recognitionsoftware based on deep learningto make itmore practical we collected samples from the real worldinstead of using the public databases We performed theCTC enrichment and imFISH experiments and screened thefluorescence images according to the figure™s quality In order toimprove the efficiency we used the machine instead of ngmanual screening First the python™s package was used to mage segmentation The obtained recognition sensitivity andspecificity are and respectively In addition therecognition sensitivity and specificity can also reach to and respectively using CNN instead of manual interventionIn the future studies we willfocus on the improvementof the accuracy and sensitivity with a more suitable deeplearning model promoting this technology to the clinic assoon as possibleDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorCTCs Recognition by Machine Learninglegal guardiannextstudy was provided by the participants™of kin Written informed consent was obtained from theindividuals and minors™ legal guardiannext of kin for thepublication of any potentially identifiable images or data includedin this AUTHOR CONTRIBUTIONSGT YL BH and QZ conceived the concept of the work BH QLJL PB HY and SL performed the experiments QL and BH wrotethe manuscript CP and HY reviewed the manuscript All authorsapproved the final version of this manuscriptFUNDINGThis research was funded by Hunan Provincial Innovation2018RS3105Platform and Talents Program NotheNaturalNo Science Foundation of Chinathe Natural Science Foundation of Hunan Province No2018JJ3570 and the Project of Scientific Research Fundof Hunan Provincial Education Department Nos 19A060and 19C0185ETHICS STATEMENTSUPPLEMENTARY MATERIALThe studies involving human participants were reviewed andapproved by The Ethics Committee of Chifeng MunicipalHospital Written informed consentto participate in thisThe Supplementary Materialonline202000897fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389fbioeatREFERENCESAnand K and Roszik J Pilot study of circulating tumor cells in earlystage and metastatic uveal melanoma Cancers Basel cancers11060856Anthimopoulos M Christodoulidis S Ebner L Christe A and MougiakakouS Lung pattern classification for interstitial lung diseases using a deepconvolutional neural network IEEE Trans Med Imaging “ 101109tmi20162535865Asante D B Calapre L Ziman M Meniawy T M and Gray E S Liquid biopsy in ovarian cancer using circulating tumor DNA and cellsready for prime time Cancer Lett “ 101016jcanlet2019Baek D H Kim G H Song G A Han I S Park E Y Kim H S Clinical potential of circulating tumor cells in colorectal cancer aprospective study Clin Transl Gastroenterol 10e00055 1014309ctgBanysPaluchowski M Schneck H Blassl C Schultz S MeierStiegen FNiederacher D Prognostic relevance of circulating tumor cellsin molecular subtypes of breast cancer Geburtshilfe Frauenheilkd “ 101055s00351545788Cristianini N and ShaweTaylor J An Introduction to Support VectorMachines and Other KernelBased Learning Methods Cambridge Cambridgeuniversity pressCristofanilli M Pierga J Y Reuben J Rademaker A Davis A A Peeters D J The clinical use of circulating tumor cells CTCs enumerationfor staging of metastatic breast cancer MBC International expert consensuspaper Crit Rev Oncol Hematol “ 101016jcritrevonc201812Dominguez C Heras J and Pascual V IJ CV combining ImageJand CV for processing images in biomedicine Comput Biol Med “ 101016jcompbiomed201703027Dong J Zhu D Tang X Qiu X Lu D Li B Detectionof circulating tumor cell molecular subtype in pulmonary vein predictingprognosis of stage IIII nonsmall cell lung cancer patients Front Oncol 103389fonc201901139Erickson B J Korfiatis P Akkus Z and Kline T L Machinelearning for medical imaging Radiographics “ 101148rg20171Ferreira M M Ramani V C and Jeï¬rey S S Circulating tumortechnologies Mol Oncol “ 101016jmolonc2016cellGabriel M T Calleja L R Chalopin A Ory B and Heymann D Circulating tumor cells a review of nonEpCAMbased approaches for cellenrichment and isolation Clin Chem “ 101373clinchem2015Grover P K Cummins A G Price T J RobertsThomson I C andHardingham J E Circulating tumour cells the evolving concept andthe inadequacy of their enrichment by EpCAMbased methodology for basicand clinical cancer research Ann Oncol “ 101093annoncmdu018Guibert N Delaunay M Lusque A Boubekeur N Rouquette I Clermont E PDL1 expression in circulating tumor cells of advanced nonsmallcell lung cancer patients treated with nivolumab Lung Cancer “ 101016jlungcan201804001Hanssen A Wagner J Ges T M Taenzer A Uzunoglu F G Driemel C Characterization of diï¬erent CTC subpopulations in nonsmallcell lung cancer Sci Rep 101038srep28010Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningHornik K Stinchcombe M and White H Multilayer feedforwardnetworks are universal approximators Neural Netw “ Janning M Kobus F Babayan A and Wikman H Determination ofPDL1 expression in circulating tumor cells of NSCLC patients and correlationwith response to PD1PDL1 inhibitors Cancers Basel cancers11060835Keller L Wernerand clinicalrelevance of EpCAM Cell Stress “ 1015698cst2019and Pantel K BiologySKloten V Lampignano R Krahn T and Schlange T Circulatingtumor Cell PDL1 expression as biomarker for therapeutic efficacy ofimmune checkpoint inhibition in NSCLC Cells 103390cells808Koudelakova V Trojanec R Vrbkova J Donevska S Bouchalova K KolarZ Frequency of chromosome polysomy in relation to CEP17copy number in a large breast cancer cohort Genes Chromosomes Cancer “ 101002gcc22337Kraeft S K Ladanyi A Galiger K Herlitz A Sher A C Bergsrud D E Reliable and sensitive identification of occult tumor cells usingthe improved rare event imaging system Clin Cancer Res “ 10115810780432ccr030361Krizhevsky A Sutskever I and Hinton G œImageNet classification withdeep convolutional neural networks in Paper Presented at the NIPS LakeTahoe Harrahs and HarveysLe N Q Ho Q T and Ou Y Y Incorporating deep learning withconvolutional neural networks and position specific scoring matrices foridentifying electron transport proteins J Comput Chem “ 101002jcc24842Le N Q Ho Q T and Ou Y Y Classifying the molecular functionsof Rab GTPasestrafficking using deep convolutionalneural networks Anal Biochem “ 101016jab2018in membraneLe N Q K Huynh T T Yapp E K Y and Yeh H Y Identificationof clathrin proteins by incorporating hyperparameter optimization in deeplearning and PSSM profiles Comput Methods Programs Biomed “ 101016jcmpb201905016Lee K Kim H J Jang M H Lee S Ahn S and Park S Y Centromere copy number gain reflects chromosomal instability in breast cancer Sci Rep 101038s4159801954471wLigthart S T Coumans F A W Attard G Cassidy A M de Bono J S andTerstappen L W M M Unbiased and automated identification of acirculating tumour cell definition that associates with overall survival PLoS One6e27419 101371journalpone0027419Lindsay C R Faugeroux V Michiels S Pailler E Facchinetti F Ou D A prospective examination of circulating tumor cell profiles innonsmallcell lung cancer molecular subgroups Ann Oncol “ 101093annoncmdx156Liu X Zhang Z Zhang B Zheng Y Zheng C Liu B Circulating tumor cells detection in neuroblastoma patients by EpCAMindependentsituhybridization EBioMedicine101016jebiom2018immunostainingfluorescenceenrichment“andinLowd D and Domingos P œNaive Bayes models for probabilityInternational ConferenceNew York NY Association for ComputingProceedings22ndtheofinestimationon Machine LearningMachineryLu S S Pan Q J Cao J Xu X Zhao H and Shen D H Fluorescence in situ hybridization combined with cytomorphology forthe detection of lung cancer in bronch

Thyroid_Cancer

"clinical development of immune checkpoint inhibitors ICIs therapy has ushered in a new era of antitumor therapy with sustained responses and significant survival advantages observed in multiple tumors mostpatients do not benefit Therefore more and more attention has been paid to the identification and developmentof predictive biomarkers for the response of ICIs and more indepth and comprehensive understanding has beencontinuously explored in recent years Predictive markers of ICIs efficacy have been gradually explored from theexpression of intermolecular interactions within tumor cells to the expression of various molecules and cells intumor microenvironment and been extended to the exploration of circulating and host systemic markers With thedevelopment of highthroughput sequencing and microarray technology a variety of biomarker strategies havebeen deeply explored and gradually achieved the process from the identification of single marker to thedevelopment of multifactorial synergistic predictive markers Comprehensive predictivemodels developed byintegrating different types of data based on different components of tumorhost interactions is the direction offuture research and will have a profound impact in the field of precision immunooncology In this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of ICIs and discuss their future directions in achievingprecision immunooncologyKeywords Neoplasm Immune checkpoint inhibitor Predictive biomarker Tumor mutation burden Programmeddeath ligand1BackgroundImmune checkpoint inhibitors ICIs therapy has usheredin a new era of antitumor therapy with sustained responses and significant survival advantages observed inmultiple tumors Antiprogrammed cell death1programmed cell deathligand PD1PDL1 antibody hasbeen approved for secondline or firstline treatment in avariety of malignant neoplasms including melanoma lungcancer renal cell carcinoma RCC head and neck squamous cell carcinoma HNSCC and gastroesophageal Correspondence cuijwjlueducnCancer Center the First Hospital of Jilin University Xinmin StreetChangchun Jilin Chinacancer [ ] However despite the breakthrough in clinical treatment with ICIs most patients do not benefitPembrolizumab or nivolumab has an objective responserate ORR of “ in firstline melanoma and insecondline nonsmall cell lung cancer NSCLC [“]Therefore in recent years more and more attentions havebeen paid to the identification and development of predictive biomarkers for the efficacy of ICIs and more indepth and comprehensive understanding has also beenobtained in recent yearsincluding new data on biomarkers of tumor genome and neoantigen tumor immune microenvironmentbiopsybiomarkers hostrelated factors and all of which havephenotypeliquid 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 0cBai Biomarker Research Page of technologyimmunohistochemicalmade many new advances in the corresponding fieldsWith the development and continuous improvement ofmultiplexhighthroughput sequencing and microarray technology a variety of biomarker strategies have emerged and graduallyrealized the process from the identification of singlemarker to the development of multifactorial synergisticpredictive markers The development of predictive biomarkers contributes to revealing the therapeutic mechanisms of ICIs and the interaction mechanisms betweentumor and host immunity achieving decisionmaking ofindividualized antitumorimmunotherapy monitoringefficacy and disease development guiding clinical trial design as well as for further understanding of drug resistance mechanisms and tumor prognosis In this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of ICIs It should be pointed out here that when reading and collating we try to read and include all therelevant s In the process of selecting s we include the authoritative s published in highleveljournals or the latest research results and objectively describe and analyze their roles in this field as well as discuss the reasons that different research results may beinvolvedAdvances of multiple predictive biomarkers toICIs efficacyi Tumor genome and neoantigen biomarkersTumor mutation burdenSignificant correlations between high tumor mutationburden TMB and response to ICIs have been reported inseveral cancer types [] including urothelial carcinoma[] small cell lung cancer SCLC [] NSCLC [“]melanoma [] and human papilloma virus HPVnegative HNSCC [] A metaanalysis of cancer typesshowed that the mean response rate was positively correlated with log TMB [] The National ComprehensiveCancer Network NCCN guidelines have adopted TMBas the recommended test for patients with NSCLC receiving immunotherapy Although the results in some clinicalstudies of RCC [] HPVpositive HNSCC [] and melanoma receiving antiPD1 after recurrence [] showedthat TMB alone also did not clearly distinguish respondersand predict OS it is still exciting that multiple studies inthe American Society of Clinical Oncology ASCOmeeting have confirmed the predictive value of TMB inimmunization or combination therapy KEYNOTE061study [ ] CONDOR study [] EAGLE study []EPOC1704 study [] etc consolidating its status ofTMB as an independent predictor And in April theUS Food and Drug Administration FDA prioritized theapproval of TMB as a companion diagnostic biomarkerfor pembrolizumabNonetheless the cutoff values of TMB were defineddifferently across studies and assay platforms such asatezolizumab mtMb in urothelial cancer pembrolizumab mtMb in NSCLC and atezolizumab‰¥ ‰¥ or ‰¥ mtMb in NSCLC [“] andnivolumab plus ipilimumab ‰¥ mtMb in NSCLC [] which needs further study to confirm the optimalcutoff value in different tumors Moreover the NGSpanels have approved by the FDA that can be used to estimate TMB include the MSKIMPACT and FoundationOne CDx panel the detection results of which arehighly consistent with whole exome sequencing WES[ ] and other solutions are under development Astudy detecting TMB cutoff value at mtMb in NSCLC patients with the FoundationOne platformcontaining a gene panel found that compared withTMBL patients overall survival OS and DCR was significantly improved in TMBH patients treated withantiPD1L1 drug [] Both WES and targeted NGS a422cancergene panel performed in patients withNSCLC treated with antiPD1L1 demonstrated thatTMBH population has a significantly better durableclinical benefitDCB and progressionfree survivalPFS [] These findings demonstrate the feasibility ofcomprehensive genomic profiling CGP but the designof optimal next generation sequencing NGS panel thatis more accurate comprehensive and costeffective isstill not clear In addition given that bTMB was identified as a predictor of PFS but failed to differentiate patients with OS benefits researchers consider the need toexplore other more precise factors eg allele frequencyAF A study that developed a new bTMB algorithm intwo independent cohorts POPLAR and OAK showedthat modified bTMB low AF bTMB LAFbTMB mutation counts with an AF was significantly associated with favorable HR 95CI “ p PFS HR 95CI “ p andORR p after immunotherapy but required tobe prospectively validated [] Finally static biomarkersare insufficient to accurately predict response due to thecomplexity of tumorimmune interactions A recent analysis of tumor genomewide dynamic detection in pretreatment and ontreatment melanomasfound thatpretreatment TMB was only associated with OS in untreated patients while early 4week ontreatment changein TMB ΔTMB was strongly associated with antiPD1response and OS in the entire cohort [] The detectionof ΔTMB is helpful for early evaluating the response totherapy of patient but its clinical usability limited by thedifficulty in obtaining tissue samples and high price whileliquid biopsy discussed below might better 0cBai Biomarker Research Page of In addition epigenetic changes are associated withTMB The latest study investigated the association between TMB and DNA methylation DNAm to explorepotential complimentary biomarkers for NSCLC immunotherapies The results showed that high TMBNSCLCs had more DNAm aberrance and copy numbervariations CNVs showing certain value in predictingefficacy such as HOX gene methylation status and TMB[] Thus the correlated exploration of epigenetics hasattracted more attention in recent years and liquidbiopsybased epigenetic studies may become a future research direction Exploration in Chinese NSCLC patientsshowed that NSCLCs with high TMB had DNAm aberrance and CNVs Some insertion and deletion indelmutations can lead to frameshifts and more immunogenic neoantigens [] In the pancancer analysis of cancer types evaluated in The Cancer Genome AtlasTCGA RCC had the highest indel mutation load andframeshift indel mutations were found to produce threetimes more candidate neoantigens per mutation thannonsynonymousnsSNVs[] Somatic copy number alterations SCNAs are another feature of the genomic landscape of tumors andpancancer TCGA analysis revealed an inverse correlation between SCNAs atthe singlearm or wholechromosomelevel and immune infiltration in tumortypes tested [] and this result was subsequently replicated in a larger study of TCGA []single nucleotide variantsDNA damage response pathwaysGenetic variation involved in DNA mismatch repairMMR pathway can lead to microsatellite instabilityMSI a specific type of high TMB tumors and increased numbers of CD8 tumor infiltrating lymphocytesTILs PD1TILs and indoleamine 23dioxygenaseIDO tumor cells have been shown in MMR deficiencydMMR colorectal cancer [] Recently five clinical trials Keynote016 including multipletumor types have shown that patients with dMMRMSIH can achieve durable responses to pembrolizmabBased on this pembrolizumab is approved by the USFDA for the treatment of any advanced solid tumor withdMMRMSIH and nivolumab in combination with ipilimumab has also shown promising response in dMMRMSIH colorectal cancer [] In addition dMMR canalso cause mutations in the DNA polymerase gene epsilondelta POLEPOLD1increasing the mutationload and neoantigen load Analysis of POLEPOLD1mutations in patients with different cancer typesshowed that patients with these mutations had significantly higher TMB and OS Therefore it may be an infordependentInidentifying patients who benefitaddition pathways of base excision repairBERand prognostic markerfrom ICIs []risk factorhomologous recombination repair HRR MMR in theDNA damage response DDR signaling network contribute more significantly to TMB or neoantigens whichhave the highest levels when comutated [] It hadbeen identified that comutations in the DDR pathwaysof HRR and MMR or HRR and BER defined as comutare associated with increased levels of TMB neoantigenload and immune gene expression signatures Comutpatients showed a higher ORR and longer PFS or OS indicating that comut can be used as predictors of response to ICIs and provide a potentially convenientmethod for future clinical practice []Specific mutated gene pathways in tumor cellsIt is worth noting that alterations of signaling pathwaysin tumor cells affect the responsiveness to immunotherapy Patients with mutations in the interferon IFNγpathway genes IFNGR12 JAK12 and IRF1 are poorlyresponsive to ICIs treatment and confer resistance []A study found that in patients receiving immunotherapytumor cells can downregulate or alter IFNγ signalingpathways such as lossoffunction alleles of genes encoding for JAK12 and changes in STAT1 to escape the influence of IFNγ [] resulting in poor efficacy andresistance Recent studies suggest that inactivating mutations in a mammalian analog of the chromatin remodeling SWISNF complex and unique genes of the PBAFcomplex Pbrm1 Arid2 and Brd7 lead to sensitivitiesto ICIs [ ] Loss of function of the PBAF complexincreased chromatin accessibility to transcription regulator elements of IFNγ“inducible genes within tumorcells and subsequently increased production of CXCL9CXCL10 chemokines leading to more efficient recruitment of effector T cells into tumors [] In human cancers expression of Arid2 and Pbrm1 are related toexpression of T cell cytotoxicity genes which confirmedin Pbrm1deficient murine melanomas with strongly infiltrated by cytotoxic T cells and responsive to immunotherapy [ ]In addition doublestranded RNAdsRNA editing enzyme adenosine deaminase acting onRNA ADAR1 protein can block the IFNγ signalingpathway and lead to poor ICIs efficacy and resistanceLoss of function of ADAR1 in tumor cells can reduce AtoI editing of interferoninducible RNA species and leadto dsRNA ligand sensing by PKR and melanomadifferentiationassociated protein MDA5 This resultsin growth inhibition and tumor inflammation respectively and profoundly sensitizes tumors to immunotherapy [] Finally demethylation positively regulates thetranscriptional activity of some immunerelated genesincluding PDL1 and IFN signaling pathway genes sensitizingto anticytotoxic Tlymphocyteassociatedprotein4 CTLA4 therapy []it 0cBai Biomarker Research Page of In addition to the IFNγrelated signaling pathway alterations in other tumor genome such as tumor oncogenes and suppressor genes pathways and pathwaysrelated to tumor cell proliferation and infiltration canalso affect immunotherapy efficacy Epidermal growthfactor receptor EGFR and anaplastic lymphoma kinaseALK mutations have been shown to be associated withreduced response rates to ICIs and low TMB and therefore the FDA does not recommend firstline ICIstreatment in patients with EGRF or ALK positive tumors[ ] certain types of mutations in MDM2MDM4and ARID1A can predict nonresponse to ICIs in highTMB tumors [] NSCLC with KRAS and STK11 comutated was associated with reduced response andshorter survival in three independent cohorts of patientstreated with antiPD1 therapy [] and STK11 deficiency was an independent indicator of poor antiPD1response in NSCLC with KRAS mutant however at the American Association for Cancer Research AACRmeeting of patients in the Keynote042 studyNCT02220894 update data were tested for STK11 andKEAP1 and the results showed that patients could benefit from pembrolizumab regardless of STK11 and KEAP1status but patients with STK11 mutations did not respond well to chemotherapy but given that only ofall patients had mutation detection the results may beaffected in initial data from studies using targeted NGSpanels suggested that duration of ICIstreatment was associated with certain BRAF and MET alterations butnot TMB status [] NOTCH signaling pathway is associated with the occurrence development and prognosisof tumors especially with the biological function of cancer stem cells Recent breakthrough findings have distinguished deleterious NOTCH mutation showing that itcan be used as a potential predictor of favorable ICI response in NSCLC potentially via greater transcription ofgenes related to DNA damage response and immune activation [] Another tumorspecific inheritance thatmay influence ICIs efficacy is the aberrant expression ofendogenous retroviruses ERVs Pancancer analysisidentified a positive correlation of transcript expressionof ERVs with Tcell activity in various tumors [] andpatient prognosis [] Furthermore with the improvement of precision detection technology the accurateanalysis of negative mutation sites helps to identify thepossibly effective ones For example the analysis of studydata of secondline PD1L1 inhibitor therapy found thatthe mPFS of patients with KRAS G12C or G12V was significantly better than that of patients with KRAS mutations at other sites []In addition several pancancer biomarkers are recentlyapproved by the FDA For example given the effectiveORR of and a disease control rate DCR of in secondline cholangiocarcinoma patients treated withanalysispemigatinib a new targeted therapy the recent FDA approval of pemigatinib for the treatment of previouslytreated patients with locally advanced or metastatic cholangiocarcinoma with fibroblast growth factor receptor FGFR2 fusion or rearrangement and the comprehensive genomicassay FoundationOne CDxdeveloped by Foundation Medicine as a companiondiagnostic Also exciting is the recent FDA approval ofthe targeted anticancer drug capmatinib for the treatment of metastatic NSCLC with MET exon skippingMETex14 mutations including firstline patients andpreviously treated patients also using FoundationOneCDx as a companion diagnostic to help detect specificmutations present in tumor tissueimmunogenicity ofNeoantigen loadNeoantigen load the number of mutations actually targeted by T cells may be directly related to the responseto ICIs [“] A retrospective study showed thatclonal neoantigen burden was associated with the longerOS in primary lung adenocarcinomas p []Traditionallycomputational neoantigen predictionshave focused on major histocompatibility complexMHC binding of peptides based on anchor residueidentities however neoantigen loads identified by thismethod are generally not superior to overall TMB inpredicting ICIs efficacy or survival [] In recent practice this neoantigen can be assessed by the difference inpredicted MHCI binding affinity between the wildtypepeptide and the corresponding mutant peptide knownas the differential agretopicity index DAI reflectingclinically relevanttumor peptide[] A high DAI value indicates that the mutant peptidesignificantly increases binding affinity to MHC compared to the wildtype sequence and can generate moreimmune responses Studies on previously published cohorts treated with three ICIs have shown that DAI outperforms TMB and the traditionally defined neoantigenload in predicting survival [ ] In additionlowneoantigen intratumour heterogeneity might also be important for ICIs response Analysis of the lung adenocarcinoma TCGA database found that combining highmutational load and low intratumoral neoantigen heterogeneity was significantly associated with OSand longer lasting clinical benefit than either variablealone [] Anotherreported method for assessingneoantigen foreignness is based on sequence homologyof experimentally validated immunogenic microbial epitopes in the Immune Epitope Database IEDB [] butit does not account for all possible human leukocyteantigen HLA contexts In addition the detection forneoantigen can be reflected from different levels such aspeptides or genomes A study developed the Neopepseealgorithm using a machine learning approach incorporating 0cBai Biomarker Research Page of integration of nine immunogenicity features and gene mutation expression levels [] and its application to melanoma and leukemia patients could improve the sensitivityand specificity of neoantigen prediction Recently it has alsobeen shown that promoter hypermethylation of neoantigengenes may be an important mechanism for immune editingand tumor immune evasion [] indicating that combineddetection of tumor genome and epigenetics may providemore information for immunotherapy efficacyii Tumor immune microenvironment phenotypebiomarkerscells is also considered separately as one of the biomarkersto distinguish the benefit population called immune positive score IPS Herbst [] showed that response toatezolizumab treatment was significantly associated withhigh levels of PDL1 expression on the surface of TILs before treatment but not with PDL1 expression on tumorcells p Finally other inhibitory immune pathways may affect the response to ICIs therapy including Tcelllymphocyte activationgene3 LAG3 and Vdomain Ig suppressor of Tcell activation VISTA which can be used as potential biomarkers for ICIs responseimmunoglobulin3 TIM3PDL1 expressionGiven that multiple studies in a variety of tumors havedemonstrated a positive correlation between PDL1 expression and response to ICIs or OS even in firstlinecombination therapy [“] pembrolizumab is currently approved by the FDA for use in patients with PDL1 PDL1 ‰¥ of tumor cells in firstline treatmentand ‰¥ in secondline treatment NSCLC and PDL1immunohistochemistry IHC as a companion diagnosticfor antiPD1 therapy in NSCLC patients [ ] However some studies have not detected a significant correlation between PDL1 expression and response to ICIs[ ] and PDL1 negative patients can still benefitclinically with treatment with ICI or combination treatment with ICIs [] with ORRs ranging from to Therefore PDL1 cannot yet be a comprehensive and independent biomarker in clinical practice in assessing efficacy with following challenges still existing FirstlyPDL1 assay and antibody are not standardized []Secondly PDL1 expression is temporally and spatiallyheterogeneous [] A study of metastatic NSCLCtreated with ICIs showed that PDL1 varies substantiallyacross different anatomic sites and during clinicalcourse being highest in adrenal liver and lymph nodemetastases and lower in bone and brain metastases Andthe predictive value of PDL1 at different biopsy sites forthe benefit of ICIs in NSCLC may vary higher PDL1 inlung or distant metastasis specimens was significantly associated with higher response rate PFS and OS whilePDL1 in lymph node metastasis biopsy was not associated with either response or survival [] Thirdly positive score and cutoff value of PDL1 expression is notstandardized [] At present PDL1 positive scoremainly focuses on the PDL1 expression level of tumorcells that is tumor proportion score TPS But PDL1is also expressed on immune cells such as lymphocytesand macrophages and stromal cells thus the investigators introduce the concept of combined positive scoreCPS which is the proportion score of the sum of PDL1 expressed by tumor cells and tumorassociated immune cells In addition PDL1 expression on immuneresponseto ICIsimmunetreatmentBiomarkers of tumorinfiltrating immune cellsOverall immune status of tumor microenvironmentThe pattern of tumor immune infiltration can be broadlyclassified into immuneinflamed immuneexcluded andimmunedesert [] Immuneinflamed is characterizedby the presence of CD8 and CD4 T cells in the tumorparenchyma accompanied by the expression of immunecheckpoint molecules [] indicating a potential antitumor[]immuneexcluded is characterized by the presence ofdifferent immune cell types in the aggressive margin orstroma of tumor but cannot infiltration into tumor parenchyma [ ] Analysis of pretreatment samples forantiPD1PDL1 revealed a relatively high abundance ofCD8T cells at the invasive margin in responders andserial sampling during treatment showed an increasedinfiltration of CD8T cells into tumor parenchyma []while immunedesert phenotype is characterized by theabsence of abundant T cells in the parenchyma orstroma of tumors and poor response to ICItreatment[] Recentlyimmunoscore has been proposed as avalid marker for characterizing the immune status oftumor microenvironment TME classifying tumors aswell as predicting treatment response and prognosis[] which involves the density of two lymphocyte populations CD8 and memory [CD45RO] T cells in thecenter and invading margin of tumor [] Mlecnik et al[] evaluated immunoscore in specimens of stageI“IV colorectal tumor and confirmed that it was significantly associated with PFS DFS and OS and multivariate analysis also showed the superiority of immunoscorein predicting disease recurrence and survival The valueof immunoscore to predicting ICIs efficacy is being validated internationally in clinical trials of melanoma andNSCLC []A wider assessment of active immune responses withinTME by immune gene expression profiling might effectively predict clinical benefit to ICIs strategies Analysisof total RNA and genes that were substantially differentbetween the patient groups in pretreatment tumor biopsies revealed atleast a 25fold increase in the 0cBai Biomarker Research Page of expression of immunerelated genes in clinically active patientsincluding cytotoxic T cell markers egCD8A perforin granzyme B Th1 cytokines or chemokines MHCII and other immunerelated genes egNKG7 IDO1 [] Ascierto [] screened morethan immunerelated genes in patients with recurrent breast cancer “ years after treatment and thosewithout recurrence more than years later and foundthat five genes IGK GBP1 STAT1 IGLL5 and OCLNwere highly overexpressed in patients with recurrencefree survival In addition IFNγinduced immune genesignatures may be effective biomarkers for predicting theclinical benefit of treatment with ICIs The study developed IFNγ scores combining multiple immune variablesbased on gene signatures which were then extendedto gene signatures in a validation set of melanomapatients including genes encoding IFNγ granzymes AB perforin IDO1 and other immunerelated genesBoth gene scores showed significant associations withbest overall response rate and PFS Optimized cutoffvalues for IFNγ scores based on receiver operatingcharacteristic curve ROC curve can achieve a positivepredictive value of for responders and a negativepredictive value of for nonresponders []Immune cells with specific phenotypes in TMEThe phenotype of TILs also influences the efficacy ofICIs The study used singlecell mRNA sequencingscRNAseq data analysis to identify two major CD8Tcell phenotypes within melanoma memorylike andexhausted [] the proportion of which is strongly correlated with response to ICIs The research furtherfound that the transcription factor TCF7 is selectivelyexpressed in memorylike T cells so the ratio ofCD8TCF7 to CD8TCF7TILs is strongly correlatedwith improved response and survival in melanoma patients treated with antiPD1 [] Balatoni []found that of immune cells in TME were positivelyassociated with OS after treatment including CD4 andCD8 T cells FOXP3 T cells CD20 B cells CD134and CD137 cells and NKp46 cells and different immune cells at different sites were differently associatedwith clinical outcomes Researchers found that only asmall proportion of CD8 TILsin tumors couldrecognize tumor mutationassociated antigens while another population bystander cells was insensitive anddifferential CD39 expression was the key molecule thatdistinguished the two populations [] Analysis of peripheral blood from a patient with colorectal cancer whoresponded rapidly to pembrolizumab treatment showedhigh expression of CD39 on CD8 TILs indicating thatCD39CD8TIL may be a promising predictive biomarker [] The fact of very low level of CD39 expression on CD8TILs in of EGFRmutant NSCLC isconsistent with their low response rate to antiPD1immunotherapyIn addition a study showed that Fc domain glycan ofthe drug and Fcγ receptor FcγR expressed by the hostbone marrow cells could determine the ability of PD1tumorassociated macrophages TAMs to capture antiPD1 drugs from the surface of T cells which leads toPD1 inhibitor resistance [] and the association ofTAMs and poor antiPD1 response was reported inmelanoma cohorts [] antiPD1 response was associated with an increase in CD8T cells and natural killercells NK cells and a decrease in macrophages [] andhigh intratumoral myeloid markers were associated witha nearly 6fold decrease in mPFS after antiPDL1 therapy in RCC emphasizing the inhibitory role of myeloidcells in response to ICIs [] In conclusion immunecells in TME show a great promise in the developmentof predictive biomarkers for ICIsimmunerepertoireDiversity of immune repertoires in TMEEffective T cell responses involve the activation and expansion of specific antigenreactive T cell clones so diversity ofin intratumoral orperipheral may correlate with ICIs responses and can bequantified as richness and clonality [] However theresults seem to be complex with some studies finding apositive correlation between TIL clonality and the response to ICIs before [] or after [] treatment whileothers showing that only an increase in TIL clonalityduring treatment is associated with the response to antiPD1 [ ] others show that intratumoral T cellclonality is not associated with survival while peripheralT cell clonality is inversely associated with PFS and OS[] Tumeh [] further investigated whetherbaseline TILs have a narrow T cell receptor TCR repertoire focusing on tumorspecific immune responsesand whether this narrow TCR repertoire correlates withpembrolizumab responses They found that respondingpatient had more restricted usage of the TCR beta chainie a more clonal less diverse population than patientswith progressive disease and showed a 10times increasein these clones after treatmentimplying a tumorspecific response to treatment in these patients Notablybaseline TCR clonality was not highly correlated withTIL density suggesting that some patients with restricted TCR clonality specific for tumor antigens maystill benefit from antiPD1 therapy even though TILdensity is low Recently researchers have proposed theimmune repertoire IRIndex the average frequency ofshared TCR clones in T clones in TILs and peripheralPD1CD8 T cells They found that neoantigenstimulated TCR agreed with IRIndex and patients withhigh IRindex had better immune activation and highergene expression profiles GEPs score subsequently they 0cBai Biomarker Research Page of confirmed the predictive value of IRindex to ICIs efficacy DCRPFS But considering that it is difficult tosort out PD1CD8 T cells in tumor tissue based ontwo separate patient cohorts a research confirmed thatTCR repertoire diversity and clonality of peripheral PD1CD8T cells may serve as noninvasive predictors ofclinical outcomes after ICIs in patients with NSCLC[] The viewpoints of T cell diversity and TCR clonality as markers of ICIs efficacy need to be further validated in a large patient populationiiiLiquid biopsy biomarkersPeripheral blood cell biomarkersPeripheral blood is a noninvasive source to explore potential biomarkers for ICIs and although associationswith clinical benefit and survival have been observed itseffectiveness has not been validated in prospective studies Analysis of melanoma treated with ipilimumabshowed that improved OS and PFS were associated withbaseline values of peripheral blood components including low absolute neutrophil countlow neutrophiltolymphocyte ratio NLR low absolute monocyte countlow frequency of myelogenous suppressor cells high frequency of FoxP3 Treg cells high lymphocyte frequencyhigh eosinophil count and clinical benefit also associated with the dynamic changes of blood markers duringincluding decreased FoxP3Treg concentratreatmenttions and increased lymphocyte and eosinophil counts[] Reports in patients with melanoma treated withpembrolizumab and in patients with NSCLC treatedwith nivolumab have shown that NLR is associated withworse tumor response [ ] Multivariate analysis inmelanoma patients treated with antiPD1 antibodiesshowed that NLR was the only factor associated withworse ORR and shorter PFS indicating that NLR is astrong predictor of worse outcome in patients treatedwith ICI [] Low baseline lactate dehydrogenase LDHlevels high relativeabsolute eosinophil counts and relative lymphocyte counts were associated with prolongedOS in antiPD1 and CTLA4 treated melanoma [] Given that previous studies have proposed the importance of baseline derived NLR dNLR and LDHlevels as prognostic markers a recent study proposed acomposite prognost

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

incidence and death rate of nonsmall cell lung cancer NSCLC in China ranks the first among the malignant tumors Circular RNA circRNA was reported to be involved in the progression of NSCLC Our study aimed to investigate the underlying mechanism of circ_0020123 in NSCLC progressionMethods Quantitative realtime polymerase chain reaction qRTPCR was used to detect the expression of circ_0020123 miR5905p and Thrombospondin THBS2 in NSCLC tissues and cells Cell proliferation and migration were examined by Cell Counting Kit8 CCK8 assay and Transwell assay respectively Flow cytometry assay was used to detect the apoptosis of NSCLC cells The protein levels of Ki67 matrix metalloprotein9 MMP9 Cleavedcaspase9 Cleavedcasp9 and THBS2 were detected by Western blot The targets of circ_0020123 and miR5905p were predicted by starBase and TargetScan and then confirmed by dualluciferase reporter assay and RNA immunoprecipitation RIP assay The animal experiment showed the effect of circ_0020123 on tumor growth in vivoResults The expression of circ_0020123 was upregulated in NSCLC tissues and cells Functionally circ_0020123 downregulation inhibited the proliferation and migration and promoted the apoptosis of NSCLC cells Interestingly circ_0020123 directly targeted miR5905p and inhibition of miR5905p reversed the knockdown effects of circ_0020123 on NSCLC cells More importantly THBS2 was a target of miR5905p and THBS2 overexpression reversed the effects of circ_0020123 knockdown on cell proliferation migration and apoptosis in NSCLC cells Finally suppression of circ_0020123 inhibited tumor growth in vivo through miR5905pTHBS2 axisConclusion Circular RNA circ_0020123 regulated THBS2 by sponging miR5905p to promote cell proliferation and migration and inhibit cell apoptosis in NSCLC cellsKeywords NSCLC Circ_0020123 miR5905p THBS2Highlights Circ_0020123 was upregulated and downregulation of circ_0020123 inhibited cell proliferation migration and promoted cell apoptosis in NSCLC cellsCorrespondence bskrju163comDepartment of Thoracic Surgery Lianyungang Second People™s Hospital No Hailian East Road Haizhou District Lianyungang Jiangsu China Circ_0020123 directly targeted miR5905p and miR5905p downregulation reversed the knockdown effects of circ_0020123 on NSCLC progression THBS2 acted as a target of miR5905p and overthe effects of expression of THBS2 reversed circ_0020123 knockdown on NSCLC progression Downregulation of circ_0020123 suppressed tumor growth in vivo through miR5905pTHBS2 axis 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 0cWang a0et a0al Cancer Cell Int Page of BackgroundLung cancer has the highest incidence of total cases and is the most common cause of cancer death of total cancer deaths in worldwide [] Lung cancer can be divided into several histological subtypes according to the location and the tendency of metastasis Small cell lung cancer SCLC accounts for about of all lung cancer cases [] However nonsmall cell lung cancer NSCLC accounts for of lung cancer and the a0years overall survival rate OS is only about [] Therefore it is important to find the effective treatment and potential molecular targets of NSCLC progressionCircular RNA circRNA is a single stranded RNA molecule with a closed circular structure Recently amounts of circular DNA have been discovered and most of which were thought to be the byproducts of typical splicing [ ] Previous reports indicated that the expression of circRNA was tissuespecific and the change of its expression intensity was associated with some diseases [“] Furthermore circRNA was involved in the occurrence and development of the disease and might be used as a potential biomarker in clinical diagnosis prognosis and treatment of diseases [ ] For example circ_0039569 facilitated cell proliferation and migration of renal cell carcinoma by sponging miR34a5p to regulate CC Chemokine ligand CCL22 [] Meanwhile hsa_circ_0043256 participated in the progression of NSCLC cells by mediating the cinnamaldehyde treatment [] A previous report suggested that circ_0020123 acted as an oncogene in NSCLC and circ_0020123 regulated zincfingerenhancer binding protein ZEB1 and enhancer of zeste homolog EZH2 by competitively binding with miR144 to induce cell progression and migration [] These reports suggested that circ_0020123 was a vital factor in the pathogenesis of NSCLC and its function and molecular mechanism need to be further studiedAs a small endogenous RNA microRNA miRNA is essential in regulating gene expression and plays a potential role in the exploitation of biomarkers [] Recently some aggregated miRNAs have been found in prostate cancer such as miR221222 miR143145 miR23b27b241 and miR1133a which were downregulated and had tumor inhibiting functions [] A previous study found that circulating miR5905p could be used as routine diagnostic tools for lung cancer and as a potential prognostic marker for liquid biopsy Besides overexpression of miR5905p reduced the development of NSCLC cells and regulated the expression of epithelialmesenchymal transformation EMTrelated proteins by targeting the signal transducers and activators of transcription STAT3 [] However the precise mechanism by which miR5905p affects NSCLC needs further investigationThrombospondin THBS2 as a secreted protein was confirmed to be highly expressed in different cancers including cervical cancer [] colorectal cancer [] and NSCLC [] A previous report suggested that THBS2 was involved in the proliferation apoptosis and antiautophagy regulation of cervical cancer cells by miR20a [] Tian et a0al found the expression and clinicopathological features of THBS2 in colorectal cancer were significantly correlated with the prognosis of cancer and might be used as a biomarker of prognosis [] However the molecular function of THBS2 in NSCLC remains poorly definedIn this study the targeting relationship between circ_0020123 and miR5905p was firstly detected The effects of circ_0020123 on cell proliferation migration apoptosis and tumor growth were performed by gain and lossoffunction experiments and molecular biology techniquesMaterials and a0methodsPatients and a0specimensNSCLC tissues and the adjacent healthy lung tissues were taken from NSCLC patients in the Lianyungang Second People™s Hospital All volunteers signed written informed consents NSCLC tissues and the adjacent tissues were immediately frozen in liquid nitrogen and kept at ˆ’ a0 °C for further experiments This research was approved by the Ethics Committee of Lianyungang Second People™s HospitalCell culture and a0cell transfectionTwo NSCLC cell lines A549 and H1299 and one normal lung cell line IMR90 were obtained from the Beijing Concorde Cell Library Beijing China A549 H1299 and IMR90 cells were cultivated in Dulbecco™s modified eagle medium DMEM HyClone Logan UT USA supplementing with fetal bovine serum FBS HyClone and cultured in an incubator at a0„ƒ with CO2Small interfering RNA siRNA targeting circ_00201231 sicirc_00201231 and sicirc_00201232 short hairpin RNA shRNA targeting circ_0020123 shcirc_0020123 miR5905pinhibitors siRNA negative control siNC shNC and NCinhibitors were all obtained from Biomics Biotech Jiangsu China Full length of THBS2 cDNA Sangon Biotech Shanghai China was subcloned into pcDNA31 plasmid EKBioscience Shanghai China Then cell transfection was performed by Lipofectamine Thermo Fisher Scientific Waltham MA USA 0cWang a0et a0al Cancer Cell Int Page of RNA isolation and a0quantitative real‘time polymerase chain reaction qRT‘PCRThe TRIzol reagent Invitrogen Carlsbad CA USA was used for extracting the total RNAs Next the reversed transcription was carried out by RTPCR kit Invitrogen The qRTPCR was performed using the ABI SYBR Green Master Mix Invitrogen The primers in our study were as follows F5²TTC GGA CGA CCG TCA AAC AT3² and R5²AGG ATC CCT GCA CCA CAA TG3² for circ_0020123 F5²TGA AAG ACG TGA TGG CAC AC3² and R5²CTT CCA TTT TGG for miR5905p F5²AGA AGG GGT TTT TGG3 ² CTG GGG CTC ATT TG3² R5²AGG GGC CAT CCA CAG TCT TC3² for glyceraldehyde3phosphate dehydrogenase GAPDH [] F5²GCG GCT GGG TCT ATT TGT C3² and R5²GCA GGA GGT GAA GAA CCA TC3² for THBS2 [] F5²ATT GGA ACG ATA CAG AGA AGATT3² and R5²GGA ACG CTT CAC GAA TTT G3² for U6 [] GAPDH and U6 were the internal parametersCell Counting Kit‘ CCK‘ assayThe proliferation viability of A549 and H1299 cells were detected by the CellCounting Kit8 MSK Wuhan China A549 and H1299 cells were cultivated into a 96well plate with a density of × a0cellswell and incubated in a0°C for or a0h Then a0μL fresh medium and CCK8 solution was added After incubation at a0°C for a0h the OD values were detected by the Multiskan Ascent microplate reader Abcam Cambridge MA USATranswell assayTranswell chamber Corning Life Sciences Corning NY USA was used to detect cell migration Firstly the serumfree DMEM Thermo Fisher Scientific was fixed with cell suspension cells and seeded into the upper chamber and the DMEM containing serum was put into the lower chamber After incubation for a0h the cells in lower surface of the upper chamber were treated with formaldehyde solution for a0 h and then thoroughly washed Finally the migrated cells were stained with crystal violet Corning Life Sciences and observed by using a microscopeFlow cytometryFirstly A549 and H1299 cells were cultured and PBS was used for washing cells Then the binding buffer was used to resuspend cells and the Annexin Vfluorescein isothiocyanate VFITCpropidium iodide PI Apoptosis Detection Kit Thermo Fisher Scientific was used to stain cells Finally cell apoptosis was detected by flow cytometry Thermo Fisher ScientificWestern blot analysisThe total proteins of NSCLC tumors or cells were collected by RIPA lysis buffer Sangon Biotech Then the proteins were separated by Sodium dodecyl sulphate polyacrylamide gel electrophoresis SDSPAGE and transferred to polyvinylidene fluoride PVDF membranes Thermo Fisher Scientific The skimmed milk was added and incubated with primary antiGAPDH antibody Invitrogen Carlsbad CA USA antiβactin antibody Invitrogen antiKi67 antibody Invitrogen antimatrix metalloprotein9 MMP9 antibody Invitrogen antiCleavedcaspase9 Cleavedcasp9 antibody Invitrogen or antiTHBS2 antibody Invitrogen at a0°C overnight Finally the membranes were incubated with the secondary antibody for a0 h at room temperature The results were viewed using Kodak film developer Fujifilm JapanDual‘luciferase reporter assaysThe wild type circ_0020123 sequences circ_0020123WT mutant circ_0020123 sequences circ_0020123MUT wild type THBS2 ²UTR sequences THBS2WT mutant THBS2 ²UTR sequences THBS2MUT were cloned into pGL3 luciferase reporter plasmid Promega Madison WI USA Then the plasmid and miR5905p or miRNC were cotransfected into A549 and H1299 cells by Lipofectamine Thermo Fisher Scientific After transfection for a0h the DualLuciferase Reporter Assay System Promega was performed to detect the luciferase activityRNA immunoprecipitation RIPFirstly the Magna RIP RNAbinding Protein Immunoprecipitation Kit gzscbio Guangzhou China was performed to verify the relationship between circ_0020123 and miR5905p In brief the magnetic beads and antiAgo2 antibody Abcam were added into cells and incubated for a0h Then the proteinase K and the phenol“chloroformisoamyl alcohol reagent were added for purifying RNAs Finally qRTPCR was used to measure circ_0020123 enrichmentAnimal experimentsThe 4weekold BALBc male nude mice Vitalriver Beijing China were raised in a sterile environment for 0cWang a0et a0al Cancer Cell Int Page of experiments Then PBS was used to suspend A549 cells × transfected with shcirc_0020123 or shNC Next the nude mice were divided into two groups n A549 cells transfected with shcirc_0020123 or shNC were shcirc_0020123 or shNC inoculated into the nude mice The tumor volume was detected every a0 days After a0days the nude mice were euthanatized and the tumor weight was detected Besides the tumor tissues from each group were collected to detect the expression of circ_0020123 miR5905p and THBS2 The animal experiment was approved by the Animal Experimentation Ethics Committee of Lianyungang Second People™s HospitalStatistical analysisThe software GraphPad Prism was performed for statistical analysis The data was displayed as mean ± standard deviation SD The significant difference was calculated by Student™s t test and oneway analysis of variance ANOVA P was considered as statistically significantResultsCirc_0020123 was a0upregulated in a0NSCLC tissues and a0cellsTo begin with qRTPCR was used to detect the expression of circ_0020123 the result showed that circ_0020123 was significantly upregulated in NSCLC tissues compared with the adjacent healthy tissues Fig a0 1a Similarly the expression of circ_0020123 in NSCLC cells A549 and H1299 was markedly higher than that in normal cells IMR90 Fig a01b From these data it is speculated that circ_0020123 might be acted as an oncogene in NSCLCFig Circ_0020123 was upregulated in NSCLC tissues and cells a QRTPCR was used to detect the expression of circ_0020123 in adjacent healthy tissues n and tumor tissues n b The expression of circ_0020123 in normal cell line IMR90 and NSCLC cell lines A549 and H1299 was detected by qRTPCR P Downregulation of a0circ_0020123 inhibited the a0proliferation migration and a0induced apoptosis of a0NSCLC cellsTo investigate the functional effects of circ_0020123 on NSCLC cells sicirc_00201231 and sicirc_00201232 were obtained and transfected into A549 and H1299 cells Firstly the transfection efficiency was detected by qRTPCR Fig a02a Next CCK8 was used to detect the proliferation and the results showed that the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was reduced Fig a0 2b Moreover the migration of A549 and H1299 cells was significantly downregulated by circ_0020123 knockdown Fig a02c In addition the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was obviously higher than transfected with siNC Fig a02d Finally the protein levels of cell proliferationrelated protein Ki67 and cell migrationrelated protein MMP9 were inhibited while cell apoptosisrelated protein Cleavedcasp9 was upregulated in NSCLC cells transfected with sicirc_00201231 or sicirc_00201232 Fig a02e These data suggested that inhibition of circ_0020123 suppressed cell proliferation migration and promoted apoptosis in NSCLC cellsCirc_0020123 directly targeted miR‘‘5pBy searching in the online software starBase the potential binding sites between circ_0020123 and miR5905p were detected Fig a0 3a To confirm that the dualluciferase reporter assay was performed the results showed that the luciferase activity of circ_0020123WT reporter plasmid was reduced by miR5905p mimic while the circ_0020123MUT reporter plasmid activity was not changed in A549 and H1299 cells Fig a03b Furthermore the expression of miR5905p was lower in A549 and H1299 cells compared with that in IMR90 cells Fig a0 3c In contrast miR5905p expression was elevated in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 Fig a0 3d Finally the RIP assay was also used to confirm the targeting relationship between circ_0020123 and miR5905p and the results showed that circ_0020123 and miR5905p were enriched in antiAgo2 group Fig a03eMiR‘‘5p downregulation reversed the a0inhibition effects of a0circ_0020123 on a0NSCLC cellsTo further explore the functional effects between circ_0020123 and miR5905p miR5905pinhibitor was established QRTPCR was used to detect the transfection efficiency Fig a0 4a Interestingly miR5905p was upregulated in A549 and H1299 cells transfected with sicirc_00201231 while the expression of miR5905p was recovered in cells transfected with 0cWang a0et a0al Cancer Cell Int Page of Fig Downregulation of circ_0020123 inhibited the proliferation and migration and induced the apoptosis of NSCLC cells a The transfection efficiency of sicirc_00201231 and sicirc_00201232 in A549 and H1299 cells was detected by qRTPCR b CCK8 assay was used to detect the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 c The migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was measured by Transwell assay d Flow cytolysis assay was used to detect the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 e The protein levels of cell proliferation related protein Ki67 cell migration related protein MMP9 and cell apoptosis related protein Cleavedcasp9 were detected by Western blot P Fig a0sicirc_00201231 miR5905pinhibitors 4b Moreover circ_00201231 knockdown inhibited cell proliferation and migration while the miR5905p inhibitor reversed these effects Fig a0 4c d In addition the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 was increased which was abolished by miR5905pinhibitor Fig a0 4e Similarly miR5905p inhibitors reversed the effects on the protein levels of Ki67 MMP9 and Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 Fig a0 4f These results that miR5905p downregulation reversed the effects of circ_0020123 downregulation on the proliferation migration and apoptosis of NSCLC cellsindicated MiR‘‘5p targeted THBS2 in a0NSCLC cellsThe THBS2 ²UTR was predicted to contain the binding sites of miR5905p through the online software TargetScan Fig a05a Then the dualluciferase reporter assay was used to confirm the targeting relationship The results showed that cotransfection of miR5905p and THBS2WT significantly limited the luciferase activity in both A549 and H1299 cells the luciferase activity was not altered in cells cotransfected with miR5905p and THBS2MUT Fig a05b Importantly the mRNA and protein level of THBS2 was enahnced in NSCLC cells Fig a05c d We further explored whether circ_0020123 affected the functions of THBS2 in NSCLC cells The mRNA and protein expression of THBS2 were repressed in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 Fig a05e f 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0020123 directly targeted miR5905p a The binding site between circ_0020123 and miR5905p was detected by the online software starBase b The luciferase activity of circ_0020123WT or circ_0020123MUT reporter plasmid in A549 and H1299 cells transfected with miRNC or miR5905p was detected by dualluciferase reporter assay c QRTPCR was used to detect the expression of miR5905p in A549 and H1299 cells d The expression of miR5905p in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qRTPCR e RIP assay was used to confirm the relationship between circ_0020123 and miR5905p P THBS2 overexpression reversed the a0effects of a0circ_0020123 knockdown on a0the a0proliferation migration and a0apoptosis of a0NSCLC cellsBased on the work ahead of us the pcDNA31THBS2 was constructed Then the qRTPCR and Western blot were used to detect the transfection efficiency and the THBS2 expression was increased in A549 and H1299 cells transfected with pcDNA31THBS2 Fig a0 6a b In addition the proliferation and migration rates of A549 and H1299 cells transfected with sicirc_00201231 pcDNA31THBS2 were higher than that transfected with sicirc_00201231 Fig a0 6c d Meanwhile a similarly phenomenon was also observed in cell apoptosis the pcDNA31THBS2 significantly reversed the promotion effect of circ_0020123 deletion on cell apoptosis Fig a0 6e Furthermore the effects of circ_0020123 deletion on Ki67 MMP9 and Cleavedcasp9 protein levels were also reversed by THBS2 overexpression Fig a0 6f These data suggested that overexpression of THBS2 could reverse the effects of circ_0020123 downregulation on cell proliferation migration and apoptosisReduction of a0circ_0020123 suppressed tumor growth in a0vivo through a0circ_0020123miR‘‘5pTHBS2 axisTo further explore the function of circ_0020123 in NSCLC cells the shcirc_0020123 was constructed and the xenograft tumor was established Then A549 cells transfected with shcirc_0020123 or shNC were injected into the nude mice The xenograft tumor volume was measured every a0 days after injection and the results showed that tumor volume was smaller shcirc_0020123 group than that in shNC group Fig a07a Moreover tumor weight was inhibited by circ_0020123 knockdown Fig a0 7b Furthermore the expression circ_0020123 and THBS2 was decreased while the miR5905p was increased in xenograft tumor transfected with shcirc_0020123 Fig a0 7c Western blot assay also revealed that the protein level of THBS2 was repressed by circ_0020123 knockdown Fig a07d Finally the digital tomosynthesis DTS was used to detect the number of lung metastatic nodules in xenograft tumor and it was reduced in shcirc_0020123 group Fig a07e The results suggested that downregulation of circ_0020123 inhibited tumor growth in a0vivoDiscussionClinically only a few NSCLC patients were diagnosed at an early stage and treated by surgical resection More than of NSCLC patients were diagnosed with the advanced stage or metastatic tumors [] Thus finding novel biomarkers and therapeutic targets were necessary for the effective diagnosis and treatment of NSCLCRecently circRNA was no longer considered as a random product in the RNA shearing process and its biological significance and function in malignant tumors 0cWang a0et a0al Cancer Cell Int Page of Fig MiR5905p downregulation reversed circ_0020123 knockdown effects in NSCLC cells a QRTPCR was used to detect the expression of miR5905p in A549 and H1299 cells transfected with miR5905pinhibitors b The expression of miR5905p in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors was detected by qRTPCR c The proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors was tested by CCK8 assay d Transwell assay was used to measure the migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors e Flow cytolysis assay was used to detect the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors f The protein levels of Ki67 MMP9 Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors were detected by Western blot P had received more and more attention Previous reports revealed that circ_0020123 was involved in the development of NSCLC [] Moreover the level of circ_0020123 was elevated in NSCLC cells [] Consistently we found that the expression of circ_0020123 was markedly higher in NSCLC tissues and cells Moreover this research indicated that inhibition of circ_0020123 suppressed the proliferation migration and induced apoptosis of NSCLC cells in a0 vitro Besides circ_0020123 promoted tumor growth in a0vivoEndogenous circRNAs could act as microRNA sponges to inhibit their function and some studies linked miRNA sponges to human diseases including cancer [] A previous study indicated that circRNA ctransferrin receptor cTFRC regulated TFRC by sponging miR107 to facilitate bladder carcinoma development [] MiR5905p was studied in different cells such as airway smooth muscle cells [] colon epithelial cells [] and NSCLC cells [] However the potential relationship between miR5905p and circRNA has not been researched In this study circ_0020123 directly targeted miR5905p and miR5905p inhibition reversed the effects of circ_0020123 knockdown on NSCLC progression These data provided a clue to the therapeutic strategy for NSCLC 0cWang a0et a0al Cancer Cell Int Page of Fig MiR5905p targeted THBS2 in NSCLC cells a The potential binding site between THBS2 ²UTR and miR5905p was predicted by the online software TargetScan b Dualluciferase reporter assay was used to measure the luciferase activity of THBS2WT or THBS2MUT reporter plasmid in A549 and H1299 cells transfected with miRNC or miR5905p c QRTPCR was used to detect the mRNA expression of THBS2 in NSCLC cells d The protein level of THBS2 in NSCLC cells was tested by Western blot e The mRNA expression of THBS2 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qRTPCR f Western blot was used to measure the protein level of THBS2 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 P Our study also confirmed that miR5905p could target THBS2 directly in NSCLC cells THBS2 is a calciumbinding protein that binds to and inactivates matrix metalloproteinase MMP genes involved in tissue formation and repair [ ] A previous document suggested that THBS2 acted as a target of miR2213p and participated in lymph node metastasis in cervical cancer [] The data in this research showed that the expression of THBS2 in NSCLC cells was markedly higher than normal healthy cells Furthermore overexpression of THBS2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of NSCLC cells suggesting that circ_0020123 promoted the progression of NSCLC cells through miR5905pTHBS2 axisConclusionIn conclusion our research showed that the expression of circ_0020123 was higher in NSCLC tissues and cells than control and downregulation of circ_0020123 inhibited the proliferation migration and promoted apoptosis of NSCLC cells and also suppressed tumor growth in a0 vivo Moreover circ_0020123 directly targeted miR5905p while miR5905p inhibition reversed the effects of circ_0020123 knockdown on NSCLC cells More importantly circ_0020123 regulated the expression of THBS2 by sponging miR5905p and upregulation of THBS2 reversed the effects of circ_0020123 knockdown on NSCLC cells Therefore our research demonstrated that circ_0020123 enhanced proliferation migration and inhibited 0cWang a0et a0al Cancer Cell Int Page of Fig Overexpression of THBS2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of NSCLC cells a b The mRNA and protein expression of THBS2 in A549 and H1299 cells transfected with pcDNA31THBS2 was detected by qRTPCR and Western blot c CCK8 assay indicated the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 d The migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 was measured by Transwell assay e The apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 was detected by Flow cytolysis assay f The protein levels of Ki67 MMP9 Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 were detected by Western blot P apoptosis of NSCLC cells by sponging miR5905p to regulate THBS2results and develop the manuscript All authors read and approved the final manuscriptAbbreviationsNSCLC Nonsmall cell lung cancer circRNA Circular RNA qRTPCR Quantitative realtime polymerase chain reaction CCK8 Cell Counting Kit8 MMP9 Matrix metalloprotein9 Cleavedcasp9 Cleavedcaspase9 Cleavedcasp9 Cleavedcaspase9 RIP RNA immunoprecipitation ZEB1 Zincfingerenhancer binding protein EZH2 Zeste homolog STAT3 Signal transducers and activators of transcription THBS2 Thrombospondin AcknowledgementsNot applicableAuthors™ contributionsLW collaborated to design the study LZ were responsible for experiments analyzed the data YW wrote the paper All authors collaborated to interpret FundingNoneAvailability of data and materialsPlease contact corresponding author for data requestsEthics approval and consent to participateThis research was approved by the Ethics Committee of Lianyungang Second People™s Hospital The animal experiment was approved by the Animal Experimentation Ethics Committee of Lianyungang Second People™s HospitalConsent for publicationAll listed authors have actively participated in the study and have read and approved the submitted manuscript 0cWang a0et a0al Cancer Cell Int Page of Fig Reduction of circ_0020123 suppressed the tumor growth in vivo through circ_0020123miR5905pTHBS2 axis a A total of × A549 cells transfected with shcirc_0020123 or shNC were injected into nude mice to establish the xenograft tumor Tumor volume was measured every d after injection b Tumor weight was measured on d c The expression of circ_0020123 miR5905p and THBS2 in xenograft tumor was measured by qRTPCR d The protein level of THBS2 in xenograft tumor was evaluated by Western blot e The number of lung metastatic nodules in xenograft tumor was detected by digital tomosynthesis DTS P Competing interestsThe authors declare that they have no competing interestsReceived April Accepted July References Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “ Abe H Takase Y Sadashima E Fukumitsu C Murata K Ito T Kawahara A Naito Y Akiba J Insulinomaassociated protein is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions validity and reliability with cutoff value Cancer Cytopathol “Li C Zhang L Meng G Wang Q Lv X Zhang J Li J Circular RNAs pivotal molecular regulators and novel diagnostic and prognostic biomarkers in nonsmall cell lung cancer J Cancer Res Clin Oncol “ Belousova EA Filipenko ML Kushlinskii NE Circular RNA new regulatory molecules Bull Exp Biol Med “ Zhang Z Xie Q He D Ling Y Li Y Li J Zhang H Circular RNA new star new hope in cancer BMC Cancer Li L Chen Y Nie L Ding X Zhang X Zhao W Xu X Kyei B Dai D Zhan S Guo J Zhong T Wang L Zhang H MyoDinduced circular RNA CDR1as promotes myogenic differentiation of skeletal muscle satellite cells Biochim Biophys Acta Gene Regul Mech “ Greco S Cardinali B Falcone G Martelli F Circular RNAs in muscle function and disease Int J Mol Sc

Thyroid_Cancer

Supraclavicular Recurrence inCompletely Resected ypN2NonSmall Cell Lung CancerImplications for PostoperativeRadiotherapyLiang Liu   Zhiqin Zheng   Juan Li Yuan Li and Jianjiao Ni Department of Radiation Oncology Fudan University Shanghai Cancer Center Shanghai China Department of OncologyShanghai Medical College Fudan University Shanghai China Department of Radiation Oncology Minhang BranchHospital Fudan University Shanghai Cancer Center Shanghai China Department of Pathology Fudan University ShanghaiCancer Center Shanghai Chinatarget volume CTVBackground The clinical value and delineation of clinicalof postoperative radiotherapy PORTin completely resected ypN2 nonsmall celllung cancer NSCLC remain controversial Investigations specifically focusing on thecumulative incidence and prognostic significance of initial disease recurrence at thesupraclavicular region SCR in this disease population are seldom reportedMethods Consecutive patients with curatively resected ypN2 NSCLC who receivedadjuvant chemotherapy from January to December at our cancer center wereretrospectively examined Disease recurrence at the surgical margin ipsilateral hilumandor mediastinum was defined as locoregional recurrence LRR Disease recurrencebeyond LRR and SCR was defined as distant metastasis DM Overall survival OS1 andOS2 were calculated from surgery and disease recurrence to death of any cause in theentire cohort and in patients with recurrent disease respectivelyResults Among the patients enrolled PORT without elective supraclavicularnodal irradiation ESRT was performed in patients and neoadjuvant chemotherapywas administered in patients With a median followup of months patientsdeveloped recurrent disease including SCRs among which were without DMand involved the ipsilateral supraclavicular region The and 5year cumulativeincidence of SCR were and respectively Chosen DM as a competingevent cN2 ypN2 not receiving lobectomy and negative expression of CK7 weresignificantly associated with SCR using the univariate competing risk analysis whileypN2 was identified as the only independent risk factor of SCR p PORTsignificantly reduced LRR p and prolonged OS1 p but didn™timpact SCR p Pattern of failure analyses indicated that the majority of LRRsdeveloped within the actuarial or virtual CTV of PORT and of the ipsilateralSCRs could be covered by the virtual CTV of proposed ESRT In terms of OS2patients who developed SCR but without DM had intermediate prognosis comparedwith those who had DM p and those who had only LRR p Edited byStephen V LiuGeetown University MedicalCenter United StatesReviewed byTim KruserNorthwestern Medicine United StatesHeloisa De Andrade CarvalhoUniversity of S£o Paulo BrazilCorrespondenceJianjiao Ninijianjiao8sinacom These authors have contributedequally to this workSpecialty sectionThis was submitted toThoracic Oncologya section of the journalFrontiers in OncologyReceived May Accepted July Published August CitationLiu L Zheng Z Li J Li Y and Ni J Supraclavicular Recurrence inCompletely Resected ypN2NonSmall Cell Lung CancerImplications for PostoperativeRadiotherapy Front Oncol 103389fonc202001414Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCConclusions SCR is not uncommon and has important prognostic significance incompletely resected ypN2 NSCLC The clinical value of PORT and ESRT in suchpatients need to be further investigatedKeywords supraclavicular recurrence postoperative radiotherapy nonsmall cell lung cancer overall survivalclinical target volumeINTRODUCTIONStage III nonsmall cell lung cancer NSCLC is a heterogeneousdisease and surgical resection with or without neoadjuvanttherapy could be carried out in selected patients Aftercurative resection disease recurrence poses a considerablethreat and it has been demonstrated that platinumbasedadjuvant chemotherapy could significantly reduce postoperativerecurrence and improve 5year survival Howeveralthough numerous retrospective studies and several populationbased investigations “ have suggested a beneficial role ofpostoperative radiotherapy PORT in reducing locoregionalrecurrence LRR prolonging diseasefree survival DFS andeven improving overall survival OS among patients withcompletely resected ypN2 NSCLC the clinical valueof PORT is still controversial due to a lack of convincing datafrom large randomized clinical trials Moreover there is no definite agreement on the delineationof clinical target volume CTV during PORT for completelyresected ypN2 NSCLC and it varies between diï¬erentinstitutions and clinical trials The rationales of CTVdelineation are mostly based on the patterns of disease recurrencein surgical resected patients who don™t receive PORT and patternsof treatment failure in those who receive PORT In these studiescumulative incidence anatomic locations and risk factors of LRRwere extensively examined However the definitions of LRR arediï¬erent some of which include the initial disease recurrencedeveloped in the supraclavicular region SCR whileothers don™t Investigations specifically focused on SCR areseldom reported and elective supraclavicular nodal irradiationESRT is not routinely performedIn the current study we investigated the cumulative incidencerisk factor and prognostic significance of SCR in completelyresected ypN2 NSCLC Additionally our recent study findscrucial prognostic value of routine immunohistochemical IHCmarkers in completely resected NSCLC Hence besidescommon clinicpathological variables a list of routine IHCmarkers were examined when investigating the risk factorsof SCRMATERIALS AND METHODSPatientsLung cancer patients who received surgery at Fudan Universityfrom January toShanghai Cancer Center FUSCCreviewed PatientsDecemberwho underwentresection withpathologically confirmed N2 disease and received standardretrospectively werecompletesurgicaladjuvant chemotherapy were included in the study Patientsreceived PORT or not as well as neoadjuvant chemotherapyor not were both allowed to be included Exclusion criteriaincluded a second primary tumor compromised resectionpositive surgical margins neoadjuvant radiotherapy receivingno adjuvant chemotherapy death due to surgical complicationsand postoperative follow up monthsinvasion perineuralFor each patient common clinicpathological parameterswere gathered from the electronic medical records including agesex smoking history the Eastern Corporative Oncology GroupECOG performance score clinical TNM stage pathologicalTNM stage primary tumor size tumor diï¬erentiation tumorlymphovascular invasion visceralhistology tumor locationpleuralinvasion and type of surgeryPathologic TNM stage was in accordance with the eighth editionLung Cancer Stage Classification Tumor diï¬erentiationand tumor histology were determined on the basis of the World Health anization Classification of Tumors of the LungPleura Thymus and Heart Besides the expression statusof IHC markers ie HER2 TTF1 ERCC1 CK20 CK56CK7 P63 NapsinA Syn RRM1 EGFR and Ki67 were collectedThe IHC staining and evaluation were routinely performedin the Immunohistochemistry Diagnostic Laboratory of ourcancer center Our study followed The Declaration of HelsinkiThe institutional review board of FUSCC approved the studyInformed consent was waived by the institutional review boardbecause this was a retrospective studyTreatmentPretreatment evaluation generally included clinical assessmentblood test bronchoscopy contrastenhanced chest computedtomography CT scan ultrasonographic examination or CTthe abdomen brain magnetic resonance imagingscan ofMRI and bone scans Patients with mediastinallymphnode enlargement cm in the short axis on CT scan orpathologically proven to be malignant were defined as harboringclinical N2 cN2 disease Of note positron emission tomographyPETCT as well as invasive staging of the mediastinum wasstrongly recommended for patients with cN2 disease at ourcancer centerNeoadjuvanttherapy generally consisted of “ cyclesof platinumbased doublet regimen and surgicaltreatmentincluded lobectomy sublobectomy and pneumonectomy withsystematic multilevel mediastinallymph node dissection oradequate mediastinal sampling no N2 stations must includethe subcarinal station PORT was performed according toour institutional protocol using the intensitymodulatedradiation therapy technique employing a linear accelerator withFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLC6MV Xrays Briefly the CTV for left lung cancers included thebronchial stump and 2R 2L 4R 4L and 11L lymphnode stations while the CTV for right lung cancers included thebronchial stump and 2R 4R and 11R stations ESRT wasnot performed The total radiation dose prescibed to the planningtarget volume PTV was generally Gy administered daily at Gy per fraction days per weekFollow UpFollowups were at the discretion of the treating physicians andwere generally scheduled at regular intervals every monthsafter surgery in the first years every months for the next years and annually thereafter During followup blood testschest CT scans and CT scans or ultrasonographic examination ofabdominal and cervical regions were routinely performed whilebrain MRI and bone scans were not mandatory Telephone callswere also implemented when necessaryPostoperative recurrence was diagnosed considering allthe evidence provided by imaging scans and pathologicconfirmation Initial disease recurrence in the supraclavicularregion was defined as SCR and first relapse developed at thesurgical marginipsilateral hilum andor mediastinum wasconsidered LRR Initial disease recurrence beyond LRR and SCRwas categorized as distant metastasis DMPattern of Failure AnalysesFor patients with LRR the PTVs were restored for thosewho received PORT and virtual PTVs were created for thosewho didn™t receive PORT by independent radiation oncologistaccording to ourinstitutional protocol mentioned aboveMeanwhile for patients with SCR individual virtual PTVs werecreated for ipsilateral ESRT PTVsc by independent radiationoncologist according to the CT atlas proposed by Lynch et al Then we plotted the sites of LRRs andor SCRs and overlaidthem with restored or created PTVs Coverage of the LRRs andSCRs by the PTVs were investigatedStatistical AnalysesRecurrence free survival RFS was calculated from surgery toinitial disease recurrence Overall survival OS1 was calculatedfrom surgery to death of any cause in the entire cohort andOS2 was calculated from initial disease recurrence to deathof any cause in patients with recurrent disease Diï¬erences andbetween clinical parameters were compared using the χFisher™s exact tests The predictors of SCR were selected usingcompeting risk methodology and Stata version softwareStataCorp College Station TX USA The associations betweenclinicpathological parameters and OS were identified using theCox proportional hazard regression model The hazard ratioHR and the confidence interval CI were calculatedusing coefficients from the model Kaplan“Meier methodwas used to estimate survival and diï¬erences among groupswere investigated by the logrank test Statistical analysis wasperformed using SPSS SPSS Chicago IL USA Allassessment is considered to be significant when twosided pvalueis RESULTSPatients CharacteristicsA total of patients were finally enrolled and a flowchartfor patient selection was presented in Supplementary Figure Detailed baseline disease characteristics of the patientswere summarized in Table The majority of patients had ahistology of nonsquamous NSCLC and received lobectomyTABLE Disease characteristicsVariablesNumber of patients Age at diagnosis years‰¤SexFemaleMaleSmoking historyEver smokerNever smokerECOG performance scoreClinical N stagecN0“cN2Neoadjuvant chemotherapyYesNoSurgery typeSublobarLobectomyPneumonectomyPathological T stagepT0“pT3“Lymphovascular invasionAbsentPresentVisceral pleural invasionAbsentPresentTumor locationLeft lower lobeLeft upper lobeRight lower lobeRight middle lobeRight upper lobeHistologySquamous cell carcinomaNonsquamous nonsmall cell lung cancerECOG Eastern Corporative Oncology Group Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCFIGURE Patterns of supraclavicular recurrence A Venn diagram demonstrating the distribution of initial postoperative recurrence B Pie chart demonstrating thedistribution of SCR SCR supraclavicular recurrence LRR locoregional recurrence DM distant metastasisFIGURE Cumulative incidence and dynamics of supraclavicular recurrence A Cumulative incidence of supraclavicular recurrence in the entire cohort andstratified by pathological status ypN2 vs pN2 B The dynamics of hazard ratio of supraclavicular recurrenceThe positive rate of HER2 TTF1 ERCC1 CK20 CK56 CK7P63 NapsinA Syn RRM1 and EGFR was and respectivelyAdditionally Ki67 ‰¥ was detected in of the patientsPretreatment PETCT was performed in patients andinvasive staging of the mediastinum was underwent in patients One hundred and sixtyfour patients were found tohave cN2 disease among whom patients receivedpretreatment PETCT and patients had invasivestaging of the mediastinum A total of patientsreceived neoadjuvant chemotherapyCumulative Incidence and Risk Factors ofSCRPost surgery patients received PORT and with a medianfollow up of range “ months patients developedrecurrent disease including SCRs Of note of the SCRswere pathologically confirmed and the rest were diagnosed byclinical assessments and radiographic findings The and year RFS were and in patients without PORTrespectively and were and in patients withPORT respectively Among the patients with SCR patients developed SCR without DM Figure 1A and patients developed SCR involving the ipsilateral supraclavicularregion Figure 1B Moreover among the patients with leftlung cancer who developed SCR seven were ipsilateral threebilateral and two contralateral Among the patients with rightlung cancer who developed SCR nine were ipsilateral threebilateral and three contralateralThe and 5year cumulative incidence of SCR were and respectively Figure 2A and the dynamicof hazard ratio of SCR was presented in Figure 2B ChosenDM as a competing event cN2 disease ypN2lobectomyand CK7 were identified as significant risk factors of SCRFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCTABLE Competing risk analyses of clinicalpathological variables associatedwith supraclavicular recurrenceVariablesUnivariate Analyses Multivariate AnalysesHR 95CIpHR 95CIpAge vs ‰¤ “ Sex Male vs Female “ Smoking Never vs Ever “ ECOG vs cN2 vs “ “ “ pT stage T3“ vs T0“ “ pN1 vs “ “ Multiple levels of pN2 vs “ “ Histology SCC vs NonSCC “ Differentiation P vs WM “ LVI vs “VPI vs “PNI vs “ypN2 vs pN2 “ “ “ “ “ Tumor Location Left vs Right “ Tumor Lobe Upper vs Others “ TLN ‰¥ vs “ PLN ‰¥ vs “ LNR ‰¥ vs “ Surgery Others vs Lobectomy “ “ PORT vs “ERCC1 vs “Her2 vs “ “ “ “ Ki67 ‰¥ vs “ TTF1 vs “CK20 vs “CK7 vs “CK56 vs “P63 vs “ “ “ “ “ “ “ NapsinA vs “ “ Syn vs “RRM1 vs “EGFR vs “ “ “ “ HR hazard ratios CI confidence intervals SCC squamous cell carcinoma LVILymphovascular invasion VPI Visceral pleuralinvasion ECOGthe Eastern Corporative Oncology Group TLN total lymph node examined PLN positivelymph node LNR positive lymph node ratio PORT postoperative radiotherapy WMwellmoderate P poor Bold values indicates statistical significantinvasion PI perineuralusing the univariate competing risk analysis Table Sincethere was a significant association between cN2 disease and testreceiving neoadjuvant chemotherapy p χwe excluded cN2 disease and included the otherthreesignificant risk factors in the multivariate competing riskanalyses The result showed that only ypN2 were identifiedas an independent risk factor of SCR Table The and 5year cumulative incidence of SCR were and among ypN2 patients respectively and were and among pN2 patients respectivelyFigure 2APattern of Failure AnalysesIn the entire cohort of the patients who receivedPORT developed LRR while of the patientswho did not receive PORT developed LRR PORT significantlyreduced the risk of LRR Figure 3A Among the six patients whoreceived PORT and subsequently developed LRR five developedLRR only within the PTV and the rest one developed LRRboth within and outside the PTV Among the patients whodid not receive PORT and subsequently developed LRR developed LRR only within the proposed PTV three developedLRR both within and outside the proposed PTV and the restone developed LRR outside the proposed PTV That patienthad adenocarcinoma in the middle lobe of right lung withpathologically proven metastatic lymph node in the right hilumand station but developed recurrent disease at mediastinallymph node stations and On the other hand of the patients who receivedPORT developed SCR while of the patients whodid not receive PORT developed SCR in the entire cohort PORTwithout ESRT didn™t reduce the incidence of SCR Figure 3BFifteen of the ipsilateral SCRs could be covered by theproposed PTVsc and the ipsilateral parts of the six bilateral SCRscould all be covered by the proposed PTVscSurvival AnalysesBy the time of data cutoï¬ patients had died and the medianOS1 was 95CI “ months PORT was found tosignificant prolong OS1 in the entire cohort Figure 3C Agesex ECOG scorelymphovascular invasion total number ofpositive lymph node positive lymph node ratio PORT and Ki67were found to be significantly associated with OS1 in univariateCox analyses while age ECOG score PORT and Ki67 wereidentified to be independent indicators of OS1 in multivariateCox analyses Table Among the patients with recurrentdisease the median OS2 was 95CI “ monthsAge sex ECOG score and DM were revealed to be significantlyassociated with OS1 in univariate and multivariate Cox analysesTable In order to investigate the prognostic significance of SCRpatients with recurrent disease were further divided into threegroups Group A consisted of patients who had DM n Group B consisted of patients who did not have DM but have SCRn and Group C consisted of patients who only had LRR n In terms of OS2 patients in Group B had an intermediateprognosis when compared with patients in Group A and GroupC Figure 3DDISCUSSIONTo the best of our knowledge this is the first comprehensivestudy specifically focusing on SCR in completely resected ypN2NSCLC with a relatively large sample size in the era of modernradiation technique SCR was not uncommon and had imperativeprognostic significance indicating that treatment modalities ableto reduce the incidence of SCR may be beneficial AdditionallyPORT without ESRT significantly reduced LRR and prolongedOS but did not decrease SCR in our study suggesting that theFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCFIGURE Prognostic significance of postoperative radiotherapy and supraclavicular recurrence The impact of postoperative radiotherapy PORT on locoregionalrecurrence LRR A supraclavicular recurrence SCR B overall survival OS1 C in the entire cohort Kaplan“Meier survival curve stratified by the diseaserecurrence patterns among patients with recurrent disease Dclinical value of ESRT may be reconsidered in selected patientswith high risks of SCRSCR is not uncommon in completely resected ypN2 NSCLCespecially among those with extra risk factors Although therewas limited historical data published that could be directlycompared the incidence of SCR in our study was reliable sincethe overall recurrence rate and the percentage of SCR amongpatients with recurrent disease were in accordance with previousfindings The cumulative incidence of postoperative recurrencein the PORT group and nonPORT group were generallycomparable with recent studies Furthermorestudies from our institution and others had reported asimilar percentage of SCR among patients with recurrent disease“ in the literature in our study Compared withtheir counterpart patients staged cN2 or ypN2 generally had amore advanced and aggressive disease and thus it was reasonablefor them to have a higher risk developing disease recurrenceincluding SCR “ Compared with those receivinglobectomy patients receiving pneumonectomy generally hada higher tumor burden and those receiving sublobectomycommonly had unfavorable prognostic factors such as morecomorbidities and poorer preoperative lung functions that madethem unsuitable for lobectomy Therefore patients whodidn™t receive lobectomy were also at a higher risk developingpostoperative recurrence which is generally consistent with arecent retrospective study using the SEER database Inaddition two recent studies found that positive expression ofCK7 were associated with more advanced disease and shorteroverall survival In our study distant metastasis waschosen as a competing event and negative expression of CK7was identified as a risk factor of SCR which need to befurther verifiedCompared with patients developing only LRR and thosedeveloping DM patients developing SCR but without DM hadintermediate OS2 highlighting the vital prognostic significanceof SCR in curatively resected ypN2 NSCLC The TNMstaging system is one ofindicators ofpatient™s prognosis in NSCLC among which patients havingsupraclavicular lymph node metastasis N3 generally haveintermediate prognosis when compared with those having distantmetastasis M1 and those harboring metastatic tumor lesionslimited to the ipsilateral hilar N1 or mediastinal N2 lymphthe most powerfulFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCTABLE Cox analyses of clinicalpathological variables associated with overallsurvival OS1TABLE Cox analyses of clinicalpathological variables associated with OS2 inpatients with recurrent diseaseVariablesUnivariate Analyses Multivariate AnalysesVariablesUnivariate AnalysesMultivariate AnalysesHR 95CIpHR 95CIpHR 95CIpHR 95CIpAge vs ‰¤ “ “ Sex Male vs Female “ “ Smoking Never vs Ever “ ECOG vs cN2 vs “ “ “ “ pT stage T3“ vs T0“ “ pN1 vs “ “ Multiple levels of pN2 vs “ “ Histology SCC vs NonSCC “ Differentiation P vs WM “ LVI vs “VPI vs “PNI vs “ypN2 vs pN2 “ “ “ “ “ Tumor Location Left vs Right “ Tumor Lobe Upper vs Others “ TLN ‰¥ vs “ Age vs ‰¤ “ “Sex Male vs Female “ “Smoking Never vs Ever “ECOG vs cN2 vs “ “ “ “pT stage T3“ vs T0“ “pN1 vs “ “Multiple levels of pN2 vs “ “Histology SCC vs NonSCCDifferentiation P vs WM “ “LVI vs “VPI vs “PNI vs “ypN2 vs pN2 “ “ “ “Tumor Location Left vs Right “Tumor Lobe Upper vs Others “TLN ‰¥ vs “PLN ‰¥ vs “LNR ‰¥ vs “PLN ‰¥ vs “ “ Surgery Others vs Lobectomy “LNR ‰¥ vs “ “ Surgery Others vs Lobectomy “ PORT vs “ERCC1 vs “Her2 vs “ “ “ “ “ Ki67 ‰¥ vs “ “ TTF1 vs “CK20 vs “CK7 vs “CK56 vs “P63 vs “ “ “ “ “ “ NapsinA vs “ “ Syn vs “RRM1 vs “EGFR vs “ “ “ “ HR hazard ratios CI confidence intervals SCC squamous cell carcinoma LVILymphovascular invasion VPI Visceral pleuralinvasion ECOGthe Eastern Corporative Oncology Group TLN total lymph node examined PLN positivelymph node LNR positive lymph node ratio PORT postoperative radiotherapy WMwellmoderate P poor Bold values indicates statistical significantinvasion PI perineuralnodes Similarly SCR represented an unfavorable sign ofsubsequent disease metastasis to distant ans and thus wasreasonable to have worse prognosis when compared with thosewho had only LRR On the other hand when compared withthose who already had DM patients who had recurrent diseaselimited to the thoracic region ie LRR and SCR could beconsidered as harboring locoregional disease and may benefitfrom aggressive locoregional treatment as well as systematictherapies and thus may still have a chance of longterm survival In fact among the patients with SCR but without DMthe 3year survival rate exceeded in our study Figure 3DHowever due to the advancement of adjuvant chemotherapy andPORT vs “DM vs “ERCC1 vs “Her2 vs “ “ “ “ “ “Ki67 ‰¥ vs “TTF1 vs “CK20 vs “CK7 vs “CK56 vs “P63 vs “NapsinA vs “Syn vs “RRM1 vs “EGFR vs ““ “ “ “ “ “ “ “ “OS overall survival HR hazard ratios CI confidence intervals SCC squamouscell carcinoma LVI Lymphovascularinvasion PIperineural invasion ECOG the Eastern Corporative Oncology Group TLN total lymphnode examined PLN positive lymph node LNR positive lymph node ratio PORTpostoperative radiotherapy DM distant metastasis WM wellmoderate P poor Boldvalues indicates statistical significantinvasion VPI Visceral pleuralPORT the number of patients who developed localized recurrentdisease ie LRR and SCR was small patients in group Band patients in group C although a total of patients wereenrolled and followed up for a median of months Hencethe prognostic significance of SCR needed to be interpreted withcaution and future investigations with larger sample size andprospective design are warrantedThe clinical value of PORT in completely resected ypN2NSCLC was demonstrated again in our study but the delineationof CTV remain controversial In the current study PORTsignificantly reduced LRR and improved OS1 which havebeen demonstrated in various studies “ However since ESRT was not routinely performed in our cancerFrontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLCindicating thatinstitution PORT failed to reduce SCRthe majority of SCRs represented the outgrowth of subclinicaltumor lesions already in the supraclavicular region and werenot originated from the locoregional recurrent disease throughlymphatic metastasis In fact of the patientswith SCR had no LRR in the current study These dataindicated a potential role of ESRT in selected patients withhigh risks Actuallyfor locally advanced NSCLC receivingchemoradiotherapy there is no significant diï¬erence of patient™ssurvival between those with or without N3 disease highlighting that the treatment efficacy of chemoradiotherapyin locally advanced NSCLC was largely dependent on theintrinsic biology of the tumor and the prognosis of patients withor without macroscopic supraclavicular tumor lesions seemedsimilar PORT with adjuvant chemotherapy has been repeatedlyshown to significantly reduce LRR indicating the beneficial roleof adjuvant chemoradiotherapy in treating microscopic N1N2disease It is possible that adjuvant chemoradiotherapy ieadjuvant chemotherapy in combination with ESRT may alsoplay a role in reducing SCR and subsequently improve patient™ssurvival Furthermore nearly of the ipsilateral SCRs couldbe covered with the virtual CTV of ESRT in our study Howeverthere are also evidence against the use of ESRT for patients withcompletely resected NSCLC Elective irradiation of mediastinalcontralateral hilar and supraclavicular lymph nodes failed toimprove patient™s survivalin unresectable stage III NSCLCwithout clinical N3 disease And pattern of failure analyses ofa prospective trial of PORT without ESRT suggested that the useof limited CTV including only the involved lymph node stationsand those with a risk of invasion was associated withacceptable risk of geographic miss Taken together PORTwithout ESRT provided significant clinical benefit for patientswith completely resected ypN2 NSCLC and the clinical valueof ESRT in highly selected patients for example those withpersistent N2 ypN2 disease after neoadjuvant chemotherapyneed to be further investigatedOur study also has some limitations Firstly since ESRT is notroutinely performed in our cancer center we could not directlyexamine the clinical value and prognostic significance of ESRTSecondly as this was a retrospectively study treatment decisionsand followup strategies were at the discretion of the treatingphysicians Diï¬erent neoadjuvant and adjuvant chemotherapyregimens were used and the protocols of followup were notidentical Moreover since brain MRI and bone scans were notmandatory asymptomatic brain andor bone metastasis may beunderestimated Despite these limitations we believe our studyprovided valuable information about the cumulative incidenceand prognostic significance of SCR in completely resected ypN2NSCLC which may guide better design of adjuvant treatmentmodalities and individualized surveillance strategiesDATA AVAILABILITY STATEMENTThe raw data supporting the conclusions of this will bemade available by the authors without undue reservationETHICS STATEMENTThe studiesinvolving human participants were reviewedand approved by the institutional review board of FudanUniversity Shanghai Cancer Center Written informed consentfor participation was not required for this study in accordancewith the national legislation and the institutional requirementsAUTHOR CONTRIBUTIONSLL ZZ and JN conceptualization LL and ZZ methodologyvalidation and writing”original draft preparation LL ZZ andJL formal analysis and investigation LL ZZ and YL resourcesand data curation LL and JN writing”review and editing Allthe authors have approved the final manuscriptFUNDINGThis study was supported by the National Natural ScienceFoundation of China No to JN and grant provided bythe Shanghai Municipal Health Commission No 20194Y0501to JNSUPPLEMENTARY MATERIALThe Supplementary Materialonline202001414fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncatSupplementary Figure Flowchart of patient enrollment NSCLC nonsmallcell lung cancerREFERENCES Eberhardt WE De Ruysscher D Weder W Le Pechoux C De Leyn PHoï¬mann H et al 2nd ESMO Consensus Conference in Lung Cancerlocally advanced stage III nonsmallcell lung cancer Ann Oncol “ 101093annoncmdv187 Tan WL Chua KLM Lin CC Lee VHF Tho LM Chan AW et alAsian thoracic oncology research group expert consensus statement onoptimal management of stage III NSCLC J Thorac Oncol “ 101016jjtho201910022 Kenmotsu H Yamamoto N Yamanaka T Yoshiya K Takahashi T Uenostudy of pemetrexed plus cisplatintoT et al Randomized phase IIIversus vinorelbine plus cisplatin for completely resected stage IIIIIA nonsquamous nonsmallcell“ 102139ssrn3460654lung cancerJ Clin Oncol Kato H Tsuboi M Kato Y Ikeda N Okunaka T Hamada C Postoperativeadjuvant therapy for completely resected earlystage nonsmall cell lungcancer Int J Clin Oncol “ 101007s101470050493x Herskovic A Mauer E Christos P Nagar H Role of postoperativeradiotherapy in pathologic stage IIIA N2 nonsmall cell lung cancer in aprospective nationwide oncology outcomes database J Thorac Oncol “ 101016jjtho201609135 Deng W Xu T Xu Y Wang Y Liu X Zhao Y et al Survival patterns for patientswith resected N2 nonsmall cell lung cancer and postoperative radiotherapya prognostic scoring model and heat map approach J Thorac Oncol “ 101016jjtho2018082021Frontiers in Oncology wwwfrontiersinAugust Volume 0cLiu et alSupraclavicular Recurrence in ypN2 NSCLC Feng W Fu XL Cai XW Yang HJ Wu KL Fan M et al Patternsof localregional failure in completely resected stage IIIAN2 nonsmallcellimplications for postoperative radiation therapyclinical target volume design Int J Radiat Oncol Biol Phys “ 101016jijrobp201312048lung cancer cases Dai H Hui Z Ji W Liang J Lu J Ou G et al Postoperative radiotherapy forresected pathological stage IIIAN2 nonsmall cell lung cancer a retrospectivestudy of cases from a single institution Oncologist “ 101634theoncologist20100343 Zou B Xu Y Li T Li W Tang B Zhou L et al A multicenter retrospectiveanalysis of survival outcome following postoperative chemoradiotherapy innonsmallcell lung cancer patients with N2 nodal disease Int J Radiat OncolBiol Phys “ 101016jijrobp200905044 Billiet C De Ruysscher D Peeters S Decaluwe H Vansteenkiste J Dooms Cet al Patterns of locoregional relapses in patients with contemporarily stagedstage IIIN2 NSCLC treated with induction c

Thyroid_Cancer

"development of cancer is a problem that has accompanied mankind for years The growing number of cases emerging drug resistance and the need to reduce the serious side effects of pharmacotherapy are forcing scientists to better understand the complex mechanisms responsible for the initiation promotion and progression of the disease This paper discusses the modulation of the particular stages of carcinogenesis by selected physiological factors including acetylcholine ACh peroxisome proliferatoractivated receptors PPAR fatty acidbinding proteins FABPs Bruton™s tyrosine kinase Btk aquaporins AQPs insulinlike growth factor2 IGF2 and exosomes Understanding their role may contribute to the development of more effective and safer therapies based on new binding sitesINTRODUCTIONThe increasing prevalence of various types of cancers is a global phenomenon that has been occurring widely and affecting increasing numbers of people In over million new cases were reported and statistics show that every fifth man and every sixth woman is at risk for developing cancer [] Carcinogenesis is a multistage process that leads to cancer development It includes three main stages initiation promotion and progression and each is characterized by different processes During the promotion stage the genetic material of cells is damaged and if not detected by repair systems before the next division the change is transferred to newly formed cells Under the control of growth factors initiated cells may move on to the second stage of cancer formation“promotion During that stage mutated cells undergo multiple divisions their growth is uninhibited as they become insensitive to apoptotic signals As a result the tumor increases its mass but the cells remain within one an The formation of metastases occurs in the next phase“progression Metalloproteinases secreted by tumor cells destroy the extracellular matrix This allows them to enter the bloodstream and reach a new an at a considerable distance from the primary lesion with the stream of flowing blood The key step for the survival of migrating cells is adhesion to the attacked an and the formation of new blood vessels thanks to which they will receive the components necessary for development Angiogenesis the formation of new blood vessels is the last element of carcinogenesis and is enhanced by tumor cell derived factors metabolic changes and a decrease in available oxygen Each of these processes is controlled by different signal pathways via proteins that naturally occur in the body Figure Dysregulation of their expression and thus activity both to an excessive and insufficient degree ensures the continuity of carcinogenesis and increases the chances cancer cells survive in a host anism Understanding the role of individual substances and components of the body in the subsequent stages of neoplastic transformation provides a more complete picture of cancer pathogenesis This allows to develop new recommendations to improve the quality and safety of pharmacotherapy which has a direct effect on an improvement in the cancer patients™ quality of life It also provides the basis for research aimed at developing new compounds that are effective weapons in the fight against cancer Therefore there is no doubt that any actions that bring scientists closer to solving the problem of insufficient cancer therapy are sensible and much needed The purpose of this paper was to discuss the role of selected physiological factors in the various stages of neoplastic transformationOncotargetwwwoncotargetcomwwwoncotargetcom Oncotarget Vol No pp 0cCrucial mechanisms involved in carcinogenesisThe carcinogenic process is regulated by many molecular and cellular mechanisms The diversity of signaling pathways exploited during cancer initiation promotion and progression is immense Many of them are common in different cancer types but there are also clearly unique molecular and cellular signaling signatures specific for particular cancers [] Pathways such as AktPI3K RasMAPKERK Wntcatenin JAKSTAT occupy crucial roles in the regulation of cell proliferation apoptosis differentiation angiogenesis cell migration and invasion immunological activities and inflammationPI3K phosphatidylinositol 3kinase is a family of enzymes that after activation by growth factors cytokines and hormones are involved in the conversion of PIP2 phosphatidylinositol45bisphosphate into PIP3 phosphatidylinositol 345trisphosphate [] The latter in turn takes part in recruiting Akt into the cell membrane where it is phosphorylated and activates other agents such as mTOR NFkB Wnt or inhibits factors like Bad p27 to enhance carcinogenesis Dysregulation of this pathway occurs in endometrial [] bladder [] or gastric cancers [] The aforementioned PIP2 can be also transformed by phospholipase C into DAG and IP3 which then activate protein kinase C PKC [] One of the PKC substrates is Raf kinase which is an element of the RasMAPKERK pathway which is involved in the pathogenesis of ovary cancer [] or nonsmall cell lung cancer [] Ras proteins activate Raf which then phosphorylates kinases MEK12 Its final substrate is kinase ERK12 which after transportation into the nuclei regulates transcription factors essential for DNA synthesis and cell cycle progression They condition cell growth proliferation and viability [] Because of that targeting those two cascades as an anticancer therapy seems to be beneficial However it is important to notice that the existing crosstalk between both of the pathways can impede treatment and inhibition of one can strengthen the activity of the other Another component that is also connected with the MAPK family Figure Modulators of individual stages of carcinogenesis Inducers red frames inhibitors green frames M1rec muscarinic receptor M3rec muscarinic receptor Nrec nicotinic acetylcholine receptor COX2 cyclooxygenase2 PPARÎ peroxisome proliferatoractivated receptors gamma Btk Bruton™s tyrosine kinase FABP fatty acidbinding protein LFABP Ltype fatty acidbinding protein AQPs aquaporins IGF2 insulinlike growth factor IGFBP3 insulinlike growth factor binding protein IGFBP5 Insulin Like Growth Factor Binding Protein IGF2BP2 Insulin Like Growth Factor2 mRNA Binding Protein2 IGF2BP3 Insulin Like Growth Factor2 mRNA Binding Protein Oncotargetwwwoncotargetcom 0cis a signaling pathway conducted by p38MAPKs This subfamily contains four kinases which are activated mainly by inflammatory cytokines and stress factors p38MAPKs control many aspects of cell physiology such as cell cycle regulation differentiation or skeleton remodelling Additional p38MPAKs can act as tumor promotors by enhancing metalloproteinase and VEGF expression []Wntcatenin signaling plays essential roles in caricinogenesis Activation of canonical Wnt signaling results in the inhibition of GSK3 kinase dissociation of catenin proteins and its transfer to the nucleus where it regulates multiple downstream genes like cMyc and cyclin D [] This axis was found to be associated with several oncogenic events including tumor cell proliferation migration epithelial“mesenchymal transition and invasion An abnormally active WntBcatenin pathway is observed among others in gastric [] colon [] breast [] or adrenocortical cancer [] Another signaling pathway involved in the pathogenesis of many cancer types is that induced by the Jak kinases family It includes four kinases which after activation impact of the STAT molecules causing their translocation into the nuclei [] STAT a family that gathers seven forms of proteins is associated with cancer cell development progression metastasis survival and resistance to treatment [] STAT3 and STAT5 factors seem to be the most important agents in light of cancer progression They have an impact on p53 protein which leads to a disruption in cycle control and apoptosis and induces cell proliferation by the increased cMyc and cyclin D expression By the induction of VEGF gene expression they take part in enhanced angiogenesis and induction of genes such as survivin Bcl2 BclXL conditioning overall survival of the tumor cells Moreover pSTAT3 acts to negatively regulate neutrophils NK cells effector T cells and dendritic cells while positively regulating populations of MDSCs myeloidderived suppressor cells and regulatory T cell leads to a highly immunosuppressive TME tumor microenvironment []Multiple endogenous factors are involved in carcinogenesis which affects particular stages of carcinogenesis in various mechanisms Some of them interact directly with the DNA intracellular signalling molecules others by specific cellular receptors Among them are well known growth factors EGF TGFα and TGF FGF or reactive endogenously generated oxygen molecules as well as less often described agents like ACh PPAR FABPs Btk AQPs IGF2 or exosomes The latter have only recently gained in importance and are more widely studied and discussed therefore are considered in this articleAacetylcholine Ach and its receptorsOne of the factors involved in the carcinogenesis process is acetylcholine and its receptors both muscarinic inhibitors and nicotinic Their expression has been demonstrated in numerous types of cancer cells [“] It has also been proven that these cells contain acetylcholinesterase AChE an enzyme that enables cells to produce ACh in the absence of agonists from the external environment Cancer cells also have the ability to produce autoantibodies that stimulate one of the muscarinic receptor subtypes M3 These facts prompted more extensive research to explain the exact role of ACh and its receptors in the carcinogenesis process Figure Muscarinic receptors can be divided into five subtypes each of which is involved in tumor development through a different mechanism The role of muscarinic M3 receptor is the most widely described subtype It is involved in the pathogenesis of lung colon gastric and breast cancer In the course of colon cancer it has been observed that stimulation of the M3 receptor causes phosphorylation of the Akt and ERK12 kinases which are factors that through the ability to regulate the activity of pro and antiapoptotic proteins affect the intensification of cell proliferation survival and motility In addition coexpression of the M3 receptor and endothelial growth factor receptor EGFR as well as EGFR transactivation after M3 receptor stimulation has been demonstrated [ ] Moreover the administration of EGFR inhibits acetylcholineinduced ERK12 kinase phosphorylation which is reflected in a significant decrease in colon cancer cell proliferation This indicates that acetylcholine is involved in the formation of colon cancer but for its full action it is necessary to activate the EGFR receptor Thus indicating that enriching colon cancer treatment with EGFR inhibitors may improve patient outcomes Acetylcholine is also involved in the formation of vascularlike structures in the vicinity of a developing tumor guaranteeing the supply of essential nutrients to the proliferating cancer cells This is called vascular mimicry The basis of this process is the acetylcholinedependent regulation of metastasisassociated in colon cancer1 MACC1 oncogene expression via the M3RAMPKMACC1 signaling pathway [] High activity of this oncogene is associated with an increase in cell invasiveness intensified epithelial“mesenchymal transition more frequent metastases and hence worse treatment prognosis [“] The first subtype of the M receptor M1 is also involved in the tumor development process It activates the hedgehog pathway [ ] whose physiological role is to regulate the transcription factors affecting the oncogenic activity of Gli zinc finger transcription factors [ ] Excessive stimulation causes acceleration of carcinogenesis intensification of proliferation growth and survival of cancer cells as well as angiogenesis by affecting the expression of genes such as cyclin D antiapoptotic Bcl2 or VEGF Furthermore stimulation of this pathway promotes the creation of a microenvironment conducive to tumor growth through increased expression of extracellular matrix components [] Nicotinic Oncotargetwwwoncotargetcom 0cacetylcholine receptors are part of the ligandgated ion channels and are created from two types of subunits α and Receptors that contain an alpha7 or alpha9 subunit in their structure are characterized by a high degree of permeability to calcium ions thanks to which they are involved in the course of numerous processes based on signaling involving secondary messengers Thus after activating N receptors Ca2 inflows into the cell causing the secretion of growth factors exerting paracrine action on neighboring cells Simultaneously apoptosis is inhibited enabling further proliferation Studies conducted on a lung cancer cell line after exposure to cigarette smoke have shown that nicotine and nitrosamines which have a higher affinity for nicotinic acetylcholine receptors nAChRs than nicotine bind to the alpha7 receptors activating the RasERKMAPK and the JAK2STAT3PI3K signaling pathways [ ] The effect of this interaction is increased proliferation and migration of cancer cells and thus facilitated metastasis creation Moreover by affecting Badrenergic receptors nicotine intensifies COX2 expression which through the p38 MAPK and the JNK pathways leads to an increase in VEGF production a highly proangiogenic factor In addition the high activity of cyclooxygenase affects the inactivation of the PTEN protein [ ] whose biological role is silencing PI3KAkt pathway signaling This leads to cellular signal transition and induces effects that contribute to the enhanced growth and proliferation of cancer cells as well as inhibits the process of their death Under the influence of the Akt kinase proapoptotic transcription factors are inactivated including kinase9 mTOR kinase intensifying cell proliferation is activated and VEGF is activated This indicates the involvement of this pathway in processes that facilitate cancer cell survival Another mechanism contributing to cancer development in which cyclooxygenase is involved is the complex process involving the COX2PGE2EP4 axis as a result of which metalloproteinase expression is stimulated [“] MMP9 causes proteolytic activation of TGF which begins the induction of epithelialmesenchymal transformation EMT During this transition the cells acquire features that increase their invasiveness This leads to epithelial cell phenotype changes the connections between adjacent cells and between cells and the basement Figure Carcinogenic effect of acetylcholine Ach Intracellular signaling pathways activated by ACh via M1 and M3 receptors Induction of process is illustrated by arrows inhibition by horizontal lines M1 muscarinic receptor M3 muscarinic receptor N receptor nicotinic acetylcholine receptor Akt protein kinase B ERK extracellular signalregulated kinase JAK2 Janus kinase PI3K phosphoinositide 3kinase STAT3 signal transducer and activator of transcription proteins MAPK mitogenactivated protein kinaseOncotargetwwwoncotargetcom 0cmembrane are loosened the cytoskeleton is rearranged cells lose contact inhibition thus causing excessive proliferation Furthermore the microenvironment of the growing tumor also changes promoting features that facilitate the movement of the cancer cells from the primary focus to other regions of the body Through the EMT process cells acquire greater migration capacity and thus their invasiveness increases In this cellular process MMP9 also modulates the activity of integrins and other molecules that ensure cell adhesion [] and secrete soluble factors into the bloodstream that facilitate the implantation of migrating cells in ans distal to the primary tumor focus [] Thanks to these processes COX2 enables the formation of cancer metastasesPPAR peroxisome proliferatoractivated receptorsThe family of peroxisome proliferatoractivated receptors PPARs includes isoforms of these proteins that function as transcription factors Each type of steroid receptor differs in its location and affinity for ligands [] The best known is the function of the PPARÎ form Agonists of this receptor are a therapeutic option in the treatment of patients with diabetes Current research provides evidence that stimulation of PPARÎ receptors inhibits cell proliferation vessel formation and induces apoptosis Figure [ ] These properties make PPARÎ agonists attractive candidates for anticancer drugs To achieve full transcriptional activity heterodimerization of PPARÎ with the retinoid X receptor is necessary [ ] This interaction enables the promoter to bind with the target gene and regulate its expression An example of a gene against which PPARÎ acts as a repressor is the vascular endothelial growth factor VEGF gene [] Thus angiogenesis is inhibited and this mechanism has been demonstrated in studies on human hepatocellular carcinoma HCC cells [] For this cancer PPARÎ is also involved in inhibiting cell growth and attenuating cell migration and invasiveness By upregulating KLF4 Krüppellike factor expression and inhibiting cyclin D1 expression the cell cycle is inhibited Furthermore PPARÎ negatively affects the expression of transcription factor STAT3 thereby inhibiting proliferation survival and metastasis Lowering STAT3 levels also negatively affects the angiogenesis process by inhibiting VEGF depriving cancer cells of nutrients PPARÎ activation in HHC cells also impairs their metastatic ability [] This is associated with the inhibition of the expression of metalloproteinases MMP9 and MMP13 the increased expression of their inhibitor and Ecadherin a protein conditioning cell adhesion In addition PPARÎ increases plasminogen activator inhibitor expression thus inhibiting the breakdown of the extracellular matrix and basement membrane proteins which also reduces cancer cell invasiveness PPARÎ has also been shown to inhibit esophageal cancer [] The mechanism responsible for suppressing this tumor is MAPK signaling pathway inhibition This process requires TLR4 Tolllike receptor inhibition which allows PPARÎ agonists to be administered TLR4 is a procarcinogenic protein and affects among others changes in the tumor microenvironment and the severity of angiogenesis Therefore in addition to the indirect effect on the MAPK cascade its inhibition is an important element of tumor suppression Studies on esophageal cancer cells indicate that PPARÎ stimulation leads to a decrease in PCNA proliferating cell nuclear antigen factor expression which indicates inhibition of DNA replication [] In addition PPARÎ activation intensifies the process of apoptosis as evidenced by an increase in the expression of the active form of caspase and the Bax gene and a decrease in Bcl2 expression In nonsmallcell lung cancer PPARÎ stimulation inhibits the neoplastic process by regulating PTEN protein expression [] This protein is a tumor suppressor and its activation causes the dephosphorylation and inactivation of the PIP3 second messenger As a result Akt kinase activity is inhibited leading to a decrease in NFkB expression Due to PPARÎ stimulation by eicosapentaenoic acid EPA a relationship is sought between PPARÎ and COX2 activity that uses EPA as a substrate for prostanoid production The combination of PPARÎ agonists with COX2 inhibitors has demonstrated a synergistic tumorsuppressing effect in studies on nonsmallcell lung cancer Researchers indicate significantly reduced thromboxane TXA2 synthesis using combination therapy compared with monotherapy as the main mechanism of this phenomenon [] A decrease in its concentration limits signaling in four pathways ERK p38 MAPK JAK and catenin limiting tumor growth [] Furthermore it affects the arrest of the cell cycle in the G2M phase preventing further cell division and contributing to the reduction of the expression of survivin an antiapoptotic protein [] The described mechanisms affect the induction of the apoptosis process and inhibit cell cycle progression which limits cancer development The above examples indicate that peroxisome proliferatoractivated receptors play a significant role in inhibiting the development of various types of cancer which is why agonist compounds can be considered potential anticancer drugs However further comprehensive and extensive research is needed to identify potential applications and treatment regimensBtk Bruton™s tyrosine kinaseBruton™s tyrosine kinase Btk also plays an important role in carcinogenesis Figure It is a key point in signal transduction from the BCR receptor leading to the activation of the NFkB signaling pathway [] Btk affects phospholipase CÎ2 [] which after phosphorylation causes PIP2 hydrolysis The result Oncotargetwwwoncotargetcom 0cis IP3 and DAG DAG activates protein kinase C which stimulates NFkB pathway factors Finally genes conditioning cell survival are expressed Btk also contributes to inhibiting the apoptosis process through interacting with the Akt kinase [] The signaling cascade includes active PI3K which recruits Akt to the plasma membrane Under the influence of Btk its phosphorylation occurs and thus its activation Then the active Akt returns to the cytoplasm and induces antiapoptotic pathways dependent on NFkB among others In addition Btk as a kinase belonging to the Tec family affects intercellular signaling causing changes in the tumor microenvironment In most cancer cells the developing microenvironment leads to the inhibition of immune processes which results in protection and protects defective cells from natural defense mechanisms One of the most common mechanisms of this type is the effect of cancer cells on the increased differentiation of T lymphocytes towards Th2 cells The biological role of these cells is the secretion of interleukins that start a humoral response dependent on B lymphocytes Due to the increased differentiation of T lymphocytes in this direction the number of formed Th1 lymphocytes directly destroying cells containing the abnormal genome decreases The tumor microenvironment also interferes with normal dendritic cell activity It inhibits their ability to migrate and to present the antigen to immune response cells Some types of cancer show increased CXCL12 expression [] which attracts immature dendritic cells and prevents their differentiation In addition this cytokine via the CXCR4 receptor causes a reanization of the cytoskeleton [] and increases cell motility [] Tec family tyrosine kinases have also been shown to have the ability to activate CXCR4 which determines the growth survival and migration of tumor cells []FABPs fatty acidbinding proteinsThe fatty acids supplied to the cell are involved in its metabolism Due to their lipid nature they are Figure Carcinogenic effect of peroxisome proliferatoractivated receptors PPARÎ The induction of the process is illustrated by arrows the inhibition by the horizontal line PTEN phosphatase and tensin homolog deleted on chromosome ten PIP3 phosphatidylinositol 345trisphosphate Akt protein kinase B PCNA proliferating cell nuclear antigen TXA2 thromboxane A2 ERK extracellular signalregulated kinases p38MAPK P38 mitogenactivated protein kinases JAK Janusactivated kinases KLF4 Kruppellike factor STAT3 Signal transducer and activator of transcription MMP314 metalloproteinase or PAI Plasminogen activator inhibitor TRL4 tolllike receptor MAPK mitogenactivated protein kinaseOncotargetwwwoncotargetcom 0cunable to enter the aqueous environment of the cellular cytoplasm Solubilizing molecules ie fatty acidbinding proteins FABPs enable their transport to various cellular structures and thus become involved in the processes taking place in the cells including their growth reproduction and inflammatory processes In cells characterized by intensive lipid metabolism such as the liver intestine heart brain etc high transporter expression is observed which is associated with their physiological role in the cell Depending on the type of cancer both the overexpression and a decrease in FABPs can be seen FABP isoforms are not specific to one type of cancer and each plays a different role in the carcinogenesis process Figure In liver cancer LFABP was overexpressed and its high concentration was shown to correlate positively with VEGFA [“] The causes of this phenomenon are sought in the direct interaction between these two proteins which in effect provokes the activation of the SrcFAKcdc42 and the AktmTORP70S6K4EBP1 signaling pathways Activation of the SrcFAKcdc42 pathway increases tumor cell migration and LFABP plays a key role in this process [ ] Moreover by stimulating the second mentioned signaling cascade LFABP enhances VEGFA expression facilitating angiogenesis This process is regulated by HIF1α which additionally induces blood vessel formation [] Due to the high homology in their construction FABP3 and FABP4 isoforms were assigned to the same protein subfamily In addition to their physical features it has been observed that the same factors ie HIF1α VEGF increase their coexpression The physiological role of both proteins is to inhibit excessive cell proliferation and increase apoptosis Excessive FABP3 expression leads to an increase in reactive oxygen species and a decrease in the mitochondrial membrane potential [] resulting in the ing of mitochondrial megachannels which play a key role in initiating apoptosis FABP4 activates the apoptosis process by mediating the response to lipidinduced endoplasmic reticulum stress Due to the biological functions of both proteins their deficiency may be the cause of cancer progression When examining the level of FABP3 expression in embryonic tumor cells breast cancer cells [] and FABP4 in breast ovarian prostate bladder or liver cancer cells low levels of both proteins were observed which was associated with a worse prognosis in terms of overall survival Furthermore high FABP4 levels slowed the development of hepatocellular carcinoma and thus contributed to its reduction in size [] This effect is explained by the inhibition of STAT3 phosphorylation via the RaspSTAT3 signaling pathway and the inhibition of the Snail protein an accelerator of the epithelialmesenchymal transition [] However the increased FABP3 expression in tumors of the stomach brain small and nonsmallcell lung cancer seems paradoxical it has been shown to increase tumor aggressiveness and poorer patient prognosis [ ] Thus far the molecular mechanism responsible for the oncogenic potential of FABP3 and FABP4 is unknown Further research is needed to clarify the reasons these proteins promote the neoplastic process Another subtype of fatty acid binding proteins is FABP5 It is involved in the development of breast prostate liver stomach and colon cancers FABP5 has been shown to transport saturated and unsaturated longchain fatty acids that act as PPARδ ligands [ ] The nature of the supplied fatty acids determines the oncogenic activity if unsaturated fatty acid or a suppressor action is supplied to PPARδ in the case of saturated ligands [] When the Figure Carcinogenic effect of Bruton™s tyrosine kinase Btk The induction of the process is illustrated by arrows the inhibiton by the horizontal line Btk Bruton™s tyrosine kinase CXCR2 CXC chemokine receptor type CÎ2 phospholipase CÎ2Oncotargetwwwoncotargetcom 0cFABP5PPARδ pathway is stimulated the transcription of the genes responsible for cell growth and survival increases Moreover one of the target genes stimulated by PPARδ is the FABP5 gene which increases the expression of the transport protein in question [] In prostate cancer cells by supplying long chain fatty acids FABP5 leads to PPARÎ activation [“] Stimulation of these receptors results in increased expression of vascular endothelial growth factor VEGF thereby accelerating the angiogenesis process This mechanism plays a key role in the development of castrationresistant prostate cancer [“] In the case of colon cancer FABP5 contributes to its development by reducing p21 activity Overexpression of FABP5 increases the expression of cMYC which inhibits the action of a cell cycle inhibitor After silencing FABP5 activity a significant reduction in the invasive capacity of colon cancer cells CRC is observed [] The molecular basis of this interaction is not yet known at the moment High expression of monoacylglycerol lipase MAGL responsible for the production of free fatty acids is observed in highly malignant colon cancer cells [ ] This fact combined with the physiological role of FABP5 consisting of transporting free fatty acids to the appropriate cell compartments prompts to seek the answer to the question whether FABP5 and MAGL come into functional interaction with each other and if so how does this affect CRC progression Another FABP isoform that may be involved in colon cancer development is FABP6 [“] It transports bile acids between the epithelial cells of the large intestine which are transformed into secondary metabolites such as deoxycholic acid DCA and lithocholic acid via cellular metabolism A correlation was demonstrated between increased exposure of intestinal epithelial cells to DCA and an increase in reactive oxygen species inside them which is associated with oxidative stress development and DNA damage [“] This mechanism contributes to the formation of mutations that in the event of a replication of defective cells initiate their malignancy Due to their detergent properties bile acids can cause damage to the cell membrane Compensatory mechanisms lead to an intensified inflammatory response and increased proliferation which is qualified as early tumor development Furthermore bile acids activate the muscarinic M3 receptor [“] Its stimulation induces Figure Carcinogenic effect of fatty acid binding proteins FABPs The induction of the process is illustrated by arrows the inhibition by the horizontal lineSTAT3 signal transducer and activator of transcription Src protooncogene tyrosineprotein kinase Src FAK focal adhesion kinase cdc42 cell division control protein Homolog Akt protein kinase B mTOR mammalian target of rapamycin P70S6K p70 ribosomal protein S6 kinase 4EBP1 eukaryotic translation initiation factor 4E eIF4Ebinding protein VEGFA vascular endothelial growth factor1 M3 muscarinic receptor MMPs metalloproteinases PPARÎ peroxisome proliferatoractivated receptors Î PPARδ peroxisome proliferatoractivated receptors or δOncotargetwwwoncotargetcom 0cmetalloproteinase activity and the WntBcatenin signaling pathway which in turn increases the metastatic potential as well as the proliferation and survival of colon cancer cells Desp

Thyroid_Cancer

Deacetylases inSkeletal Muscle Physiology andSystemic Energy HomeostasisImplications for Metabolic Diseasesand TherapyHaili Tian1  Sujuan Liu2  Jun Ren3 Jason Kai Wei Lee456 Ru Wang1 and Peijie Chen1 School of Kinesiology Shanghai University of Sport Shanghai China Department of Anatomy and Histology Schoolof Basic Medical Sciences Tianjin Medical University Tianjin China Department of Cardiology Shanghai Instituteof Cardiovascular Diseases Zhongshan Hospital Fudan University Shanghai China Department of Physiology Yong LooLin School of Medicine National University of Singapore Singapore Singapore Global Asia Institute National Universityof Singapore Singapore Singapore N1 Institute for Health National University of Singapore Singapore SingaporeSkeletal muscle is the largest metabolic an in the human body and is able torapidly adapt to drastic changes during exercise Histone acetyltransferases HATs andhistone deacetylases HDACs which target histone and nonhistone proteins are twomajor enzyme families that control the biological process of histone acetylation anddeacetylation Balance between these two enzymes serves as an essential elementfor gene expression and metabolic and physiological function Genetic KOTG murinemodels reveal that HDACs possess pivotal roles in maintaining skeletal muscles™metabolic homeostasis regulating skeletal muscles motor adaptation and exercisecapacity HDACs may be involved in mitochondrial remodelinginsulin sensitivityregulationturn onoff of metabolic fuel switching and orchestrating physiologicalhomeostasis of skeletal muscles from the process of myogenesis Moreover manymyogenic factors and metabolic factors are modulated by HDACs HDACs areconsidered as therapeutic targets in clinicaltreatment of cancer‚ammation and neurological and metabolicrelated diseases This review will focuson physiological function of HDACs in skeletal muscles and provide new ideas for thetreatment of metabolic diseasesresearch forKeywords histone deacetylases exercise capacity skeletal muscle metabolism muscle physiologyINTRODUCTIONSkeletal muscle is the largest metabolic an in the human body consuming about of theentire body daily expenditure of energy Durnin It produces various secrete factors andparticipates in the interplay among multiple tissues and ans Pedersen and Febbraio Mizgier Muscular contraction is one of the main physiological functions of skeletalmuscles and plays a role in maintaining an and systemic metabolic homeostasis Guo Proper exercises help build up body defense to combat various diseases including obesitytype diabetes Alzheimer disease osteoarthritis and so on Luan These importantEdited byBrian James MorrisThe University of Sydney AustraliaReviewed bySean L McGeeDeakin University AustraliaViviana MoresiSapienza University of Rome ItalyCorrespondenceRu WangwangrutysuseducnPeijie Chenchenpeijiesuseducn These authors have contributedequally to this workSpecialty sectionThis was submitted toIntegrative Physiologya section of the journalFrontiers in PhysiologyReceived May Accepted July Published August CitationTian H Liu S Ren J Lee JKWWang R and Chen P Roleof Histone Deacetylases in SkeletalMuscle Physiology and SystemicEnergy Homeostasis Implicationsfor Metabolic Diseases and TherapyFront Physiol 103389fphys202000949Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise Capacityfunctions require delicate regulations in muscle from thelevel of genome to signal transduction establishing muscleplasticity and responses to environmental stress AcetylCoAas a central metabolite of amino acidfatty acidglucose notonly serves as fuel for energy expenditure but also bridgesthe gap between environmental stress and anismal responseLempradl Liu This control occursthrough posttranslational modification on nucleosomes histonelysine residues and other signal transducing proteins whichin turn controls accessibility of DNA to regulatory factorsand protein activity Acetylation one of the most prevalentmodifications of proteinis believed to function as a keymodulator of chromatin structure and signal transduction andprovides an avenue to couple extracellular stimuli with genomeduring muscle metabolism process by regulating acetylation anddeacetylation Haberland histone deacetylasesHDACs as a family of proteindeacetylases have been demonstrated to moderate physiologicalhomeostasis and development by deacetylation Haberland Table In humans there are types ofHDACs which are classified into four categories based onhomologous proteins in yeast namely Class I Rpd3like proteinHDAC1 Class II Hda1like proteins are classifiedas Class IIa HDAC4 and Class IIb HDAC6 Class III Sir2plike nicotinamide adenine dinucleotideNADdependent SIRT1 and ClassIV HDAC11 containing homologous domain with bothRpd3 and Hda1 Seto and Yoshida HDACs modulategene expression and protein activity through deacetylatingproteins When histone lysine εamino acid in the nucleosomeis acetylated it can neutralize positive charge before looseningchromatin structuretranscriptionfactors to DNA and expression of downstream target genesBy contrast histone deacetylation compresseschromatinstructure thereby inhibiting transcriptional gene expressionShahbazian and Grunstein to promote binding ofBiochemical properties of HDACs have been comprehensivelyreviewed Shahbazian and Grunstein Howeverthephysiological functions of HDACs have yet to be well examined inskeletal muscles A series of researches shed light on the potentialof drugs targeting HDACs to improve muscle fitness and cardiacmuscle disease which needs further clarification of HDACsphysiological function to understand the mechanisms Bai Galmozzi Xie Zhang and Ren Gaur Ample work has revealed the role of HDACin muscle physiology such as skeletal muscles me olism and thusexercise capacity McGee and Hargreaves Table Class IHDACs can interact with myocyte enhancer factor MEF2MyoD regulating myogenesis and exercise capacity Mal Puri Ohkawa Gregoire HDAC3 participates in myocytes diï¬erentiation and is linked toskeletal muscles metabolic fuel switching Gregoire Hong Class II HDACs can be phosphorylated byHDAC kinases and are transduced from nucleus to cytoplasmin response to cellular stress mediating muscle fiber switch andaï¬ecting the pathway of insulin sensitivity such as Glut4 andAKT Haberland McGee and Hargreaves Class III HDACs target a crucial mitochondrial biogenesis factorperoxisome proliferatoractivated receptor gamma coactivator alpha PGC1α in skeletal muscles liver and fat tissue Whiteand Schenk Therefore we will further discuss howthese HDACs ‚uence respective downstream targets exercisecapacity and therapeutic eï¬ects in human diseasesCLASS I HDAC HDAC123Regulation of Myogenesis by Class IHDACsIn previous studies HDAC123 was shown to be associated withthe process of myogenesis or myocyte diï¬erentiation Mal Puri Ohkawa Gregoire HDAC1 tightly binds to MyoD and deacetylates specific sites toinhibit the expression of musclespecific genes such as MHC andMCK in myoblasts Mimicking muscle diï¬erentiation conditionby serum deprivation HDAC1 protein gradually decreasesduring myocyte diï¬erentiation and transfers to bind to the tumorsuppressor pRb accompanied by isolation of HDAC1MyoD andtranscriptional activation of musclespecific genes Puri A HDAC1 H141A mutant incapable of binding withMyoD loses its inhibitory property on musclespecific genes inmyoblast state Mal In the late stage of myocytediï¬erentiation activating factor gradually switches from MyoDto myogenin occurring with a decrease in the MEF2D inhibitoryregulator HDAC2 Simultaneous overexpression of myogeninand MEF2D can enhance the expression of the musclespecificgene MHC in the absence of MyoD Ohkawa MEF2D a key factor that controls myocyte diï¬erentiationcan only be eï¬ectively deacetylated by HDAC3 rather thanHDAC128 Gregoire In addition HDAC3 caninhibit autoacetylation of acetyltransferases p300 and p300CBPassociated factor PCAF Thus HDAC3 impedes MyoDMEF2PCAF to form a multicomplex disturbing MEF2dependentmyogenic transcription Gregoire Figure Redundant Roles of HDAC12 inMaintaining Sarcomere Homeostasis inMuscleLoss of function of HDAC12 inhibits autophagy flux in skeletalmuscles tissue accompanied by decreased LC3 III ratio andp62 accumulation under fasting condition Moresi Toxic autophagy intermediates accumulate in muscle fibersin which class I HDACs deficiency led to impaired exercisecapacity Moresi This oï¬ers convincing evidenceon the role of HDAC12 in stabilizing basic structure andfacilitating development in muscles Genetic models suggestthat Class I HDACs control the physiological homeostasis ofskeletal muscles A germline deletion shows that wholebodyknockout of either HDAC1 or HDAC2 leads to mortalityin mice prior to the perinatal period Montgomery However a tissuespecific single deletion of HDAC1or HDAC2 in myocardium does not cause any phenotypeMontgomery Double knockout HDAC12 in theFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityTABLE The targets of HADCs in skeletal muscles and their physiological functionsClassMembersDown targetsGenetic KOTG phenotypeReferencesIHDAC1MyoD†“ PTEN†“ FoxOHDAC2HDAC3HDAC8HDAC4HDAC5HDAC9HDAC7HDAC6MyoD†“ MEF2D†“ NFκBp65†“MEF2D PCAF†“ Ampd3†“RCAN1 MEF2ACpt1b†“MEF2†“ Glut4†“ Myosin HeavyChain PGC1α and Hsc70“Pax7†“MEF2†“ Glut4†“ Baf60c†“Atg7 Beclin1 LC3†“Dach2Myog Gdf5†“MEF2†“Pax7 MFN1†“ Fam65bdysferlin and MAFbxHDAC10SIRT1“PGC1α STAT3†“IIAIIBIII“MnSOD Hexokinase II PDHsubunit E1αMalonyl CoA decarboxylase†““NFκBMstnSIRT2SIRT3SIRT4SIRT5SIRT6SIRT7Functionally redundant HDAC12 DKO mice causesmitochondrial abnormalities and sarcomeredegenerationPuri Ohkawa Beharry Cho Zhu Bin mKO mice Enhanced amino acid\\lipid metabolism andendurance exercise Causes IR under basal conditionGregoire Han Yuan Hong “Functionally redundant HDAC45 DKO HDAC59 DKOHDAC459 TKO increases type I fiber percentage andMcKinsey 2000a Choi Niu Luo oxidation metabolismYuan Meng Niu Macpherson Zhang Dressel “KO mice Causes mitochondrial oxidative damagemitofusion defect Protect against muscle wastingBalasubramanian Lee Ratti “mKO mice Mediates CR induced insulin sensitivity hasno effect on glucose homeostasis or exercise capacityTG mice no effect on glucose homeostasis or exercisecapacityKO mice Exacerbates obesity and IR in HFDKO mice Exacerbates obesity and IR in HFDKO mice Increased exercise tolerance and protectagainst HFDinduced obesity“Rodgers Schenk Boyle Jing Lantier Laurent KO mice Abnormal hypoglycemia TG mice Protectagainst HFD mKO mice Impaired insulin sensitivity lossKanfi Xiao Cui Samant of muscle mass““HDAC11IVActivation inhibition†“ unknown KO wholebody deletion TG wholebody overexpression DKO double knockout TKO triple knockout mKO musclespecificknockout CR caloric restriction HFD high fat diet IR insulin resistance““““heart leads to severe cardiomyopathy in mice indicating theobligatory role for HDAC12 in specific tissues Montgomery In skeletal muscle double knockout of HDAC12by myogeninCre causes mitochondrial abnormalities andsarcomere degeneration disrupting fundamental structural unitsof myofibers Moresi Therefore these findingsdemonstrate that HDAC12 redundantly maintains sarcomerehomeostasis in skeletal muscleHDAC3 Functions as a Fuel Switch inMuscle Energy Metabolismthe enzymatic core of nuclearHistone deacetylases isreceptor corepressorNCoR and silencing mediator ofretinoic acid and thyroid hormone receptors SMRT corepressor correspondingly NCoR and SMRT corepressoractivate HDAC3 through its SANT domain enzyme activityKaragianni and Wong Mice suï¬er from insulin resistanceafter specifically knocking out HDAC3 in skeletal musclesmKO while their endurance exercise abilities are enhancedHong It seems a selfcontradictory phenomenonbecause former studies pointed out that increased enduranceexercise capacity enhances insulin sensitivity The study showsthat insulin signaling cascades including pAKTAKT pIRS1S1101 and pGSKGSK have no change in HDAC3 mKO musclewhile glucose uptake and insulin sensitivity are impaired inglucose tolerance test GTT and insulin tolerance test ITTHong which is called the œdissociation eï¬ect byresearchers Lipid tends to be used as energy fuel in HDAC mKOmice which inhibits glucose absorption but does not ‚uenceinsulin signaling sensitivity Hong Further workfound that the HDAC3 knockout upregulates the expressionof AMP deaminase AMPD3 the first ratelimiting enzymein purine metabolism in skeletal muscles Fortuin Hong AMPD3 can deaminate AMP to form IMPfacilitating aspartic acid to transmit into fumarate and malate theintermediate metabolites of tricarboxylic acid cycle TCA cycleHong Research studies found that exercise inducedglucose labeled 13C6 expressed a lower glycolysis flux rate inmuscles of HDAC3 mKO but a higher expression in the TCAFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Controls of myogenesis by Class I HDACs The inhibitory effects of HDACs on myogenic factors MEF2 and MyoD maintain a primary myoblast stateDuring myocyte differentiation and myogenic process the inhibition is lifted by other factors facilitating MEF2MyoD complex formation to promote myogenesis“Ac deacetylationcycle intermediates Hong Gong Thus ageneral increase in TCA cycle metabolites activates the oxidationin mitochondria In vitro radioactive aspartic acid isotope tracertest showed that inhibiting HDAC3 or overexpressing AMPD3can increase amino acid metabolism rate and enhance fatty acidmetabolism thereby downregulating glucose metabolism Hong Figure In liver knockout HDAC3 causes severeliver steatosis and can be rescued by wildtype or catalyticallyinactive mutants of HDAC3 indicating an enzymatic activityindependent function Sun However the musclefuel switching in HDAC3 mKO mice cannot be rescued by anenzymatic inactivity mutant HDAC3 Song Thisfinding demonstrates that HDAC3 controls the fuel utilizationin skeletal muscles and is dependent on its enzymatic activityThe relationship between exercise and glucose uptake is far morecomplex than one could expectCLASS II HDACs HDAC45796Phosphorylation and NucleusCytoplasmShuttle of Class II HDACsThe MEF2 family is a class of transcription factors that areclosely associated with myogenic basic helixloophelix domainsincluding MyoD Taylor and Hughes MEF2 may interactwith Class IIa histone deacetylase HDAC45 to inhibit thetranscription of MEF2dependent genes thereby impeding thediï¬erentiation from myoblast to myotube McKinsey et al2000a During exercise Ca2 flows out from sarcoplasmicreticulum and activate calciumcalmodulindependent proteinkinase CaMK which phosphorylates HDAC45 and mediatesHDAC45 shuttle from the nucleus to the cytoplasm thusreleasing the repressive eï¬ect of HDAC45 on MEF2dependentgenes McKinsey 2000b Yuan Further researchdemonstrates that the nucleoplasmic shuttle of HDAC4 relies on binding while the shuttle of HDAC5 depends on CaMKphosphorylation at serine and sites first and thenexporting from the nucleus by binding with McKinsey 2000b As expected HDAC7 in Class IIa also possessesthe ability to be exported from nucleus to cytoplasm Gao suggesting that there may be redundant functions of ClassIIa HDACs Except for Class IIa knocking out Class IIb HDAC6in embryonic stem cells ESCs can promote ESCs to diï¬erentiateinto myoblast cells and transplantation of ESCs with HDAC6knockdown can also help the regeneration of wound skeletalmuscles Lee type I fiberthe proportion ofRegulation of Class II HDACs onFiberType Switch MitochondriaRemodeling and Energy Stress inSkeletal MuscleEvidence showsinthatskeletal muscles has no change in animal knocked out aloneany member of Class IIa Potthoï¬ Howeverthe proportion of type I fiber is significantly increased inHDAC45 DKO HDAC59 DKO HDAC459 TKO miceas well as exercise capacity of skeletal muscles Potthoï¬ Altogether the finding indicated a functionalredundant mechanism in Class II HDACs family Previousstudy found that Class IIa HDACs are closely associated withthe MEF2 family and can repress their downstream targetgenes McKinsey 2000a Therefore similar to Class IIaHDACs DKO the proportion of type I fiber is decreased in theMEF2C and MEF2D knockout mouse models overexpressingMEF2C results in increasing type I fiber and exercise capacityHDAC45 not only regulates exercise capacity but also governsmotor adaptation Several studies had shown that exerciseFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class I HDACs regulates sarcomere structure and exercise capacity in mature muscle A schematic diagram shows Class I HDACs regulate exercisecapacity on different levels HDAC12 have redundant roles in modulating autophagy flux in muscle eliminating toxic factors in muscle HDAC3 serves as a fuelswitch and promotes muscle to use fatty acid to adapt endurance exercisepromotes phosphorylation of HDAC45 mediated by CaMK andadenosine monophosphateactivated protein kinase AMPKthen increasing a MEF2dependent transcription of Glut4 andmusclespecific genes which participates in the adaptationand plasticity of skeletal muscles McGee McGee and Hargreaves Besides exercise glucose can alsoactivate KATP channeldependent calcium signaling and CaMKwhich induce phosphorylationdependent nuclearcytoplasmtransportation of HDAC5 Meng The leavingHDAC5 releases its repressive eï¬ect on Baf60c and enhancinginsulinindependent AKT activation Meng Usingspecific inhibitor of class IIa HDAC activity myosin heavy chainPGC1α and heatshock cognate HSC70 are confirmed to beone of the substrates of HDAC4 in denervationinduced atrophymuscle in comparison with acetylated protein enrichment withuntreated group Luo Taken together Class IIaHDACs play critical roles in exercise capacity remodeling andnutrient sensing of skeletal muscles Figure For Class IIbit was revealed that glucose deprivationinduced mitochondrialfusion mediated by mitofusion1MFN1 is significantly disrupted in HDAC6 KO mice causingmitochondria degeneration which isreversed followingapplication of MFN1 acetylationresistant mutant Lee This study suggested that MFN1 deacetylation by HDAC6plays an important role in mitochondrial adaptive energyproduction and remodeling of skeletal musclesCLASS III HDACsSir2HOMOLOGSIRT1634NuclearLocated SIRT16Numerous studies have found that caloric restriction CRextends lifespan of mammals Caenorhabditis elegans and fruitflies while such eï¬ect is lost in SIR2 or NPT1 mutant C elegansstrains Imai Lin SIR2 encodes thesilencing protein Sir2p and NPT1 participates in the synthesis ofNAD NAD is an essential cofactor for Sir2pClass III HDACsto exert enzyme activity rather than Class I II and IV relayingon the zinc Imai Lin In mammalsSir2 has homologous proteins and these proteins are essentialto mitochondrial energy homeostasis antioxidant defense cellproliferation and DNA repair VargasOrtiz SIRT1 Mediates Energy Stress Adaptation in SkeletalMuscles reported that SIRT1 promotes theRodgers et altranscriptional activity of PGC1α by deacetylating the PGC1αat K13 site and mediates CRinduced gluconeogenesisrelatedgenes G6P PEPCK expression in liver Further research showedthat the NADNADH ratio and the enzymatic activity of Sir2 asensor of redox state were decreased in diï¬erentiating myocyteswhich relieved the inhibitory eï¬ect on MyoD and then promotedcell diï¬erentiation Resveratrol RSV a compound that maytarget SIRT1PGC1α can improve mitochondrial function andmetabolic homeostasis owning a potential to prolong lifespanRSV loses its activating eï¬ect on PGC1α in SIRT1 ˆ’ˆ’ MEFsLagouge Moreover RSVtreated mice at a dose of gkg increased their exercise capacity oxygen consumptionand improved insulin sensitivity against highfat induced obesityLagouge In addition RSV could enhance thedeacetylation of PGC1α in brown fat tissue BAT and skeletalmuscles which promotes mitochondrial production oxygenconsumption and thus improves metabolic syndrome Lagouge The function of SIRT1 in regulating skeletal musclesmetabolic homeostasis and exercise capacity has attracted greatattention from researchers Researchers further generated skeletalmusclespecific SIRT1mKO mice and found that mKO miceFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Control of muscle exercise adaption and mitochondrial function by Class II HDACs A Schematic of the Class IIa HDACs nucleoplasm shuttle Understress condition CaMK and AMPK could be activated and phosphorylate Class IIa HDACs Phosphorylated HDACs then bind with and shuttle to cytoplasmrelieving inhibitory effects on MEF2 and Baf60c B Class IIb HDAC6 can deacetylate MFN1 and facilitates its mitofusion function P phosphorylationlost the eï¬ect of CRinduced increasing insulin sensitivity andwere unable to deacetylate and inactivate STAT3 resulting inupregulating the phosphatidylinositol3kinase PI3K inhibitoryregulator p55αp50α expression Schenk Althoughknockout or overexpression of SIRT1 in skeletal muscles hasno eï¬ect on endurance capacity or glucose homeostasis in miceGurd Philp White Svensson some studies have shown that AMPK regulatesenergy metabolism of skeletal muscles partially mediated bySIRT1 and SIRT1 is significantly increased after enduranceexercise Suwa Canto Overexpressionof Sirt1 in skeletal muscles by adenoassociated virus AAV1promotes the expression of oxidationrelated genes includingPpargc1a Tfam Cpt1b and Pdk4 while it has no eï¬ect on insulinsensitivity of body Vila But overexpressing Sirt1in liver by AAV8 can protect from fatty liver induced by highcarbohydrate food HCD Vila Taken togetherthe aforementioned studies suggest that SIRT1 possesses limitedregulation capacity of skeletal muscles motility and SIRT1may modulate mitochondrial homeostasis and mediate skeletalmuscles adaptation under certain physiological conditions suchas CR aging and regeneration Gomes Ryall Figure In addition there may be more beneficial eï¬ectsof SIRT1 on metabolism in the liver or fat tissue Lagouge Vila Stefanowicz SIRT6 Improves Muscle Fitness andExercise CapacitySIRT6 is another Sir2like deacetylase located in the nucleusWhole body deletion of SIRT6 causes mice death around weeks owning to hypoglycemia Xiao Neverthelessthe skeletal musclespecific knockout of SIRT6 causes insulinresistance and impairs glucose homeostasis of mKO mice Cui Because the activity of AMPK is reduced inSIRT6mKO mice the glycolipids absorption and utilizationof skeletal muscles are impaired and the exercise capacity ofthe mice was also attenuated Cui Furthermoreresearchers constructed the overexpressing SIRT6 Sirt6BACmice and found that their body weight and fat content werenormal but the Sirt6BAC mice could protect from HCDinducedhyperglycemia via increasing pAKTAKT induced by insulinstimulation and glucose absorption Anderson Vitroexperiments further demonstrated that the ability of glucoseuptake was enhanced mainly in skeletal muscles not in brainiWAT eWAT tissues of Sirt6BAC mice Anderson These results shows the potential eï¬ects of SIRT6 targeted inskeletal muscles on improving exercise performance and treatingmetabolic diseases Figure MitochondriaLocated SIRT34SIRT34 Maintains Mitochondria Function in SkeletalMusclesSIRT34 are mitochondrialocalized deacetylases and responsiblefor regulating the deacetylation of proteins in mitochondria andmaintaining mitochondrial homeostasis After feeding highfatdiet HFD accelerated obesity attenuated insulin sensitivityworsened fatty liver and increased acetylation of proteins inmitochondria were observed in SIRT3 KO mice Hirschey Multitissue proteomics and physiological examinationreveals that SIRT3 is responsible for mitochondrial acetylatedFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class III HDACs promote a stressinduced adaptation in muscle and regulate mitochondrial function Class III HDACs are NADdependentdeacetylases and may function as a redox sensor in muscle cell SIRT1 could activate PGC1alpha by directly deacetylasing K13 site and regulate insulin sensitivityby targeting STAT3 Mitochondrialocated Class III HDACs control mitochondrial function by deacetylating certain mitochondrial proteins modulating muscle fuelutilization and exercise capacityproteome regulation and metabolic fuel switching in brainheart kidneyliver and skeletal muscles DittenhaferReed Protein enrichment analysis discovers that itsregulatory proteins were mainly concentrated in lipid metabolismDittenhaferReed Knocking out SIRT3 leadsto reduction in the levels of deacetylation of manganesesuperoxide dismutase MnSOD mitochondrial complex II andpyruvate dehydrogenase PDH subunit E1α a downregulationof hexokinase II HK II binding with mitochondria resultingin impaired glucose and lipid metabolism of skeletal musclesLantier Interestingly SIRT3 liverspecific knockouthepˆ’ˆ’ and skeletal musclespecific knockoutskmˆ’ˆ’mice did not aï¬ect glucose homeostasis under chow or HFDconditions FernandezMarcos The above resultsindicate that SIRT3 has an important role in metabolic regulationbut in specific physiological processes such as redox stateexercise and aging and its regulatory mechanism is yet definedin skeletal muscles Kong Robin Williams A recent study found that SIRT4 knockoutcan resist HFDinduced obesity and increase endurance exercisein mice by repressing malonyl CoA decarboxylase which wasa key enzyme controlling fatty acid betaoxidation and wasreported to regulate muscle fuel switching between carbohydratesand fatty acids Koves Laurent Moreover knockdown of SIRT4 by adenoviral shRNA canincrease the mRNA and protein content of SIRT1 therebyenhancing the expression of fatty acid oxidation genes andmitochondrial oxidation capacity in hepatocytes and myotubesNasrin Table THERAPEUTIC TARGETS OF HDACsAND THEIR POTENTIAL FORMETABOLIC DISEASESA comparative analysis used HDAC paninhibitor SAHA aclass I HDAC selective inhibitor MS275 and a class IIHDAC selective inhibitor MC1568 to treat C2C12 separatelyIt was found that only MS275 could significantly stimulatemitochondria biogenesis and oxygen consumption Galmozzi In obese diabetic mice it was found that specificallyinhibiting Class I rather than Class II HDACs improvedGTT and insulin sensitivityincreased oxidative metabolismof skeletal muscles and adipose tissue and reduced bodyweight Galmozzi Similar to the eï¬ect of MS275knockdown HDAC3 in C2C12 could also increase Pgc1αGlut4 Tfam Idh3α transcription suggesting that HDAC3 andits target genes played an important role in the above eventsGalmozzi For Class II HDACs studies have found that scriptaid a ClassIIa HDAC inhibitor has similar eï¬ects to exercise Six weeksof scriptaid administration significantly improved enduranceperformance and significantly increased the wholebody energyexpenditure and the expression of lipid oxidation related genesPdk4 Cpt1b Pgc1α Pparδ etc of C57BL6 mice Gaur One possible explanation of above observation is thepotential target of scriptaid inhibition of titin a structure proteinof sarcomere deacetylation and downstream genes of Class IIaHDACs Gaur Huang Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityIn mice treated with SPT1720 another activator of SIRT1overtly enhanced endurance exercise ability was noted Moreoverthese mice were protected from HFDinduced obesity and insulinresistance owing to an upregulation of the oxidative metabolismin skeletal muscles liver and BAT tissues Pacholec In order to further explore the principle of SIRT1 activation theresearchers purified SIRT1 in vitro and added SRT1720 and itsanalogs SRT2183 SRT1460 and RSV and they found that theenzymatic activity of SIRT1 was not enhanced suggesting thatthese drugs may indirectly activate SIRT1 Pacholec The enzymatic activity of SIRT1 largely depends on the contentof cofactor NAD This implied that the enhancement of SIRT1activity may be achieved through indirect upregulation of NADStudy has discovered that knocking out poly ADPribosepolymerase1 PARP1 which is a NAD consuming enzymecould increase NAD content and enhance the activity of SIRT1in BAT and skeletal muscles Bai PARP1 inhibitorscan upregulate the proteins of mitochondrial respiratory chaincomplexes in mice enhance the aerobic oxidation capacity ofmitochondria and improve mitochondrial defects in the primarymyotubes of obese humans Bai Pirinen et alResveratrol was initially reported as an SIRT1 activatorthat improves mitochondrial function and exercise capacity inmice and resists HFDinduced obesity Lagouge However subsequent studies have discovered that administeringthe same dose of RSV to rats or mice does not increasemitochondrial protein content Higashida Bycontrast overexpression of SIRT1 in the triceps muscle of ratsdecreases the mitochondrial protein content Higashida In a doubleblind human trial healthy and obesemen were supplied for 30day RSV in which the data showedthat RSV can improve systolic blood pressure and homeostasismodel assessment HOMA indexindicating glucose metabolismability simulating the eï¬ect of CR Timmers However some researchers found that the overexpression ofSIRT1 alone cannot mimic the CR eï¬ect in transgenic mice andthe transcriptomic changes in various tissues were quite diï¬erentor even opposite Boutant Such an opposite situationmay explain that the genetic model and compound stimulationare not completely consistent RSV as a potential metabolicsyndrome treatment drug still needs largescale populationsample verificationCONCLUSIONThe very first mammalian histone deacetylase HDAC1 wascloned and isolated by Taunton and sabout HDACs have been published in the last years CurrentlyREFERENCESAnderson J G Ramadori G Ioris R M Galie M Berglund E D CoateK C Enhanced insulin sensitivity in skeletal muscle and liver byphysiological overexpression of SIRT6 Mol Metab “ 101016jmolmet201509003we know at least HDAC proteins They are responsible foreradicating epigenetic modifications establishing an epigeneticoï¬ chromatin state and regulating heritable gene expressionYang and Seto Haberland In these processeseach HDACs may play a diï¬erent role Table What kind ofgeneprotein is the specific downstream target of these HDACsIs its enzyme activity related to intracellular localization andthe formation of multiprotein complex It is still one of thekey and difficult issues in this field Based on previous researchexperience some techniques may be used to further experimentsas follows HDACs interacting proteinscomplex coIP Proteinacetylation western Histone acetylation target geneChIP High through put proteomicsacetylome with specificHDACs inhibitor etctherapeuticare potentialHistone deacetylasestargetsand clinical drugs such as SAHA Vorinostat and FK228romidepsin have been used for antitumor treatment Bolden However they have two disadvantages greattoxic and side eï¬ects and difficulty in specific inhibition ofHDACs activity With the development of computer simulationtechnology and structural biology it is believed that more specificHDACs inhibitorsactivators can be constructed And researchesshould attach importance to HDACs regulatory factors likePARP1 when direct targeting on HDACs fails to show its eï¬ectsFurther development of their inhibitors with more specificityand trials for the treatment of metabolic diseases may have greatpotential as well

Thyroid_Cancer

immune‘related genes pairs signature predict the prognosis of cervical cancer patientsHan Nie1 Fanqin Bu2 Jiasheng Xu1 Taoshen Li1 Jun Huang2To screen the key immune genes in the development of cervical cancer construct immune related gene pairs IRGPs and evaluate their influence on the prognosis of cervical cancer Tumor Genome Atlas TCGA database and geo database were downloaded as training set and validation set respectively and immune related gene data were downloaded from immport IRGPs model is established by machine learning and the model is analyzed and evaluated Using the Uclcan to analyze the immune genes expression in cervical cancer and to further explore the association with the expression level and the clinical stage and prognosis of cervical cancer According to the analysis of training set we identified IRGPs as key gene pairs and constructed the model The AUC value of the model was greater than and the model group survival rate was conspicuous different P The reliability of the model was confirmed in the validation group Our IRGPs play an important role in the occurrence and development of cervical cancer and can be used as a prognostic marker and potential new target of cervical cancerCervical cancer is one of the four most common gynecological tumors1 Every year at least women in the world are diagnosed with cervical cancer and more than people are killed2 In recent years the incidence rate of cervical cancer has decreased significantly through universal screening and health knowledge However the incidence rate of cervical cancer is still high in developing countries3 For women with low education in less developed areas the coverage rate of cervical cancer screening is still very low4 Squamous cell carcinoma is the most common type of cervical cancer accounting for of cervical cancer cases while adenocarcinoma only accounts for about In developing countries of cervical cancer patients have local infiltration or metastasis which has led to the high mortality of cervical cancer in developing countries Early cervical cancer is usually treated by radical hysterectomy When there are risk factors such as lymph node metastasis and endometriosis that may lead to recurrence they will be treated with chemotherapy5 The standard treatment for patients with locally advanced cervical cancer is conventional radiation therapyCRT6 The fiveyear survival rate of patients with locally advanced cervical cancer can be as high as “ after surgical resection radiotherapy chemotherapy CRT and so on7 However at present all treatment methods are not effective for patients with paraaortic lymph node metastasis and their threeyear progression free survival time PFS and total survival time OS are and respectively The fiveyear survival rate of cervical cancer patients with recurrence and metastasis was as low as Limited treatment is the main reason for this situation Now palliative chemotherapy is the most commonly used for patients with metastatic and recurrent cervical cancer10 The median survival time of patients with metastatic or recurrent cervical cancer treated with platinumtaxane chemotherapy and bevacizumab can be extended to a0months11 However these treatments are far from enough for most locally advanced and metastatic cervical cancer patients with positive lymph node metastasisIn recent years immunotherapy has been developed and increasingly used in cancer patients For example PDL1 is overexpressed in a variety of tumor cells including liver cancer cells and lung cancer cells and plays an important role in regulating the immune response of tumor cells12“ Currently there are several clinical trials involving FDAapproved immunosuppressive checkpoint inhibitors which attack tumor cells expressing PDL1 by blocking the PDL1PD1 signaling pathway so as to improve the treatment and prognosis of patients From the current situation immunosuppressive therapy has achieved good results in many solid tumors16 The 1Department of Vascular Surgery The Second Affiliated Hospital of Nanchang University No Minde Road Nanchang Jiangxi Provence China 2Department of Gastrointestinal Surgery The Second Affiliated Hospital of Nanchang University No Minde Road Nanchang Jiangxi Provence China email junhuangncu163comScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cresults of PD1PDL1 inhibition in cervical cancer are also satisfactory However at present immunoassay sites with a therapeutic effect are scarce and the research on tumor immunotherapy is far from sufficient In this study we screened immune genes that are significantly related to the prognosis of cervical cancer constructed an immune gene pair IRGP model based on these genes and used it to verify the unique prognostic markers of cervical cancerMethodData acquisition Gene expression profile data of patients with cervical squamous cell carcinoma were obtained from cancer and tumor gene map TCGA wwwtcga and gene expression profile data set gse4400116 was obtained from gene expression compilation GEO wwwncbinlmnihgovgeo database including samples of cervical cancer patientsAcquisition of sample immune gene expression immune related genes were downloaded from immport wwwimmpo rthome including antigen presenting cells chemokines and their receptors cytokines and their receptors interferon interleukin etc Using limma package in R we compared the gene expression data of cervical cancer samples downloaded from TCGA database and geo database as training set and verification set with immune related genes and extracted the expression amount of immune related genes in cervical cancer samplesConstruction of immune related gene pairs IRGPs In the two groups of data processed in the previous step the IRGP of the sample is calculated and the relatively high change is selected according to the standard of media absolute deviation IRGP values are calculated by comparing gene expression levels in specific samples or profiles in pairs The immune related genes are matched to compare the IRGPs If the first IRG is larger than the second IRG the output of the IRGP is otherwise the output is If the ratio of IRGP score of or in training set and verification set is higher than then remove the IRGP and retain the remaining IRGP as candidate IRGP for prognosis prediction The logistic rank test was used to screen the prognosis IRGP FDR Cox risk regression analysis and glment in R were used to perform tenfold cross validation to analyze the candidate IRGP and obtain the IRGP index We constructed the best gene pairs as immune gene pair model We use ROC to calculate the optimal cutoff value of IRGP index and use it as the basis to distinguish high and low risk groupsIRGPs model validation The single factor and multi factor Cox proportional risk analysis and survival analysis of TCGA and gse44001 cervical cancer samples were carried out with IRGPs modelInfiltration of immune cells in cervical cancer samples In order to study the infiltration of immune cells in the high and low risk groups of cervical cancer we used CIBERSORT17 to evaluate and predict the enrichment of immune cells in the samples CIBERSORT is a tool for deconvolution of the expression matrix of immune cell subtypes based on the principle of linear support vector regression RNA SEQ data can be used to estimate the infiltration of immune cells CIBERSORT can analyze the relative abundance of immune infiltrating cells in each sample including NK cells T cells B cells and macrophagesFunctional enrichment analysis of GSEA go and KEGG Gene set enrichment analysisGSEA enrichment analysis was carried out for each gene related to immune prognosis using the fgsea package in R Cluster profiler19 was used to enrich Gene ontologyGOfunction and KEGG pathway Significant enrichment criteria the absolute value of NES is greater than the nomp value is less than and the fdrq value is less than Expression of immune gene in cervical cancer Ualcan were used to analyze the expression of immune genes in cervical cancerStatistical analysis Measured data were expressed as mean ± standard deviation x ± s and data were compared using t test Kaplan Meier method was used for survival analysis The receiver operating characteristic curve ROC curve and ROC analysis were completed by survivalROC103 Single factor and multi factor analysis using Cox proportional risk regression model P was statistically significant P a0 as the difference has very significant statistical significanceEthical approval and consent to participate This article does not contain any studies with patients or animals performed by any of the authorsResultsExpression of immune related genes and construction of IRGPs in cervical cancer samples We obtained gene expression data of cervical cancer samples from TCGA database as training set cervical cancer samples from gse44001 as verification set immune related genes from immport and immune related genes from cervical cancer samples by comparing the two Through these immune related genes we constructed IRGPs We remove more than of the IRGP with a score of or from the training set and validation set leaving IRGP as candidates Combine TCGA clinical data with training set data Scientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cTCGA clincial dataAge ‰¥ ‰¥ GradeG1G2G3G4TT1T2T3T4MM0M1NN0N1Table TCGA clinical dataIRG1APOBEC3HARG2BTCCCL2CCL20CCL20CCL20CCL28CXCL1CXCL2DESDESDLL4FLT3LGHCKHCKHLADQA2IL1BIL1BJAK1NOD1NRP1PLXNB3PSMD7RBP7RBP7STC1TLR3VAV3Immune processesAntimicrobialsAntimicrobialsCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesAntimicrobialsCytokinesAntimicrobialsAntimicrobialsAntigen_Processing_and_PresentationAntimicrobialsAntimicrobialsAntimicrobialsAntimicrobialsCytokine_ReceptorsCytokine_ReceptorsAntigen_Processing_and_PresentationAntimicrobialsAntimicrobialsCytokinesAntimicrobialsBCRSignalingPathwayTable Model information about IRGPIImmune processesCytokinesCytokinesCytokinesCytokine_ReceptorsIRG2BTCCLCF1IL16FGFR3APOBEC3C AntimicrobialsARAFPLXNA1MAP3K14TNFSF10PTAFREPORVEGFCDESINHBASAA2STC2LTB4R2DUOX1EDN1APOBEC3C AntimicrobialsCSF2RBCD3DFGFR2SHC1CXCR3DESTNFRSF18CXCR6NRP1NaturalKiller_Cell_CytotoxicityChemokine_ReceptorsTCRsignalingPathwayTNF_Family_MembersChemokine_ReceptorsCytokine_ReceptorsCytokinesCytokinesTGFb_Family_MemberChemokinesCytokinesCytokine_ReceptorsAntimicrobialsChemokinesCytokine_ReceptorsTCRsignalingPathwayCytokine_ReceptorsNaturalKiller_Cell_CytotoxicityChemokine_ReceptorsCytokinesCytokine_ReceptorsAntimicrobialsCytokine_ReceptorsCoefficient““““““““““Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure A Timedependent ROC curve for IRGPI in the training cohort B Timedependent ROC curve for IRGPI in a0year C Timedependent ROC curve for IRGPI in a0year D Timedependent ROC curve for IRGPI in a0yearTable a0 prognosis related IRGPs were screened by lasso Cox proportional risk regression analysis After iterations we selected optimal IRGPs to build the immune prognosis model Table a0IRGPs model validation The immune prognosis model was applied to the training set and the patients in each training set were scored According to ROC curve analysis the optimal cutoff value for patients to be divided into high and low risk groups is Fig a01A After evaluating the model we found that AUC value of model and a0years is Fig a01B Fig a01C and Fig a01D The results show that our immune prognosis gene has a high reliability for the model The training set was divided into highrisk group Fig a02A and highrisk group Fig a02B The results showed that the overall survival rate OS of highrisk group was significantly lower than that of lowrisk group For TCGA training set data single factor and multi factor Cox risk regression analysis showed that only IRGPs model showed significant prognostic effect in single factor Cox Fig a02C while age and IRGPs could be significant independent prognostic factors in multi factor Cox Fig a02D Applying this model to the validation set of gse44001 Fig a03A Table a0 survival analysis showed that the OS of patients in the highrisk group was significantly lower than that in the lowrisk group Fig a03B In the single factor and multi factor Cox analysis IRGPs model and tumor size were significantly correlated with prognosis Fig a03CDInfiltration of immune cells in cervical cancer samples Most studies believe that the occurrence and development of tumor are closely related to immune cells so it is an ideal method to study the infiltration of immune cells in tumor We used CIBERSORT to analyze the infiltration of kinds of immune cells in patients with high and low risk groups Figure a04A shows the expression of immune cells in different risk groups Macrophage M0 Fig a04B activated mast cells Fig a04C were significantly overexpressed in the highrisk group while stationary dendritic cells Fig a04D stationary mast cells Fig a04E activated CD4T cells Fig a04F and cd8t cells Fig a04G were overexpressed in the lowrisk groupScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A The model divides the training set patients into lowrisk or highrisk groups B Kaplan Meier curve between high and low risk groups C Training set single factor Cox regression analysis forest map D Training set multivariate Cox regression analysis forest mapFunctional enrichment analysis of GSEA go and KEGG We analyzed the function enrichment of IRGP in the model The results of go analysis showed that IRGP in the model was mainly enriched in the binding of cytokines and their receptors the binding of chemokines and their receptors the binding of growth factors and their receptors the binding of epidermal growth factor receptors the binding of fibroblast growth factors and the activity of tyrosine kinase Fig a05AB KEGG results showed that these IRGP were involved in cytokine cytokine receptor interaction chemokine signaling tumor necrosis factor signaling MAPK signaling NF kappa B signaling natural killer cellmediated cytotoxicity viral proteins and cytokines and Th1 and Th2 cell differentiation Fig a05CD The results of GSEA Fig a06A showed that these IRGP were significantly enriched in trace ribonucleoprotein complex Fig a06B neurotransmitter transporter activity Fig a06C endopeptidase activity Fig a06D fibroblast growth factor receptor binding Fig a06E hormone activity Fig a06F fibroblast cell proliferation Fig a06G and growth factor receptor binding Fig a06HExpression of immune gene in cervical cancer We explored the expression of IRGP in cervical cancer using the ualcan model Table a0 There were lowlevel expression of IRGP in cervical cancer Fig a0 and highlevel expression of IRGP Fig a0 There were differences in the expression of low expression IRGP and high expression IRGP in different age groups Fig a0 and there were differences in the expression of IRGP in different stages of cervical cancer Fig a0DiscussionCervical cancer is one of the most common gynecological malignancies HPV infection is considered to be the main cause of cervical cancer2122 although the incidence rate of cervical cancer has been significantly decreased due to the development and promotion of HPV vaccine23 But incidence rate of cervical cancer is still high in developing countries and China™s low income countries24 At present for cervical cancer patients without invasion and lymphatic metastasis the effect of surgery combined with radiotherapy and chemotherapy is better If metastasis and infiltration occur the treatment effect of cervical cancer patients will become very unsatisfactory In recent years immunotherapy has performed well in a variety of cancers including cervical cancer25“ Blocking PDL1 PD1 signaling pathway to attack tumor cells expressing PDL1 is the current mainstream method28 Although the anticancer activity of PD1 and PDL1 inhibitors is exciting such immunotherapy is not effective for all patients and a metaanalysis shows that patients who receive PD1 PDL1 inhibitors have a Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure A The model divides the validation set patients into lowrisk or highrisk groups B Kaplan Meier curve between high and low risk groups C Validation set single factor Cox regression analysis forest map D Validation set multivariate Cox regression analysis forest mapGSE44001 clincial dataStageLargest diameter cm ‰¥ ‰¥ ‰¥ Table GSE44001 clincial datahigher risk of rash thyroid dysfunction pruritus pneumonia and colitis29“ Therefore it is of great significance for the detection and treatment of cervical cancer to predict and find more biomarkers that may be related to immune prognosisAt present most of the prognostic genes need to be standardized to reduce the errors caused by sequencing platform and samples In this study the scores of IRGPs constructed by us are calculated from the gene expression data of the same sample which can not only ignore the impact of different platforms but also do not need to standardize and scale the data This method has been used in many studies including cancer molecular classification with high reliability3233In this study we screened pairs of IRGP to construct the immune prognosis model related to the overall survival rate of cervical cancer patients The AUC values of the model in and a0years were all greater than According to these pairs of IRGP they were divided into highrisk group and lowrisk group In TCGA training group and GSE44001 verification group the OS of highrisk group was significantly lower than that of lowrisk group P These pairs of IRGP have a good effect on sample discrimination We found that macrophage Mo and activated mast cells were significantly over expressed in highrisk group by immunocyte infiltration analysis of samples The existing research shows that mast cells and macrophages play an important Scientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Immune infiltration status within IRGPI risk groups B Expression of Macrophage M0 C Expression of Mast cells activated D Expression of Dendritic cells resting E Expression of Mast cells resting F Expression of T cells CD4 memory activated G T cells CD8role in cervical cancer which can promote the development of cervical cancer by promoting lymphangiogenesis and angiogenesis34“ However in the lowrisk group the expression of static dendritic cells static mast cells activated CD4T cells and cd8t cells is high Although the effect of CD4T cells on cervical cancer has not been agreed the cd8t cells are closely related to the better prognosis of cervical cancer patients37“ there is evidence that dendritic cells will decrease in patients with high HPV infection which indicates that high expression of dendritic cells is beneficial to resist cervical cancer40 which is consistent with our results The enrichment analysis of go and GSEA showed that these immune genes were mainly involved in the binding of cytokines and their receptors the binding of chemokines and their receptors the binding of growth factors and their receptors the binding of epidermal growth factor receptors the activity of metalloendopeptidase the binding of fibroblast growth factors and their receptors hormone activity fibroblast proliferation and the binding process of growth Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Histogram graph of Immunerelated genes GO analysis results B Point graph of Immunerelated genes GO analysis results C Histogram graph of Immunerelated genes KEGG pathway analysis results D Point graph of Immunerelated genes KEGG pathway analysis resultsfactor receptorsAs we all know cytokines and chemokines are the key factors in the immune response for example In cervical cancer IL10 can interfere with the differentiation of dendritic cells and thus play a strong immunosuppressive effectTGF”β can inhibit T cell proliferation and attenuate immune response41 Research shows that growth factors and epidermal growth factors are closely related to the growth of cervical cancer and the survival rate of cervical cancer patients High expression of growth factors and epidermal growth factors often predict poor prognosis42“ Growth of fibroblasts can stimulate angiogenesis at the early stage of tumor The proliferation and invasion of cancer cells and the remodeling of extracellular matrix promote the growth of cervical cancer4546 KEGG results showed that these immune genes were mainly enriched in chemokine signaling pathway tumor necrosis factor signaling pathway MAPK signaling pathway NF kappa B signaling pathway natural killer cellmediated cytotoxicity viral protein and cytokine and Th1 and Th2 cell differentiation Th1 and Th2 may be involved in the pathogenesis and growth of cervical cancer Th1 may be the target of predicting chemotherapy response of advanced cervical cancer47“ while other pathways are classical signal pathways related to cancer Immune cytokines play an important role in cervical lesions Torres et a0al Found that IL10 is highly expressed in the cervix of women with persistent HPV which may be related to the persistence of HPV and the promotion of disease progression Further research by their team showed that copy individuals of IL4 IL6 IL10 and TGFB1 were significantly associated with cervical cancer and could be used as biomarkers for susceptibility to the disease5152These pairs of IRGP have different immune genes most of which are cytokines antimicrobial agents and natural killer cells which are involved in various stimulation reactions and play a key role In cervical cancer HPV can inhibit the apoptosis of cervical cancer cells by down regulating NOD153 In our sample we also found that the expression of NOD1 in tumor tissue is low and there are differences in different ages and stages Figs a07D 9C 10I Sang Yeon Cho et a0al Found that duox1 is highly expressed in cervical squamous cell carcinoma and can play a good prognostic role by increasing the amount of innate immune cells54 The analysis also showed that DUOX1 is highly expressed in tumor tissues and related to age and grade Figs a08B 9G 10D Stc2 can promote the proliferation of cervical cancer cells and increase the resistance to cisplatin55 while high expression of DDL4 is usually associated with low pelvic lymph node metastasis and survival rate of cervical cancer56 Therefore we believe that the IRGP constructed in this study plays an important role in the development and prognosis of cervical cancerThere are also some deficiencies in our research Although we select data samples from two databases for analysis and use more advanced methods to reduce the errors caused by platforms samples etc this is still a retrospective analysis If we can carry out a prospective study or obtain clinical samples and evaluate them with Western blot or immunohistochemistry it will be more convincingScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A GSEA analysis of immune signature genes B“H In the high immune risk group of cervical cancer cancer marker genes were abundant P FDR Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cCLCF1DLL4INHBANOD1NRP1RBP7CXCR3DUOX1FGFR3AgeNormalvsAge4160YrsAge2140YrsvsAge4160YrsAge2140YrsvsAge6180YrsAge2140YrsvsAge81100YrsAge4160YrsvsAge6180YrsAge4160YrsvsAge81100YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsAge2140YrsvsAge81100YrsAge4160YrsvsAge6180YrsAge4160YrsvsAge81100YrsAge6180YrsvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsNormalvsAge2140YrsHLADQA2NormalvsAge4160YrsLTB4R2STC2TNFSF10VAV3NormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge4160YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsAge4160YrsvsAge6180YrsPval777E05123E02483E04115E07209E02433E07270E06354E05281E02982E03260E03384E04322E02163E05600E04868E03106E02109E02122E04396E03281E04459E02421E04876E03441E02105E02384E04566E05218E05803E09139E06225E08111E16204E13725E10305E02422E07693E10174E05433E15162E12550E10341E02803E04159E06458E04192E02524E10415E11559E10162E12162E12934E14108E02208E03StageNormalvsStage2NormalvsStage3Stage1vsStage2Stage1vsStage3Stage1vsStage4NormalvsStage1NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage4Stage3vsStageNormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Pval185E06220E04542E03304E02460E04696E07546E03252E03210E02439E02390E03186E02395E02317E02122E03164E03396E04984E03222E02315E06226E02387E07734E03433E01118E04173E05355E04222E02160E05259E09131E09258E04162E12199E12176E05202E04663E12390E05241E04162E12860E10479E09175E04148E02240E04435E05119E04463E02230E13297E09263E07840E04170E02162E12493E12284E12125E04Table P value of IRGPs expression in different ages and stagesScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues B Expression of DLL4 in cervical cancer and normal tissues C Expression of INHBA in cervical cancer and normal tissues D Expression of NOD1 in cervical cancer and normal tissues E Expression of NRP1 in cervical cancer and normal tissues F Expression of RBP7 in cervical cancer and normal tissuesScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Expression of CXCR3 in cervical cancer and normal tissues B Expression of DUOX1 in cervical cancer and normal tissues C Expression of FGFR3 in cervical cancer and normal tissues D Expression of HLADQA2 in cervical cancer and normal tissues E Expression of LTB4R2 in cervical cancer and normal tissues F Expression of STC2 in cervical cancer and normal tissues G Expression of TNFSF10 in cervical cancer and normal tissues H Expression of CESC in cervical cancer and normal tissuesScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues at different ages B Expression of DLL4 in cervical cancer and normal tissues at different ages C Expression of NOD1 in cervical cancer and normal tissues at different ages D Expression of NRP1 in cervical cancer and normal tissues at different ages E Expression of RBP7 in cervical cancer and normal tissues at different ages F Expression of CXCR3 in cervical cancer and normal tissues at different ages G Expression of DUOX1 in cervical cancer and normal tissues at different ages H Expression of FGFR3 in cervical cancer and normal tissues at different ages I Expression of HLADQA2 in cervical cancer and normal tissues at different ages J Expression of LTB4R2 in cervical cancer and normal tissues at different ages K Expression of STC2 in cervical cancer and normal tissues at different ages L Expression of TNFSF10 in cervical cancer and normal tissues at different ages M Expression of VAV3 in cervical cancer and normal tissues at different agesConclusionWe constructed an immune gene pair model which is closely related to the prognosis of cervical cancer patients The model contains IRGP and immunerelated genes The biological functions of these immunerelated genes are closely related to the occurrence and development of cervical cancer Therefore we think that these IRGPs may be the target of predicting or diagnosing cervical cancer and suggest that immunotherapy can improve the prognosis of cervical cancer patients by regulating these IRGPsScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues at different stages B Expression of CXCR3 in cervical cancer and normal tissues at different stages C Expression of DLL4 in cervical cancer and normal tissues at different stages D Expression of DUOX1 in cervical cancer and normal tissues at different stages E Expression of FGFR3 in cervical cancer and normal tissues at different stages F Expression of HLADQA2 in cervical cancer and normal tissues at different stages G Expression of INHBA in cervical cancer and normal tissues at different stages H Expression of LTB4R2 in cervical cancer and normal tissues at different stages I Expression of NOD1 in cervical cancer and normal tissues at different stages J Expression of NRP1 in cervical cancer and normal tissues at different stages K Expression of RBP7 in cervical cancer and normal tissues at different stages L Expression of STC2 in cervical cancer and normal tissues at different stages M Expression of TNFSF10 in cervical cancer and normal tissues at different stages N Expression of VAV3 in cervical cancer and normal tissues at different stagesData availabilityAll data are available Please contact us to access if it is neededReceived May Accepted July References Stewart Bernard W et al Cancer prevention as part of precision medicine œplenty to be done Carcinogenesis “ 101093carci nbgv16 Bray F et al Global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “ 103322caac21492 Passos Camila M Sales Jacqueline B Maia Emanuella G Caldeira Thaís C M Rodrigues Roberta D Figueiredo N Claro Rafael M Trends in access to female cancer screening in Brazil “ J Public Health Oxf 101093pubme dfdaa0 Asrabuddhe V V Parham G P Mwanahamuntu M H Vermund S H Cervical cancer prevention in low and middleincome countries feasible affordable essential Cancer Prevent Res “ 10115819406207CAPR110540 Koh WuiJin AbuRustum Nadeem R Bean Sarah Bradley Kristin Campos Susana M Cho Kathleen R Chon Hye Sook Chu Christina Clark Rachel Cohn David Crispens Marta Ann Damast Shari Dorigo Oliver Eifel Patricia J Fisher Christine M Frederick Peter Gaffney David K Han Ernest Huh Warner K Lurain John R Mariani Andrea Mutch David Nagel Christa Nekhlyudov Larissa Fader Amanda Nickles Remmenga Steven W Reynolds R Kevin Tillmanns Todd Ueda Stefanie Wyse Emily Yashar Catheryn M McMillian Nicole R Scavone Jillian L2019 Cervical Cancer Version NCCN Clinical Practice Guidelines in Oncology J Natl Compr Canc Netw “ 106004jnccn Chen J et al Nanotechnology in the management of cervical cancer Rev Med Virol 25Suppl “ 101002 Varia M A et al Cervical carcinoma metastatic to paraaortic nodes extended field radiation therapy with concomitant 5fluorouracil and cisplatin chemotherapy a Gynecologic Oncology Group study Int J Radiat Oncol Biol Phys “ 101016s0360 Randall Leslie M Monk Bradley J Darcy Kathleen M Tian C Burger Robert A Liao SY Peters William A Stock Richard J Fruehauf John P Markers of angiogenesis in highrisk earlystage cervical cancer a Gynecologic Oncology Group study Gynecol Oncol 101016jygyno Boussios S et al Management of patients with recurrentadvanced cervical can

Thyroid_Cancer

"Pluripotent stem cellsDirected differentiationIn vitro disease modellingLungAirwayMechanical cuesContentsChronic lung diseases remain major healthcare burdens for which the only curative treatment is lung transplantation In vitro human models are promising platforms for identifying and testing novel compounds to potentiallydecrease this burden Directed differentiation of pluripotent stem cells is an important strategy to generate lungcells to create such models Current lung directed differentiation protocols are limited as they do not recapitulate the diversity of respiratory epithelium generate consistent or sufficient cell numbers for drug discoveryplatforms and establish the histologic tissuelevel anization critical for modeling lung function In this review we describe how lung development has formed the basis for directed differentiation protocols and discussthe utility of available protocols for lung epithelial cell generation and drug development We further highlighttissue engineering strategies for manipulating biophysical signals during directed differentiation such that futureprotocols can recapitulate both chemical and physical cues present during lung development Elsevier BV All rights reservedOverview of key developmental stages Lung anogenesis molecularly defining lung fate in the embryo Branching morphogenesis and other mechanical cues generated during lung development Introduction Human embryology as a blueprint for lung directed differentiation Directed differentiation of lung epithelia inspired by embryology Mouse embryonic stem cell derived lung epithelia Modeling airway and lung diseases for drug discovery Opportunities to exploit mechanical cues for improving directed differentiation protocols in the future Micropatterning in 2D Stem cell behaviour on substrate topographies Micropatterning in 3D anoid systemsSubstrate textureHuman pluripotent stem cellderived lung epithelia Creation of human proximal lung epithelia Comparisons of proximal airway directed differentiation protocols Creation of human distal lung epitheliaComparisons of distal lung directed differentiation protocols Limitations of current directed differentiation protocols Ž Correspondence to G Karoubi Latner Thoracic Surgery Research Laboratories Toronto General Hospital College St Toronto ON M5G 1L7 CanadaŽŽ Correspondence to A P McGuigan Institute for Biomaterials and Biomedical Engineering University of Toronto College Street Toronto ON M5S 3G9 CanadaEmail addresses golnazkaroubiuhnresearchca G Karoubi alisonmcguiganutorontoca AP McGuigan101016jaddr2020080050169409X Elsevier BV All rights reservedPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxAcknowledgementsReferencesFuture outlook IntroductionEndstage lung disease is the third leading cause of morbidity andmortality worldwide [] and produces a significant burden onhealthcare systems due to extensive resource expenditures for diseasemanagement and as lung transplantation is the only curative treatmentoption Such diseases include acute respiratory distress syndromechronic obstructive pulmonary disease cystic fibrosis and pulmonaryfibrosis Chronic pulmonary diseases result in million global deathsper year [] Patients who receive transplants face continued complications associated with chronic immunosuppression and graft rejectionwith the transplant survival rates at and years being and respectively [] Furthermore since lungs function as an important barrier between the internal and the external environments they are a critical site for bacterial and viral infections and disease transmissionparticularly relevant given the current COVID19 pandemic There istherefore a critical need to better elucidate the mechanisms of infectiondisease progression host response and cellular repair in the lung to enable the development of novel targeted therapeutics for lung diseaseTissueengineered models have emerged as a technology to addressthis challenge and shown some success in drug identification and toxicology studies For example commercially available airway epithelialmodels such as EpiAirwayTM MatTek Life Sciences serve as convenientplatforms with airliquid interface culture capabilities for assessing theeffect of chemical and physical stimuli [“] Other examples includethe Alveolus LungChip and Airway LungChip systems Emulate Incoriginally developed in the Ingber laboratory which mimic theepithelialendothelial interface of the airway and provide a more dynamic platform for testing new anti‚ammatory compounds inasthma [] and new small molecule targets to decrease cancerassociated pulmonary edema [] More complex models have alsobeen reported which involve selfassembly of heterogeneous progenitor cells into 3D structures termed anoids [] These anoidmodels can recapitulate aspects of human lung development in termsof tissue structures and protein expression and therefore present apromising opportunity for drug screening []A challenge in developing such human in vitro lung models to screenfor drugs however is the requirement for large batches of similarhuman cells as a starting population for tissue manufacturing to ensureminimal heterogeneity between test wells [] Achieving this is especially challenging when using primary human lung cells which exhibitconsiderable heterogeneity across donors and have a limited ability togrow and differentiate reliably [] Furthermore primary cells areoften extracted from diseased donors which is not ideal for conductingcontrolled studies due to the wide range of therapeutic and environmental factors these cells have already been exposed to Directed differentiation of pluripotent populations has the potential to create vastnumbers of cells from either healthy or diseased patients It allows introduction of specific diseaseassociated mutations via CRISPRCas9gene editing to recapitulate and understand pathologies in a controlledmanner As such directed differentiation enables the generation of anattractive cell source for drug screening platforms and personalized disease models that may provide insight into tissue regeneration mechanisms [“]Directed differentiation protocols to manufacture specific cell populations from pluripotent stem cells PSCs have been developed to meetthe need for a homogeneous human cell source Older lung directed differentiation protocols from the late 2000s have been proven inefficientdue to the nonstandardized methods through which they derive lungendoderm from embryoid bodies [“] A series of more standardizedstepwise protocols have since emerged in the last decade that provideavenues for developing airway and lung epithelia albeit with variableefficiencies [“] The first ‚uential directed differentiation protocol to produce lung epithelia used human PSCs in [] whichwas further supported by two prominent studies conducted usingmouse PSCs in [] These protocols have continued to be enhanced through adaptations related to the selection of growth factorsand small molecules the chronology of morphogen delivery as wellas innovations in enabling platforms such as cell sorting 3D cultureand singlecell analyses to efficiently derive normal and diseased lungepithelia from human PSCs [“] Despite such advancements limitations pertaining to heterogeneity in the resultingpopulations still exist which are likely attributed to variability across directed differentiation trials PSC cell lines or the persistence of contaminating cell populations belonging to other lineages While protocolshave progressed to some degree in differentiating proximal airwayand distal alveolar epithelia they remain limited Overall many unanswered questions remain with regards to the identity maturity andfunctionality of resulting cell types as well as their utility for tissue engineering and drug testing approaches Therefore these protocols mustbe optimized further to reliably produce large numbers of spatially relevant and functional lung and airway epithelial cells that appropriatelyrespond to both chemical and mechanical stimuli in the context of disease modeling and drug discoveryIn this review we discuss the directed differentiation protocols thatattempt to recapitulate lung development and disease and highlightpossible opportunities to enhance these protocols in the future Wefirst describe development of native lung tissue and the patterningevents that occur that differentiation models attempt to mimic andhighlight how human lung embryology has served as the blueprint tocreate the common pathway of lung directed differentiation protocolsWe then discuss the evolution of directed differentiation protocols tofind opportunities for creating specific populations of airway and lungepithelia through targeted manipulation of key signaling pathways in2D and 3D models We further describe how these models have beenused to recapitulate different airway and lung diseases Finally we discuss how tissue engineering and biophysical cues using biomaterialscan be utilized during lung directed differentiation to mimic patterningcues present in development to augment current differentiationprotocols Human embryology as a blueprintdifferentiationforlung directed Overview of key developmental stagesDirected differentiation protocols have been designed to mimicin vivo human lung development [] Indeed in vitro models of lungdevelopment have provided unique insight into human lung development [] As human lung development has been described at greatlength in earlier reviews [] we provide a brief overview as followsschematically represented in Fig During early embryogenesis at days post fertilization a process called gastrulation begins with the appearance of a structure called primitive streak through which cells migrate to form the primary embryonic germ layers definitive endodermmesoderm and ectoderm [“] Definitive endoderm expandsthereby forming the primitive gut tube comprised of three endodermalregions foregut midgut and hindgut [] This is when lungPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxFig Schematic of human lung development from an epithelial perspectivedevelopment begins at approximately four weeks into embryonic lifewith the outgrowth of foregut endoderm [] and continues througheight years of postnatal life [] There are five stages to lungdevelopment Embryonic weeks “ The future lung buds emerge from the ventral side of the primitive foregut endoderm into the surroundingmesenchyme and develop into embryonic lung buds with early trachea and bronchi [“] Pseudoglandular weeks “ Branching of the airway continuesleading to formation of conducting and terminal bronchioles whilethe proximal airway epithelium begins to develop [] Canalicular weeks “ Development of the respiratory or gasexchanging airways is initiated primitive alveoli form and the future distal epithelium begins to thin as distal epithelial markers areexpressed [] Saccular weeks “ Emergence of sacshaped distal airwayswhich develop crests with muscle and elastin to create indentationsThese distal airways extend to form alveoli by weeks [] The developing epithelium and vasculature within the future alveolus continue to merge closer together to facilitate future gas exchange andfurther differentiation of alveolar epithelial cells AEC I and II takesplace Alveolar periods week “ years True alveoli are seen in week and the majority of alveolarization takes place through sacculeseptation a process by which the sacshaped distal airways changetheir internal architecture and create thin walls intraluminallySeptation leads to an increase in surface area of the gas exchangingportion of the developing lung and prepares the fetus to breath airduring this stage [] Lung anogenesis molecularly defining lung fate in the embryoDuring the embryonic period early lung is genetically defined by theexpression of transcription factor NK2 Homeobox NKX21 and Srybox SOX2 [“] During human lung development it has beenfound that the lung buds and branches given off during thepseudoglandular period are mostly SOX2SOX9 [] BothSOX2 and SOX9 are individual markers of the early proximal or distal lineage respectively [“] Over the course of the canalicular and saccular periods of development weeks “ these double positivepopulations downregulate one SOX protein and maintain expressionof the other as these cells mature towards proximal or distal lineages[] The proximal airway closer to the mouth is comprised of apseudostratified columnar epithelium that is responsible for theconducting airway function debris and pathogen removal ciliatedcells mucus production goblet cells prevention of airway ‚ammation club cells and humidification of air as it passes through to the distal lung compartment [“] The squamous distal epitheliumcomposed of alveolar epithelial cells AEC I and II facilitates the respiratory function of the lung as air in the epithelial compartment is broughtinto close apposition to blood from the pulmonary vasculature it alsosecretes surfactants which play an immunologic role and decrease thesurface tension present at the airliquid interface thereby preventing alveolar collapse [] In humans a number of cell types are found in theproximal airway each identified with specific markers Table This includes basal cells tumor protein p63P63 keratinKRT5 nerve growthfactor receptorNGFR integrin α6ITGA6 integrin β4ITGB4 ciliatedcells Forkhead BoxJ1FOXJ1 acetylated tubulinAcTUB goblet cellsmucin 5ACMUC5AC mucin 5BMUC5B club cells club cell secretoryproteinCCSP or SCGB1A1 and pulmonary neuroendocrine cellsPNECs synaptophysinSYP chromogranin ACHGA On the otherhand homeodomainonly protein HOPX identifies the distal lungalong with AEC I cells T1α podoplaninPDPN aquaporin 5AQP5while AEC II cells are recognized via surfactant protein B SPC prosurfactant protein C proSPC or SPC and HT2280 []One mechanism by which lung epithelia begin to mature is based onchemokine secretions from the surrounding mesenchyme and the developing heart field which are well reviewed here [] Key players including fibroblast growth factors FGFs [“] WNTs [“]and bone morphogenetic proteins BMPs [“] are known to inducethe differentiation of early lung progenitors in a controlled manner Forexample in mouse it has been found that FGF10 plays a role in bud outgrowth [] and drives lung progenitors towards a distal fate []through canonical WNT signaling [] Proximal epithelia developbecause they are located further away from distally located FGF reservoirs in the mesenchyme in a mechanism that appears dependent onconcentration gradients [] BMP4 plays a key role in lung bud formation from foregut endoderm and establishment of both dorsoventralback to front and proximodistal top to bottom patterning in the nascent lung [] BMP4 is also present at high levels in distal bud tips andepithelia including AEC II cells [] however its inhibition promotesa proximal fate and along with BMP2 inhibition ciliated cell development [] Branching morphogenesis and other mechanical cues generated duringlung developmentWhile the cell fate of early proximal and distal lineages is directedthrough chemical signals the lung epithelium itself undergoes markedPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxTable Epithelial populations in native human airways and lungsRegionProximal AirwayDistal LungCell TypeAssociated Markers for Cell CharacterizationCiliated CellGoblet CellClub CellBasal CellAlveolar epithelial cell type I AEC IAlveolar epithelial cell type II AEC IIFOXJ1 AcTUBMUC5AC MUC5BCCSP SCGB1A1 SCGB3A2P63 KRT5 NGFR ITGA6 ITGB4HOPX PDPN AQP5SPB SPC HT2280Cell Proportions in Native Lung“ []“ []“ []“ [] [] []changes in architecture a process known as branching morphogenesis[] From the simple tube of the anterior foregut endoderm to thecomplex tubular structure of the adult a highly stereotyped mechanismof branching morphogenesis facilitates the outgrowth division placement and structure of lung airway [] Branching morphogenesis ofthe lung is driven by three simple and iteratively used processes domain branching planar and orthogonal bifurcation [] The first formof branching is domain branching along a primary branch buds formin a linear and sequential fashion from proximal to distal The nextform of branching is planar bifurcation in which the tip of the formingtube bifurcates to create two new tips which subsequently elongateand bifurcate again creating four tips The last process of branching isknown as orthogonal bifurcation In this process the initial planar bifurcation is followed by a rotation around the planar axis which createstwo new tips through bifurcation A critical gene in this processSprouty has been found to attenuate Erk12 signaling thereby alteringthe orientation of cell division and future tube elongation [] Othercritical genes and regulatory networks associated with FGF signalingalso contribute to controlling the periodicity of the branched network[] Although elements such as domain specification bifurcation rotation and branch generation remain largely undetermined [] newtechnologies involving highresolution live imaging tension sensingand forcemapping are opening paths to further explore and explainthe branching morphogenesis phenomenon []The early structure of the lung gives rise to a striking architecturalseparation of future SOX2 proximal lineages and SOX9 distal lineages at least in mice [] The diameter of tube generated duringbranching morphogenesis in the pseudoglandular and canalicularstages has a small degree of variance within each stage as measuredfrom electron micrograph sources of fetal human tissue [] This suggests that the branching program is rigorous in its control of lung structure and that tubes themselves may have instructive potential on thedeveloping epithelia Once the basic an structure has formed thelung continues to be exposed to mechanical cues as it continues to mature In several cases these cues have been shown to be essential forcorrect an function In utero the fetal lung is a secretory an thatonly converts to an absorptive one to prepare for breathing afterbirth through a change in the activity of chloride and sodium channelslate in development Fetal lung secretions result in a static fluid pressureof around cmH2O in the developing terminal sacs of the fetus whichpropels branching morphogenesis outwards into the developing thoracic cavity [] Lack of amniotic fluid in the developing lung alters the expression of distal epithelial markers and consequentlyresults in the creation of smaller than normal lungs pulmonary hypoplasia [] highlighting the importance of this mechanical pressureduring lung development In addition cyclic strain is generated fromfetal breathing movements FBM in utero that prime the airway foruse after birth FBM are detectable from the tenth week of pregnancyand begin as infrequent and erratic activity with long quiescent periodsAs development continues these quiescent periods decrease andsustained periods of fetal breathing occur These breathing movementsvary with the fetal sleep cycle and can be chemically tuned [] andalter the volume of terminal sacs by around [] againhighlighting the importance of mechanical signals ‚uencing lung development Finally a novel FGF10FGFR2dependenttensionalmechanism has been shown by which distal epithelial cells in the lungaccumulate motor proteins at the apex of the cell thereby becoming resistant to compression from increasing fluid pressure within the tubelumen Cells under this tension are more likely to become AEC II cellswhile those under compression become AEC I cells [] Interestinglywhile the above examples highlight the importance of specific mechanical signals in the growth development and differentiation of the lungPSC directed differentiation protocols of the lung are primarily based onmimicking the sequential chemical changes that occur during lungdevelopment Directed differentiation of lung epithelia inspired by embryologyEarly attempts to create lung epithelia from PSCs began in mouseand did not attempt to mimic the stepwise changes in chemical signaling that occur during development Rather groups focused on applyinglunglike physical cues such as airliquid interface [] These protocols while successful in generating NKX21 positive populationsalso produced contaminating cells expressing pluripotency markersOCT4 NANOG SSEA4 TRA160 TRA181 These early attempts solidified that further optimization particularly related to the chemical cuesapplied was needed to reliably create lung progenitors from pluripotentsources without remnant pluripotent contaminating cells More successful directed differentiation protocols were rationalized from the detailed understanding of the chemical changes during lung embryologyIn this section we describe in detail the different differentiation protocols currently available that evolved from this approach Mouse embryonic stem cell derived lung epitheliaAlthough mouse models do not fully recapitulate human lung development they have served as guides for earlier iterations of PSC directeddifferentiation protocols and have identified critical chemical cues forlung anogenesis Broadly speaking these protocols begin by drivingstem cells towards a definitive endoderm fate SOX17 and FOXA2mimicking the preembryonic period of human lung developmentweeks “ through high doses of the nodal activating molecule ActivinA [“] Foregut endoderm is then induced via transforminggrowth factor beta TGFβ inhibition either alone [] or with BMP inhibition [] for a short period a process called anteriorization as duringthe embryonic period of human lung development weeks “ Thisforegut endoderm FOXA2SOX2 is subsequently induced to generate NKX21 cells putative lung progenitors by stimulating theretinoic acid RA BMP WNT and FGF signaling pathways []These lung progenitors are further matured as demonstrated by increased NKX21 expression through application of corticosteroids[] In brief each protocol begins with PSCs guided through definitiveendoderm followed by anteriorization to foregut endoderm and subsequent ventralization to generate NKX21 cells [] These protocolsformed the basis and backbone for the creation of human lung epitheliafrom PSCsGiven the structural and cellular complexity of the lung it is reasonable that the earliest protocols focused on mouse However there aresubtle differences that highlight how human models are different interms of structure patterning and differentiation For example thePlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxentire human conducting airway is comprised of a pseudostratified epithelium even at diameters less than 05mm [] In contrastconducting airways in mice only exhibit pseudostratified epitheliumwith accompanying submucosal glands and cartilage in the most proximal portion of the airway and transition directly into alveolar sacs []This difference in histology affects the residing cell populations as evidenced by the lack of basal cells in the lower portion of the proximal airways of mice [] Similarly mouse models suggest thatSOX2SOX9 progenitors are quite rare and their cell fate is ambiguous [] However evidence from directed differentiation of humanlung epithelia [] which has been confirmed in vivo []reveals that SOX2SOX9 progenitors are common in the developinglung buds and that branch tips of the pseudoglandular staged lunggive rise to both proximal and distal epithelia [] Moreover specificprotein markers have been found to differ in both timing and locationof expression between human and mouse models proSPC in mouseis expressed early and throughout the developing mouse epithelium[] while in human proSPC is rarely detected early in development and is only robustly found later in distal epithelia [] These examples highlight that while there are similarities development andpatterning of mouse and human lungs is different and these differencesrequire human models to be fully appreciated Human pluripotent stem cellderived lung epitheliaHuman PSC protocols have generally followed the same differentiation chronology as that of mouse directed differentiation wherein definitive endoderm anterior foregut endoderm and NKX21 lungprogenitors are produced sequentiallyDifferent groups have adhered to their own methods of generatingdefinitive endoderm which primarily involves exposing PSCs to highconcentrations of Activin A Slight variations such as introducing WNTagonism through WNT3a or CHIR99021 prior to [] or alongside[] Activin A or additional exposure to BMP4 and FGF2[] during this stage exist across protocols for differentially inducing primitive streak and its anteriorization towards producing definitive endoderm In addition the use of embryoid bodies which arelimited by user experience and technique has resulted in a widerange of production efficiencies for achieving this stage from CKITCXCR4EPCAM cells [] to CKIT CXCR4 cells []Recent advances in commercial products have led to development ofstandardized 2D culturebased media STEMdiff Definitive EndodermKit STEMCELL Technologies which allow reliable derivation of of definitive endoderm []Similarly generation of both anterior foregut endoderm andtoventralized lung progenitor populations has been subjectmuch investigation and modification Earlier work suggested thatSOX2FOXA2 ± in their case anterior foregut endoderm canonly be induced by subjecting definitive endoderm to TGFβ and BMP inhibition [] Subsequent studies however attempted to anteriorize definitive endoderm to foregut endoderm through TGFβ inhibition alone SOX2 a combination of endogenous WNT TGFβ and BMP inhibition not quantified [] and via Sonic Hedgehog SHH and FGF2signaling FOXA2 EPCAM [] A comparison of the lattertwo strategies demonstrated that SHH and FGF2 are insufficient inproducing reliable NKX21 lung progenitors [] possibly becauseFGF2 is involved in promoting thyroid lineages [] In general TGFβand BMP inhibition [] is the basis for currently applied endodermanteriorization strategies [“]Factors involved in early versions of ventralization in directed differentiation protocols included WNT3a FGF7 FGF10 BMP4 epidermalgrowth factor EGF and RA have now been reduced based on elimination studies [] As such CHIR99021 CHIR WNT agonist BMP4and RA are necessary and sufficient for producing lung progenitorsfrom anterior foregut endoderm derived from both mouse and humanPSCs [] Despite finding that FGF7 and FGF10 are nonessential for inducing NKX21 expression they continue to be used forventralization in some protocols [] Although each protocol differsin terms of the duration of each phase NKX21 lung progenitors aregenerally achieved by days with the exception of a study by deCarvalho in which they maintained their cultures for an additional days in FGF7 FGF10 and CHIR99021 to attain NKX21FOXA2 lung progenitors In all cases these lung progenitors are theneither sorted or directly guided towards proximal or distal progeny in2D or 3D culture systems Ideally products of directed differentiationprotocols should mimic the cell proportions present in human airwaysand lungs Table however current protocols have not progressedthat far While these protocols continue to be refined the percentageof select cell populations generated from these protocols have beensummarized in Table Creation of human proximal lung epitheliaProtocols to create proximal lung epithelia have focused on the production of the four major cells types present ciliated goblet club andbasal cells see Table for a summary of markers for each cell type Motivation for creating proximal epithelia in the field has primarily been todevelop patientspecific cystic fibrosis CF models [] andor toproduce epithelia with multiciliated cell populations for protocol validation [] A shift towards human PSCderived CF models hasbeen critical as mouse models do not accurately represent CF diseaseprogression and phenotypes seen in humans [“] As such thefirst evidence of human PSC proximalization using CF patientderivedPSCs was shown by Mou who exposed anterior foregut endodermto BMP4 GSK3iXV WNT agonist FGF2 and RAsupplemented B27 togenerate NKX21 cells by Day Although contaminatingneuroectodermal and distal lung NKX21SOX9 cells were presentday populations included proximal NKX21SOX2 progenitorsSubcutaneous implantation of this population in immunodeficientmice for days resulted in emergence of NKX21P63 cells howeverno mature epithelial markers for ciliated goblet and club cells werefoundWong employed a longer 2D differentiation approach to produce mature proximal airway epithelia in vitro Through a processthey called œproximal specification they generated day lung progenitors via low levels of BMP4 mimicking signaling gradients in theairway FGF7 and FGF10 which began expressing proximal genes Further culture with FGF7 FGF10 and FGF18 resulted in upregulated geneexpression of KRT5 P63 FOXJ1 SOX17 cystic fibrosis transmembraneconductance regulator CFTR and SCGB1A1 to a lesser extent alongwith low levels of distal SOX9 and SPC by day Protein expressionamounted to NKX21 panKRT P63 FOXJ1 cells These cells were subsequently matured in airliquid interface ALI culture for weeks week of submerged culture withFGF18 followed by weeks of ALI culture to generate CFTRpanKRT FOXJ1 with coexpressing CFTR and CFTRLHS28 cells The resulting epithelium ranged from being squamous to cuboidal with sparse pseudostratified regions implying thatthis protocol lacked specific maturation cues Contaminating thyroidthyroglobulin and PAX9 liver HNF4 and AFP and pancreaticPDX1 lineages were detected through quantitative PCR while percentages of goblet club and basal cell populations barring gene expression analysis were not evaluatedA similar 2D culture approach was employed by Firth to generate proximal lung progenitors which were subsequently matured intomulticiliated epithelia They optimized lower concentrations of BMP4required during the ventralization phase day “

Thyroid_Cancer

"Handling EditorAdrian CovaciKeywordsNuclear accidentsHealth surveillancePreparednessCommunicationStakeholdersRecommendationsSerious accidents at nuclear power plants have been rare but theirstories can teach us how to prevent or mitigate the eï¬ects of futurenuclear catastrophes The accidents at the Fukushima Daiichi nuclearpower plant and Chernobyl nuclear power plant occurred years and years ago respectively and there are still lessons to learn from themregarding numerous issues including radiation exposure assessmentand medical followup of emergency responders evacuees and residents decisions to lift evacuation orders and communication withresponders and stakeholders Bazyka Callen and Homma Lester Soï¬er Some of the lessons from theseaccidents have been extensively reviewed and taken into considerationby national and international anizations such as the InternationalAtomic Energy Agency the International Commission on RadiologicalProtection and the World Health anisation and are reflected inpublished literature Bennett Carr Clarke IAEA 2015a 2015b Nisbet SGDSN This hasallowed the development of various recommendations and guidancedocuments targeting specific issues of radiation protection training andcommunication and socioeconomic aspects in order to prepare andimprove decision making processes in the early and intermediatephases eg Carr IAEA 2015b Nisbet However the majority of these texts focus on technical issues andare directed towards radiation protection experts rather than for thesupport of aï¬ected populations The traditional approaches of emergency response and recovery including evacuation relocation andhealth surveillance are largely based on dose levels Although manyrecognise the importance of psychosocial or human factors it has beendifficult to adapt the approaches to better address the social economicethical and psychological factors These include the health and welfareeï¬ects that may arise from the accident from the concerns about thepresence of radiation in the environment from the mitigation actionstaken and from the information mixed or absent provided to the population Changes in the ethical and legal requirements for personaldata collection use and storage raise additional challenges particularlyin the area of health surveillance and epidemiologyAbbreviations COVID19 Coronavirus Disease EJP CONCERT European Joint Programme for the Integration of Radiation Protection Research OPERRA Project for the European Radiation Research Area SGDSN Secrtariat gnral de la dfense et de la scurit nationale France SHAMISEN NuclearEmergency Situations Improvement of Medical and Health Surveillance SHAMISEN SINGS SHAMISEN Stakeholder INvolvement in Generating Science UN UnitedNations UNDRR United Nations Office for Disaster Risk Reduction101016jenvint2020106000Received July Accepted July Published by Elsevier Ltd This is an access under the CC BYNCND license httpcreativecommonslicensesBYNCND40 0c Main components of the SHAMISEN projectThe SHAMISEN project started in late at a time when somedeleterious eï¬ects of evacuation and ultrasound thyroid screening inFukushima had started to be reported The project therefore aimed toreview the lessons learned from major nuclear accidents in particularfrom experiences of populations aï¬ected by the Chernobyl andFukushima accidents to develop recommendations for medical andhealth surveillance of populations aï¬ected by previous and future radiation accidents The ultimate motivation was to minimise the negative impacts of the accident and improve the health of aï¬ected populations The holistic WHO definition of health was used in this contextie œa state of complete physical mental and social wellbeing and notmerely the absence of disease or rmity WHO The Recommendations were to address in particular the following complementary aspects dose assessment supporting all phases of an accident including emergency response clinical decisionmaking recoveryactions and health surveillance improvement of living conditions ofaï¬ected populations engaging them and responding to their needs andminimising unnecessary anxiety and health surveillance and wherefeasible improvement of estimates of radiationinduced risk for radiation protection and communication with aï¬ected populationsTo achieve this and recognising the need for a holistic approach toaccident management and health surveillance SHAMISEN brought together a team of researchers from institutions including RadiationProtection Authorities Universities Research Centres and Associationsin Europe and Japan with complementary experience and a long trackrecord in post accidental management dosimetry radiation protectionmedical followup and screening population health surveillance healtheconomics epidemiology ethics and sociology of radiation protectionThe project also drew upon additional expertise from Belarus RussiaUkraine Japan and the UK as well as from outside of the radiationresearch field and established contacts with major international anizations including the World Health anisation and the NuclearEnergy Agency of the anisation for Economic Cooperation andDevelopmentDetails of the SHAMISEN Project are provided in the paper by Ohbaand collaborators Ohba this issue Briefly the approach involved in particular challengingevaluating the eï¬ectiveness of measures taken after Chernobyl or Fukushima accidents in particularcid129 Systematic thyroid screening with ultrasound for early detection ofpotential thyroid cancer casescid129 Criteria for evacuation and their consequencescid129 Measures taken to contribute to the wellbeing of aï¬ected populations and develop a radiological protection culture and resilience inaï¬ected populationscid129 Challenges and good practice in communication and training withthe objective of regaining the trust of the population and engagingthem in retaking control of their livescid129 Role of ethics in disaster preparedness response and health surveillance autonomy and dignity respect of privacy beneficence¦cid129 Role of health professionals in the diï¬erent phases of the accidentmanagementcid129 Cost eï¬ectiveness of the measures takenAll of this was brought together to develop practical recommendations for preparedness and the diï¬erent phases of the accident SHAMISEN results and the way forwardAlthough the SHAMISEN project was developed during a limitedperiod of months in response to the second call of the EuropeanOPERRA project a series of key results has been achieved The mainresults are several topical reports and a set of recommendations dividedinto five main topics eg Evacuation Communication and trainingEnvironment International Dosimetry Health surveillance Epidemiology These topics focusspecifically on the health surveillance of people following a nuclearaccident combining natural and social sciences values and practice tohelp health professionals decisionmakers and local stakeholders to setup protective actions and health programmes responding to the concernof aï¬ected populations Therefore the SHAMISEN recommendationsare not intended to cover all aspects of emergency and recovery response and preparednessThe formulation of the recommendations is generic enough to beapplied in diï¬erent countries recognising that cultural diï¬erences willbe important The structure describes the general context and the mainreasons for developing each recommendation provides explanations onhow to develop it and indicates who would be involved in the development of the recommendation Depending on the context specificarrangements have to be made for the implementation of these recommendations during the diï¬erent phases eg preparedness earlyand intermediate longterm recoveryThe recommendations provide advice on the values to be consideredwhen addressing the issue at stake and what type of tools and protocolsare needed rather than the tools themselves Due to the duration of theproject it was not manageable to develop them However they providea significant input for further developments for practical tools in different domains and identify the main expectations from stakeholderswith regards to health surveillance in postaccident situationsDeveloped as a research project it was not the intention to specifyabsolute dose criteria for the implementation of actions Of coursediscussions on the feedback experience from the management of theChernobyl and Fukushima accidents point out some challenges associated with the use of specific dose criteria but the spirit of the recommendations is to provide indications and guidance for the decisionmakers and health professionals with regard to the choice to be madeon the adoption of dose criteria for the diï¬erent actions to be implementedBesides the management of the direct radiation induced health effects the report underlines the need to develop a multidisciplinaryapproach to identify measure assess and alleviate psychological andother indirect health impacts of socioeconomic and social upheavals ofthe consequences of the accident For this purpose it is recommendedto promote the engagement process of local stakeholders since thepreparedness phase targeting the overall wellbeing of populations withdue considerations of the ethical principles of respect for autonomydignity and justiceThis special issue of Environmental International combines a series ofscientific papers and is an opportunity to emphasize the main analysesdeveloped during the SHAMISEN project combining advanced scientific research analyses of feedback experience from the Chernobyl andFukushima accidents and applying a multidisciplinary approachAlthough the topics presented in this special issue have already beenaddressed in general in several papers the originality of the approachadopted in the SHAMISEN project provides new insights for healthsurveillance issuesIt is worth mentioning that following the SHAMISEN project otherresearch projects have been launched A first series of projects arededicated to the development of Apps with and for citizens as recommended in the SHAMISEN project This has notably been done withthe European research project SHAMISEN SINGS as part of the EJPCONCERT as well as with an ongoing project developed by FukushimaMedical University In addition several projects are currently underdevelopment in diï¬erent countries and at the European level thatpromote a citizen science approach for addressing health and radiological monitoringAs an example of the multidisciplinary approach the SHAMISENproject has identified a series of recommendations calling for furthercooperation with diï¬erent European Research Platforms combininglow doses eï¬ects dosimetry radioecology emergency and recoverymanagement social sciences and humanities and medical research 0cEnvironment International and Thierry SchneideraŽ Deborah Oughtonb Elisabeth Cardiscdea CEPN Nuclear Protection Evaluation Centre Rue de la Redoute FontenayauxRoses Franceb Centre for Environmental Radioactivity CERAD NMBU „«s Norwayc Barcelona Institute for Global Health ISGlobal Barcelona Spaind Pompeu Fabra University Barcelona Spaine Spanish Consortium for Research and Public Health CIBERESP Institutode Salud Carlos III Madrid SpainEmail address thierryschneidercepnassofr T SchneiderReferencesBazyka D Belyi D Chumak A Lessons from chornobyl considerations forstrengthening radiation emergency preparedness in Ukraine Radiat Prot Dosim “ 101093rpdncw196Bennett B Repacholi M Carr Z Eds Health eï¬ects of the Chernobyl accidentand special health care programmes Report of the UN Chernobyl Forum expert groupœHealth WHO Press GenevaCallen J Homma T Lessons learned in protection of the public for the accident atthe Fukushima Daiichi nuclear power plant Health Phys “ 101097HP0000000000000666Carr Z Clarke M Akl EA Schneider R Murith C Li C ParrishSprowl J StenkeL CuiPing L Bertrand S Miller C Using the grade approach to supportthe development of recommendations for public health interventions in radiationemergencies Radiat Prot Dosim “ 101093rpdncw234Clarke R Valentin J International Commission on Radiological Protection Task Group ICRP publication Application of the Commission™s Recommendations forthe protection of people in emergency exposure situations Ann ICRP “101016jicrp200905004Croua¯l P Camps J Raskob W Schneider T NERIS Roadmap on medium andlongterm research on preparedness for nuclear and radiological emergency responseand recovery Version eunerisnetlibrarysra259nerisroadmap2020htmlIAEA 2015a The Fukushima Daiichi Accident No Technical Volume IAEAInternational Atomic Energy Agency Vienna Austria wwwiaeapublications10962thefukushimadaiichiaccidentIAEA 2015b Preparedness and Response for a Nuclear or Radiological Emergency NoGSR7 Safety Standards Series IAEA International Atomic Energy AgencyVienna Austria httpwwwpubiaeaMTCDPublicationsPDFP_1708_webpdfImpens N Salomaa S Second joint roadmap for radiation protection researchDeliverable No EJPCONCERT European Joint Programme for the Integrationof Radiation Protection Research H2020 wwwconcerth2020euDocumentashxdtwebfileListsDeliverablesAttachments206D37_Second20joint20roadmap_draft_reviewed_20052020_approved03062020pdfguid01b5ac77b2ec4cda9c98917dba396f0fLester MS Public information during a nuclear power plant accident lessonslearned from Three Mile Island Bull N Y Acad Med “Nisbet AF Jones A Turcanu C Camps J Andersson KG H¤nninen RRavantaara A Solatie D Kostiainen E Julien T Pupin V Ollagnon HPapachristodoulou C Ioannides K Oughton D Generic Handbook forAssisting in the Management of Contaminated Food Production Systems in Europefollowing a radiological emergency v2 No CAT1TN0901 EURANOS eunerisnetlibraryhandbookshtmlOhba T Liutsko L Schneider T Tanigawa K Fattibene P Laurier D Sarukhan ABarquinero J Kesminiene A Skuterud L Cardis E this issue The SHAMISENProject challenging historical recommendations for preparedness response andfollowup of nuclear accidents lessons learnt from Chernobyl and FukushimaSGDSN National response plan Major nuclear or radiological accident httpwwwgouvernementfrsitesdefaultfilesrisquespdfnational_plan_nuclear_radiological_accidentspdfSoï¬er Y Schwartz D Goldberg A Henenfeld M BarDayan Y Populationevacuations in industrial accidents a review of the literature about four major eventsPrehosp Disaster Med “UNDRR The Sendai Framework for Disaster Risk Reduction “ UnitedNations Office for Disaster Risk Reduction wwwundrrpublicationsendaiframeworkdisasterriskreduction20152030WHO WHO Definition of Health World Health anisation Geneva httpwwwwhointaboutdefinitionenprinthtmlThese recommendations have already been considered in the development of the European joint roadmap for radiation protection researchImpens and Salomaa and of the strategic research agenda ofdiï¬erent European platforms notably NERIS on emergency and recovery Croua¯l The results of the SHAMISEN project have been presented and discussed in several national and international workshops and meetingsRecommendations are being disseminated to decision makers and radiation protection authorities for translation into strategy and policy aswell as to scientific medical and nonexpert audiences They are nowreferred and used as basis of the reflections and the initiatives of national and international anizations for both preparedness NuclearEnergy Agency World Health anisation International Commissionon Radiological Protection ICRP National committee for postaccident management CODIRPA in France and the management of theFukushima situation with a key role of the Japanese partners involvedin the SHAMISEN project Fukushima Medical University NagasakiUniversity Hiroshima UniversityMore broadly the approach adopted in the SHAMISEN project andits results may contribute to address other hazards including naturaldisasters industrial accidents or even pandemic crisis Similarities canbe emphasized with the Sendai Framework for Disaster Risk Reduction“ UNDRR adopted at the 3rd UN World Conference in This framework underlines the importance of improving theunderstanding of disaster risk better addressing vulnerability and hazard characteristics strengthening risk governance reinforcing accountability for risk management with development of preparednessinvolvement of stakeholders and due considerations of resilience ofhealth infrastructureFinally the pandemic crisis of COVID19 highlights a series of issuesquite similar to those addressed in the SHAMISEN project confinementversus evacuation psychosocial aspects communication and dialogueanisation of the transition phases and of course the preparation ofhealth surveillance strategies and structures of epidemiological studiesThese diï¬erent issues would benefit from crosscomparison analysisand the s presented in this special issue could certainly contributeto the reflectionDeclaration of Competing InterestThe authors declare that they have no known competing financialinterests or personal relationships that could have appeared to ‚uence the work reported in this paperAcknowledgmentsSHAMISEN was supported by the EURATOM European AtomicEnergy Community program of the European Commission in the framework of the OPERRA Project for the European RadiationResearch Area project FP7 grant agreement No The authors are grateful to all partners experts and stakeholderscontributed to theandorwho participated in the projectRecommendationsISGlobal acknowledges supportfrom the Spanish Ministry ofScience Innovation and Universities through the œCentro de ExcelenciaSevero Ochoa “ Program CEX2018000806S and supportfrom the Generalitat de Catalunya through the CERCA Program NMBUacknowledges the support of the Research Council of Norway RCNthrough its Centres of Excellence funding scheme project numberŽ Corresponding author 0c"

Thyroid_Cancer

"dramatic spread of Coronavirus Disease COVID19 has profound impacts on every continent and life Due to humantohuman transmission of COVID19 nuclear medicine staffs also cannot escape the risk of infection from workplaces Everystaff in the nuclear medicine department must prepare for and respond to COVID19 pandemic which tailored to the characteristics of our profession This provided the guidance prepared by the Korean Society of Nuclear Medicine KSNM incooperation with the Korean Society of Infectious Disease KSID and Korean Society for HealthcareAssociated InfectionControl and Prevention KOSHIC in managing the COVID19 pandemic for the nuclear medicine department We hope that thisguidance will support every practice in nuclear medicine during this chaotic periodKeywords Coronavirus COVID19 Nuclear medicine Prevention and control Practice guideline HoYoung Leedebobkrgmailcom Department of Nuclear Medicine CHA Bundang Medical CenterCHA University of Medicine Professor Pocheon Republic ofKorea Department of Nuclear Medicine Seoul National UniversityBundang Hospital Professor Seongnam Gyeonggido Republic ofKorea Department of Nuclear Medicine Samsung Medical CenterSeoul Republic of Korea Department of Nuclear Medicine Seoul National UniversityHospital Seoul Republic of Korea Department of Nuclear Medicine Chosun University HospitalGwangju Republic of Korea Department of Nuclear Medicine Korea University Guro HospitalSeoul Republic of Korea Department of Nuclear Medicine Hanyang University Guri HospitalSeoul Republic of Korea Department of Nuclear Medicine National Cancer CenterGoynag Republic of Korea Department of Nuclear Medicine Seoul Medical CenterSeoul Republic of Korea Division of Nuclear Medicine Department of RadiologyEunpyeong St Mary™s Hospital College of Medicine The CatholicUniversity of Korea Seoul Republic of Korea Department of Nuclear Medicine Soonchunhyang University SeoulHospital Bucheon Republic of Korea Department of Nuclear Medicine Inje University Haeundae PaikHospital Busan Republic of Korea Department of Nuclear Medicine Keimyung University DongsanMedical Center Daegu Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityCheonan Hospital Cheonan Republic of Korea Department of Nursing Soonchunhyang University BucheonHospital Bucheon Republic of Korea Division of Infectious Disease Department of Internal MedicineKangdong Sacred Heart Hospital Hallym UniversityChuncheon Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityBucheon Hospital Bucheon Republic of Korea Department of Nuclear Medicine Korea University Anam Hospital Korean Society of Nuclear Medicine Quality Control CommitteeSeoul Republic of KoreaBucheon Republic of Korea 0cIntroductionSince the first reports of Coronavirus Disease COVID in Wuhan China the infection had spread worldwiderapidly and COVID19 has reached pandemic levels InSouth Korea since its outbreak in February COVID has affected profoundly every aspect of communities Thehumantohuman transmission of COVID19 provides challenges for all healthcare facilities and healthcare providersIn the face of the COVID19 pandemic the Korean Societyof Nuclear Medicine KSNM Korean Society of InfectiousDisease KSID and Korean Society for HealthcareAssociated Infection Control and Prevention KOSHIC haveprepared the guidance for the nuclear medicine department tominimize confusion and ensure that nuclear medicine physicians and technicians continue to provide their services whileprotecting the patients and workers and preventing the transmission of the virus The Quality Control Committee ofKSNM reviewed several reports and recommendations previously published by the European Association of NuclearMedicine EANM [] Society of Nuclear Medicine andMolecular Imaging SNMMI American Society of NuclearCardiology ASNC [] International Atomic Energy AgencyIAEA and others [“] This guidance is basically in compliance with the COVID19 guidelines of the Korea Centersfor Disease Control and Prevention KCDC [“] Finallythis document was prepared in cooperation with KSID andKOSHIC KSNM emphasize that this guidance must be considered in the context of following the state and hospital infection control policies and flexibly applied according tochanges in circumstances and evidenceGeneral Principles During COVID19PandemicIn a pandemic situation such as COVID19 if necessarythe condition of the scheduled patient can be checked inadvance to adjust the examination schedule Nonurgent elective studies or therapy should be postponed in COVID19confirmed or COVID19suspectedpatients Rescheduling the studiestherapy must be donein a discussion with the referring clinicians Only urgent studies or therapy could be performed inCOVID19confirmed or COVID19suspected patientswhenever clinically appropriate The priority of studytherapy should be based on a casebycase indepth discussion between nuclear medicine physicians and referring clinicians In case of performing the urgent studiestherapy consult with the infection control offices of eachinstitution to comply with the infection control rules ofownNucl Med Mol Imaging “ COVID19suspected patients should undergo COVID testing before performing the studiestherapy Lung ventilation scan should not be performed in anyCOVID19confirmed or COVID19suspected patients Lowdose radioiodine therapy may be considered in caseof acute hyperthyroidism patients who are unable to tolerate antithyroid medications As lowdose radioiodinetherapy lower than GBq of I131 can be performedin an outpatient setting in South Korea COVID19infected patient can be administrated lowdoseradioiodine in the isolation room or negative pressureroom without any additional monitoring related toradioiodine therapyConsideration During the StudyTherapy Patient transportationScheduling COVID19confirmed or COVID19suspected patient as last study of the day to preventcrossinfection in the nuclear medicine department Ensure that other patients or caregivers should notaccess the nuclear medicine department to minimizethe exposure to COVID19 patient during the studytherapy Transfer the COVID19infected patient to the nuclear medicine department using negative pressuretransport bag to minimize exposure and contact todroplet COVID19 patients should wear masks at all timesof procedures If necessary add gowns gloves etc Devices and scanner management Mainly use disposable instruments or items Do notreuse disposable items such as oxygen masks nasalprongs suction tubes or suction lines The protocolfor reusable devices is as follows Cleaning After use the equipment contaminated with blood bodyfluids secretions and feces should be delivered to awashing room with care not to contaminate the surrounding environment The washing place should be separated from the spaceused for cleaning other items or other patients After immersing the contaminated equipment in a washing spacewash the product carefully to avoid splashing Wash enough to remove blood body fluids secretionsand feces from remaining 0cNucl Med Mol Imaging “ Staff undertaking cleaning should wear KF94 or N95masks longsleeved waterproof gowns goggles or faceshields hats shoe covers or rubber boots and doublegloves outer gloves are rubber gloves Disinfection and sterilization Depending on the risk level of the device according tothe Spaulding Classification of medical equipmentdevices noncritical devices require lowlevel disinfectionsemicritical devices require highlevel disinfectionsterilization and critical devices must be sterilized Disinfectants and sterilization methods by device classification should be followed in accordance with the notificationof the Ministry of Health and Welfare Be sure to check the disinfectant manufacturer™s recommendations The recommended disinfection process suchas dilution and application time of disinfectant and theeffective period and concentration of disinfectant arestrictly followed Laboratory and scan room management Only the minimum number of staffs should be placedin the nuclear medicine department All participatingstaffs should wear appropriate personal protectiveequipment PPE eye protection with goggles or faceshield medical protective masks N95KF94 or equivalent respirator disposable latex gloves disposablegown disposable shoe covers etc Cover the scanner couch or other equipment with aplastic cover to prevent contamination Every effort should be made to minimize theCOVID19 exposure to medical staff during injection of radiopharmaceuticalsSelect the protocol with the shortest duration of uptake time and scan time to minimize the time spentby the COVID19 patient in the departmentIn case of studies requiring an uptake phaseCOVID19 patients should be waiting in separatespace If possible COVID19 patients wait in negative pressure transport bag If negative pressuretransport bag is not available use bed or stretcherin waiting room with disposable cover Considerusing standard radiopharmaceutical dose to shortenthe procedure time After the completion of image acquisition the scanroom and patient™s space area should be disinfectedaccording to the standard protocol After image acquisition remove the plastic cover ofthe scanner and disinfect the scanner surface Remove and discard PPE adequately when leavingthe camera room or care area and immediately perform hand hygieneIn case of performing the radiolabeling of theCOVID19 patients™ blood products every processwith infectious materials openingstirringmixingdispensing COVID19 patient™s blood sampleradiolabeling etc should be done in class II biosafety cabinet according to the Biosafety Level Regulation Disinfection of laboratory with properdisinfectants ethanol hydrogen peroxide or ppm sodium hypochlorite should bedone Used PPE and disposable covers are removed withcaution not to contaminate the clean area and disposed in a container for biosafety waste Employee management All employees should be trained in the preventionand management of COVID19 infection and adhereto the rules of infection prevention Considering the skill level fatigue etc of the working staff sufficient personnel are allocated to securethemPriority from exemption is given to employees withhighrisk underlying diseases such as diabetesmellitus chronic obstructive pulmonary diseaseCOPD endstage renal disease ESRD chroniccardiac disease etc or pregnant women Cleaning and environmental management General principle Personnel responsible for cleaning or disinfectionshould complete the infection preventioneducation Employees should wear PPE KF94 or N95 respirators fullbody protective clothing or aprons goggles or face shields shoe covers or rubber bootsdouble gloves outer gloves are rubber gloveswhen cleaning or disinfectingIf there are organic substances on the surface of theenvironment it cannot be properly disinfectedTherefore wipe the surface before disinfecting theenvironmentIn order to prevent the possibility of microbialspraying cleaning should be performed using acleaning solution or a mop moistened with a disinfectant rather than a cleaning method using abroom or a vacuum cleanerInstead of spraying disinfectants thoroughly cleanthe surface of the environment using a clean towelmoistened with the disinfectant or a commerciallyavailable disinfecting tissue towel 0cNucl Med Mol Imaging “ Use cleaning tools as disposable as possible or exclusively However when the cleaning tool isreused the used cleaning tool is sterilized usingan appropriate disinfectant and then dried andstored Disinfection of a patient™s space areaIn the case of the space area used by the patientmark the place where contamination was confirmedbefore cleaning and disinfecting the surface andseal the contaminated object to prevent others frombeing exposed Ventilation before during and after cleaningdisinfection disinfection after ventilation for hbased on air cycles per hour Wear PPE Wipe with a cloth cloth etc wet withthe diluted disinfectant Wipe the touched wall surface and all frequently used areas and keep it for atleast min After then wipe the surface with acloth dampened with clean water cloth etcResumption of use Consider the characteristics ofeach type of disinfectant used and the purpose of thefacility After disinfection the virus is killed but thedecision at the time of resumption of use cannot beapplied in batch due to different characteristics ofdisinfectants so it is necessary to consider the precautions for each productFor details on disinfecting methods such as surfacedisinfection and washing refer to œDisinfectionGuidelines to Prevent the Spread of COVID19 atPublic and Multipurpose Facilities 3rd editionRefer to the method of disinfecting the patient spaceareaSelect an environmental disinfectant Select an approved or declared disinfectant by the Ministry ofEnvironment and follow the usage usage and precautions for each product Disinfectant list of the Ministry of Environmenthttpecolifemegokr Precautions when using environmentaldisinfectants Select the disinfectant after confirming informationsuch as approval from the Ministry of Environmentand Environment When using environmental sterilizers make sure tofollow the manufacturer™s recommendations suchas checking the expiration date safe usage for eachproduct and precautions and preparing the diluentaccording to the manufacturer™s instructions The disinfecting method of sprayinginjecting disinfectant is not applied to surface disinfectionbecause it causes aerosol infection increased riskof inhalation and the range of contact between thedisinfectant and the surface is insufficient so thedisinfecting effect is insufficient Disinfectant hazard information must be checkedand used carefully Do not mix different disinfectants Do not placenear flammable materials Disinfectant should beused in a wellventilated area As the disinfection effect may decrease over timedilute as much as necessary and use it immediatelyDo not store the remaining amount and discard itimmediately Laundry managementStore clean laundry in a separate space Employees handling laundry should be trained toprevent infection Employees handling contaminated laundry shouldwear PPE N95 masks or equivalent respiratory protection gowns gloves overshoes etc and performhand hygiene after removing PPE The laundry used for the patient is disposed of according to the relevant regulations see WasteManagement Act Medical Institution LaundryManagement Rules etc Thoroughly ensure that pathogens are not exposed topersonnel handling the laundry or surrounding environment during the entire process of collectingtransporting and washing laundry Waste management Waste related to COVID19 patients is managed bythe rules of hospital infectious control policySharp tools such as needles or blades are collectedin containers for impervious and nonpermanentwaste and containers should be stored in the placewhere the items are usedSimple infectious waste contaminated or possiblycontaminated with COVID19 patients™ sample isautoclaved and discarded Radioactive waste shouldbe discarded in compliance with national regulationwith caution not to contaminate the staff or areaConclusionConsidering that outbreaks of novel viruses have been periodically appearing these days nuclear medicine staffs should getused to guidance and policies for infectious disease in working 0cNucl Med Mol Imaging “place to protect patients worker themselves and furthermorevaluable medical resources Basically this guidance can beapplied in case of any other humantohuman transmissiondisease for operating the nuclear medicine department Alsoalways bear in mind the rapid change in the situation thisguidance should be used in conjunction with the currentgovernment and local hospital policiesCompliance with Ethical StandardsConflict of InterestJiIn Bang HoYoung Lee Young Seok ChoHongyoon Choi Ari Chong Jae Sun Eo Ji Young Kim Tae SungKim HyunWoo Kwon Eun Jeong Lee Eun Seong Lee Hye LimPark Soo Bin Park Hyekyung Shim BongIl Song Ik Dong YooKyung Jae Lee Hong Jae Lee Su Ha Han Jin Seo Lee Jung Mi Parkand Sung Hoon Kim declare that they have no conflict of interestEthical Approval This work does not contain any studies with humanparticipants or animals performed by any of the authorsInformed Consent Not applicableReferences Paez D Gnanasegaran G Fanti S Bomanji J Hacker M SathekgeM et al COVID19 pandemic guidance for nuclear medicine departments Eur J Nucl Med Mol “ Skali H Murthy VL AlMallah MH Bateman TM Beanlands RBetter N et al Guidance and best practices for nuclear cardiologylaboratories during the coronavirus disease COVID19 pandemic an information statement from ASNC and SNMMI J NuclCardiol “ httpsdoiorg101007s12350020021232 Huang HL Allie R Gnanasegaran G Bomanji J COVID19“nuclear medicine departments be prepared NuclMedCommun MossaBasha M Medverd J Linnau K Lynch JB Wener MHKicska G et al Policies and guidelines for COVID19 preparedness experiences from the University of Washington Radiology httpsdoiorg101148radiol2020201326 Zhang X Shao F Lan X Suggestions for safety and protectioncontrol in Department of Nuclear Medicine during the outbreak ofCOVID19 Eur J Nucl Med Mol “ Buscombe JR Notghi A Croasdale J Pandit M O'Brien J GrahamR et al COVID19 guidance for infection prevention and controlin nuclear medicine Nucl Med Commun “ Standard guideline for healthcareassociated infection control andprevention Korean Center for Disease Control and Prevention andKorean Society for HealthcareAssociated Infection Control andPrevention httpcdcgokrCDCcmscontentmobile2675626_viewhtml Accessed 2nd Jun Korean Society for HealthcareAssociated Infection Control andPrevention Korean Center for Disease Control and Preventionhttpwwwcdcgokrboardesmida20507020000bid0019actviewlist_no366579 Accessed 2nd Jun Guidelines in response to coronavirus disease for local governmentKorea Centers of Disease Control and Prevention2020 httpswwwcdcgokrboardboardesmida20507020000bid0019actviewlist_no367279tagnPage1 Accessed 2ndJun Disinfection guidelines to prevent the spread of COVID19 at public and multipurpose facilities Korea Centers of Disease Controland Prevention httpswwwcdcgokrboardboardesmida20507020000bid0019 Acessed 15th Jun Publisher™s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c"

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

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

expanding cancer predisposition genes with ultra‘rare cancer‘exclusive human variationsRoni Rasnic1 nathan Linial1 Michal Linial2It is estimated that up to of cancer incidents are attributed to inherited genetic alterations Despite extensive research there are still gaps in our understanding of genetic predisposition to cancer It was theorized that ultrarare variants partially account for the missing heritable component We harness the UK BioBank dataset of individuals of which were diagnosed with cancer to detect ultrarare possibly highpenetrance cancer predisposition variants We report on cancerexclusive ultrarare variations and nominate variants with additional independent evidence as cancer predisposition variants We conclude that population cohorts are valuable source for expanding the collection of novel cancer predisposition genesDiscovery of cancer predisposition genes CPGs has the potential to impact personalized diagnosis and advance genetic consulting Genetic analysis of family members with high occurrences of cancer has led to the identification of variants that increase the risk of developing cancer1 In addition to familybased studies efforts to identify CPGs focus on pediatric patients where the contribution of environmental factors is expected to be small Forty percent of pediatric cancer patients belong to families with a history of cancer2Tumorigenesis results from misregulation of a0one or more of the major cancer hallmarks3 Therefore it is anticipated that CPGs overlap with genes that are often mutated in cancerous tissues Indeed CPGs most prevalent in children TP53 APC BRCA2 NF1 PMS2 RB1 and RUNX12 are known cancer driver genes that function as tumor suppressors oncogenes or have a role in maintaining DNA stability4 Many of the predisposed cancer genes are associated with pathways of DNArepair and homologous recombination5 The inherited defects in cells™ ability to repair and cope with DNA damage are considered as major factors in predisposition to breast and colorectal cancers6Complementary approaches for seeking CPGs are largescale genomeexome wide association studies GWAS which are conducted solely based on statistical considerations without prior knowledge on cancer promoting genes7 Identifying CPGs from GWAS is a challenge for the following reasons limited contribution of genetic heritability in certain cancer types low effect sizerisk associated with each individual variant lowpenetrance in view of individual™s background8 and low statistical power Large cohorts of breast cancer show that of cancer cases are associated with mutations in BRCA1 and BRCA2 which are also highrisk ovarian cancer susceptibility genes Additionally TP53 and PTEN are associated with earlyonset and highrisk familial breast cancer Mutations in ATM and HRAS1 mildly increase the risk for breast cancer but strongly increase the risk for other cancer types and a collection of DNA mismatch repair genes MLH1 MSH2 MSH6 PMS2 are associated with high risk of developing cancer9 A large cohort of Caucasian patients with pancreatic cancer reveal high risk CPGs that overlap with other cancer types CDKN2A TP53 MLH1 BRCA2 ATM and BRCA110Estimates for the heritable component of predisposition to cancer were extracted from GWAS familybased and twin studies11“ These estimates vary greatly with maximal genetic contribution associated with thyroid and endocrine gland cancers and a minimal one with stomach cancer and leukemia14 Current estimates suggest that as many as of cancer incidents can be attributed to inherited genetic alterations eg single variants and structural variations1516 The actual contribution of CPGs varies according to gender age of onset cancer types and ethnicity17“ It is evident that high risk variants with large effect sizes are very rare21 Actually based on the heritability as reflected in GWAS catalog it was estimated that only a fraction of existing CPGs is presently 1The Rachel and Selim Benin School of Computer Science and Engineering The Hebrew University of Jerusalem Jerusalem Israel 2Department of Biological Chemistry Institute of Life Sciences The Hebrew University of Jerusalem Jerusalem Israel email ronirasnicmailhujiacilScientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0cFigure a0 UK Biobank CUVs collection The Caucasian filtered UK Biobank UKBB data set include individuals who had cancer and the nonCaucasian include such individuals a Cancer type distribution for the Caucasian data set b Cancer type distribution for the nonCaucasian data set c The data of UKBB participants was used for this study of which were confirmed Caucasian d Out of UKBB variants we curated heterozygous and homozygous CUVs total CUVs known22 Therefore instances of extremely rare mutations with high risk for developing cancer remain to be discoveredA catalog of CPGs was compiled from a0years of research1 with about half of the reported genes derived from family studies representing highpenetrance variants An extended catalog was reported with a total of CPGs that were tested against rare variants from TCGA germline data covering cancer patients from cancer types and included known pediatric CPGs23 The contribution of BRCA12 ATM TP53 and PALB2 to cancer predisposition was confirmedIn this study we report on known and novel cancer predisposition candidate genes We benefit from the UKBiobank UKBB an invaluable resource of germline genotyping data for individuals The UKBB reports on cancer patients and cancer free individuals considered as control group We challenge the possibility that CPGs can be identified from very rare events henceforth called cancerexclusive ultrarare variants CUVs These CUVs are expected to exhibit high penetrance Notably the presented CUVs were extracted from UKBB DNA array and therefore only cover the array preselected SNPs We report on exome variations of which are heterologous The majority of the matching genes are novel CPG a0candidates We provide indirect genomic support for some of the CUVs that occur within coding genes and discuss their contribution to tumorigenesisResultsThe primary UKBB data set used in the is comprised of Caucasian UKBB participants see Methods Fig a01c cancerfree and diagnosed with at least one malignant neoplasm Among participants with cancer were diagnosed with either skin or breast cancer The clinical ICD10 codes assembly is summarized in Supplementary Table a0S1 A total of of the cancerdiagnosed individuals had two or more distinct neoplasms diagnosed The validation UKBB data set includes nonCaucasian participants among them are cancerfree Figure a01ab provide further details on different cancer type prevalence in these setsNonmelanoma skin cancer is mostly attributed to environmental factors rather than genetic association24 However based on evidence for hereditary links for nonmelanoma skin cancer predisposition2526 we included these individuals in our analysis In addition focusing on extremely rare variations enables the identification of existing yet overlooked genetic associationsCompilation of cancerexclusive ultrarare variants CUVs We scanned genetic markers in our prime data set for cancerexclusive variations variations met our initial criteria appearing at least twice in individuals diagnosed with cancer and not appearing in cancerfree individuals Among them were heterozygous and were homozygous variations In order to target variations with additional supporting eviScientific RepoRtS 101038s41598020704940Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Exomic CUVs are mostly gene disruptive The partition of variant types for the compiled list of exomic CUVs The list is dominated by transcript disruptive variations that include missense frameshift stop gain and splicing sites a Distribution of variation types among the exomic CUVs b Dispersion of variant types among heterozygous and homozygous CUVsdence we considered only coding exome and spliceregion variants To assure the CUVs rarity in the general population we applied an additional filter based on the gnomAD data set see Methods The resulting final list is comprised of variants associated with genes heterozygous and homozygous Fig a01d The detailed list of all CUVs can be found in Supplementary Table a0S2Most of the CUVs are missense variants There is a strong enrichment for loss of function LoF variants ie frameshift splicing disruption and stop gains which account for of the CUVs Only a single homozygous CUV is synonymous Fig a02a The distribution of variation types varies greatly between homozygous and heterozygous CUVs Fig a02b Missense variants are of the homozygous variant set but only of the heterozygous CUVs The heterozygous CUVs are highly enriched for LoF variants which constitute the other Cancerexclusive ultrarare variants overlap with known cancer predisposition genes From the listed CUVs variants were previously defined as cancer inducing genes in genes Table a0 Specifically CUVs within genes appear in the updated list of CPG catalog23 and CUVs within genes are known cancer driver genes Fig a03a as determined by either COSMIC27 or the consensus gene catalog of driver genes listing genes coined C29928 More than half of the cancer associated variants result in LoF Many of the affected genes are tumor suppressor genes TSGs among which are prominent TSGs such as APC BRCA1 and BRCA2 Table a0 each identified by two distinct CUVs Notably of the variants had at least one appearance in nonmelanoma skin cancerThe heterozygous CUVs are enriched for known cancer predisposition genes Twentyfive of the cancer associated CUVs are heterozygous and one is homozygous However there is an inherent imbalance in the initial variant sampling performed by the UKBB As the UKBB use DNA arrays for obtaining genomic data the identifiability of ultrarare exome variants is restricted by the selection of SNP markers and the design of the DNA array There are heterozygous ultrarare exome variants from genes which pass our biobankethnic and the gnomAD allele frequency filtration A total of of the filtered ultrarare variants overlap with known CPGs as some genes are overrepresented among the ultrarare variants Supplemental Table a0S3 For example the exomic region of BRCA2 is covered by such SNP marker variants while most genes have noneIn order to account for the disproportional number of the ultrarare variant of some CPGs we calculated the expected number of cancer predisposed genes when gradually removing highlyrepresented genes from the collection of heterozygous ultrarare variants As shown in Fig a03b there is an enrichment towards CPGs and even more so as we remove variants of overrepresented genes eg BRCA2 The statistical significance estimates pvalues for each datapoint are available in Supplemental Table a0S3 see MethodsIndependent genetic validation Due to the extremely rare nature of the CUVs we require additional support for the collection of the CPG candidates We seek independent genetic validation of the noncancer related CUVs We apply three sources for validation the filtered Caucasian UKBB cohort the matched filtered nonCaucasian UKBB cohort the collection of germline variants from TCGA as reported in gnomAD The complete list of genetically validated novel CPG candidates is listed in Table a0 Ten out of the novel CPGs were identified based on appearances in individuals with nonmelanoma skin cancerWithin the Caucasian cohort we consider the following as additional genomic evidence a gene with CUVs or any CUV seen in more than two individuals diagnosed with cancer We found genes that have distinct CUVs of which are already known CPGs BRCA1 BRCA2 and APC The other genes are likely novel Scientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0cRefEffecthg19TMissenseGMissenseMissenseTSplice region GSplice region AFrameshiftFrameshiftStop gainMissenseFrameshiftMissenseStop gainMissenseMissense MissenseFrameshiftMissenseFrameshiftFrameshiftMissenseMissenseFrameshiftStop gainFrameshiftSplice region CMissenseTAlt GeneGBACMSH6AVHLGTGFBR2AMLH1GAPCAAGGA APCGTCTGTCC CTG AG TCTTCCGCACAGGCGAACAAGAGCTGGGCCACCGTCTGFBR1SPTAN1RETBMPR1APTENEXT2NUMA1ATMBRCA2BRCA2RB1ERCC5TSC2NF1BRCA1BRCA1TGIF1RUNX1NF2COSMIC C299 CPG FunctionaYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYEnzymeDNA repairUbqcomplexKinaseTSGTSGTSGKinaseCytoskeletalKinaseKinaseTSG PhosphataseTSG EnzymeMT Spindle poleDDR KinaseTSG DNA repairTSG DNA repairTSGDNA repairTSGRAS regulatorTSG DNA repairTSG DNA repairTGF ligandTFCytoskeletalYYYYYYYYYYYYYYYYYYYYTable CUVs overlap with known cancer predisposition or driver genes a Function abbreviation DDR DNA damage response TSG tumor suppressor gene TF transcription factor MT microtubule Ubq ubiquitin Variants with at least one appearance in nonmelanoma skin cancerFigure a0 CUVs list is enriched with cancer predisposition genes Out of the genes in the CUVs list are known cancer genes a Venn diagram of the genes associated with CUVs known cancer driver genes as reported in COSMIC and the consensus CPGs b Expected number of known CPG CUV orange versus the actual number of known CPG in heterozygote CUVs blue An unbalanced representation of genes in ultrarare variants of UKBB results in overrepresentation of some genes We therefore ranked the genes based on number of ultrarare variants Supplementary Table a0S3 For each rank we present the expected number of CUVs from CPGs and the actual number observed for CUVs from CPGsScientific RepoRtS 101038s41598020704940Vol1234567890wwwnaturecomscientificreports 0cGene SymbolZygote form People per CUV Distinct CUVs NonCaucasian cohortTCGA germlineAGR2AKR1C2DNAH3DSPEGFLAMENDOUHIST1H2BOHSPB2ICAM1ISLRKCNH2MAP3K15MRPL39MYBPC3MYO1ENAV3PCDHB16SARDHSCN5AWDFY4ZFC3H1HeteroHeteroHomoHeteroHeteroHomoHeteroHeteroHomoHomoHeteroHeteroHeteroBothHomoHeteroHomoHomoHeteroHeteroHomoYYYYYYYYYYYYYYYYYFunction in tumorigenesisAffects cell migration transformation and metastasis Wnt signaling tumor antigenExerts an inhibitory effect on oncogenesisCancer predisposed genes in Tunisian familyAffects cell adhesion Suppressed by TGFβPromotes matrix assemblyCancer biomarkerAffects major signaling pathwaysEpigenetically regulatedBiomarker under a clinical trialMarker for mesenchymal stem cells Deregulated gene in cancerAffects proliferation and migrationContributes to cell migrationTumor suppressor by targeting miR130Cytoskeletal modifierStimulates upregulation of motility and invasionActs as a suppressor of breast cancerActs as tumor suppressorPromotes breast cancer possess antipancreatic cancerPresentats viral tumor antigen on dendritic cellsIndirect activating DNA repairRefTable Novel validated CPG candidates Variants with at least one appearance in nonmelanoma skin cancerCPG candidates DSP KCNH2 MYBPC3 and SCN5A There are CUVs which we detected in three individuals with cancer Three of them are known predisposition or driver genes NF1 ATM and TGFBR2 The other genes are CPG candidates that were not previously assigned as such This set includes PCDHB16 DNAH3 ENDOU AGR2 HIST1H2BO and NAV3 Interestingly a certain homozygous CUV in the gene ICAM1 appeared in individuals with cancer in our filtered Caucasian cohortThe nonCaucasian UKBB cohort provides additional independent genomic evidence There are CUVs that appear at least once in an individual with cancer from the nonCaucasian cohort CUVs from the genes MYO1E SARDH and ISLR appeared in two distinct individuals with cancer from this nonCaucasian cohort while CUVs from PCDHB16 and known CPG BMPR1A appeared in a single individual with cancerTCGA germline variants were obtained using exome sequencing and thus offer an additional separate source for CUV validation Clearly the appearance of CUVs in TCGA germline data is not anticipated as we discuss variants that are ultrarare in both UKBB and gnomAD The TCGA collection within gnomAD includes only samples We identified CUVs that were also observed in TCGA gnomAD germline data one of a known cancer driver gene TGIF1 and novel CPG candidates PCDHB16 EGFLAM AKR1C2 MAP3K15 MRPL39 DNAH3 WDFY4 HSPB2 and ZFC3H1Based on the above support we compiled a list of validated CPGs which includes genes that are novel CPGs Among these genes CUVs are heterozygous are homozygous and MYBPC3 is supported by both heterozygous and homozygous CUVs Two of these genes have multiple validation evidence DNAH3 with a homozygous CUV which appears in individuals with cancer in the Caucasian cohort and within TCGA germline variant collection PCDHB16 with a homozygous CUV which appeared in individuals in the Caucasian cohort one individual in the nonCaucasian cohort and in the TCGA gnomAD resource In addition nonCPG cancerdriver genes with validated CUVs include TGFBR2 and TGIF1 that are also very likely CPG candidatesSome of the prominent genes in our list were signified by additional independent studies For example a novel oncolytic agent targeting ICAM1 against bladder cancer is now in phase of a clinical trial29 Additionally DNAH3 was identified as novel predisposition gene using exome sequencing in a Tunisian family with multiple nonBRCA breast cancer instances30Somatic mutations in novel CPGs significantly decrease survival rate There is substantial overlap between CPGs and known cancer driver genes Fig a03a This overlap suggests that somatic mutations in validated CPG candidates may have an impact on patients™ survival rate We tested this hypothesis for the novel CPG candidates Table a0 using a curated set of nonredundant TCGA studies compiled in cBioPortal3132 that cover patients By testing the impact of alteration in the novel CPGs in somatic data we expect to provide a functional link between the germline CPG findings and the matched mutated genes in somatic cancer samples Altogether of the patients had somatic mutations in one or more of the genes The median survival of patients with somatic mutations in these genes is a0months while the median for patients without Scientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Somatic mutations in CPG candidate effect cancer patient survival and disease progression The effect of somatic mutations in the novel CPG candidate Table a0 on the survival rate of TCGA cancer patients was tested via cBioPortal a Meier“Kaplan survival rate estimate b Meier“Kaplan diseaseprogressionfree estimatesomatic mutations in any of these genes is much longer a0months Applying the Kaplan“Meier survival estimate yields a p value of 178eˆ’ in the Logrank test Fig a04a The Kaplan“Meier diseaseprogressionfree estimate was also worse for patients with somatic mutations in the novel CPGs with a p value of 603eˆ’ Fig a04b Cancer types in this analysis are represented by varied number of patients and percentage of individuals with somatic mutations in any of the novel CPGs Supplemental Table a0S4 The trend in most cancer types match the presented pancancer analysis Survival and diseaseprogression estimate for each cancer type are available in Supplementary Figures a0S1“S24 Hazard Ratios and confidence intervals were calculated see Materials and Methods and Supplemental Table a0S4We conclude that the CUVbased CPG candidate genes from UKBB carry a strong signature that is manifested in patients™ survival supporting the notion that these genes belong to an extended set of previously overlooked CPGsHomozygous variations are mainly recessive In order to ascertain whether the homozygous variations found are indicative of the heterozygous form of the variant as well we viewed the heterozygous prevalence within the UKBB Caucasian population In only a single variant in the gene MYO1E was the prevalence in healthy individuals significantly lower than in individuals with cancer p value As most of the variations have a strong cancer predisposition effect as homozygous variations it seems that their influence is explained by a recessive inheritance mode This phenomenon might explain the significant depletion of known CPGs within the homozygous variations in our listInspecting the heritability model of previously reported CPGs1 is in accord with our findings showing that while about twothirds of the genes comply with a dominant inheritance the rest are likely to be recessive Notably in the most updated CPG catalog of the genes were assigned with both inheritance patterns In our ultrarare list only MYBPC3 is associated with both heterozygous and homozygous variationsDiscussionWe present a list of CUVs from genes Among them variants from genes are associated with known cancer genes Most of these variants overlap with known cancer predisposition genes Expanding the number of currently identified CPGs is crucial for better understanding of tumorigenesis and identifying various processes causing high cancer penetrance Genetic consulting family planning and appropriate treatment is a direct outcome of an accurate and exhaustive list of CPGsKnown cancer predisposition variants only partially explain the cases of inherited cancer incidents CPGs identification has already impacted cancer diagnostics therapy and prognosis1 Genomic tests and gene panel for certain cancer predisposition markers are commonly used for early detection and in preventative medicine3334 It is likely that CPGs based on ultrarare variants are not saturated For example additional CPGs including CDKN2A and NF1 were associated with an increased risk for breast cancer35 Specifically CDKN2A has been also detected as a CPG in families of patients with pancreatic cancer36 Inspecting the function of genes associated with Scientific RepoRtS 101038s41598020704940Vol1234567890wwwnaturecomscientificreports 0cthe identified genes further supports the importance of protein modification eg kinases and phosphatase function chromatin epigenetic signatures37 membrane signaling DNA repair systems and moreNumerous CUVs are present in individuals with nonmelanoma skin cancer For the most part nonmelanoma skin cancers are attributed to environmental factors Nevertheless studies show that there are in fact genetic components associated with the majority of nonmelanoma skin cancers2526 Accordingly CUVs can unveil such rare genetic associationsWe chose to focus on cancerexclusive variants to shed light on mostly overlooked ultrarare cancer predisposition variants Naturally additional ultrarare variants in the dataset are presumably cancer inducing Detecting these variants requires developing a broader model expanding the scope to somewhat less rare possibly lowerpenetrance variants The impending availability of UKBB exome sequencing exomes will enable us to revisit the identified variants to further refine the list of candidate CPGs ie removing falsepositives and adding evidence to support true CPGs and to develop a less strict detection modelThe inheritably rare nature of CUVs raise concerns on the reliability of their initial identification38 We overcome this hurdle by only considering as candidate CPGs those genes that are supported by additional independent genomic evidence from either the UKBB or the TCGA cohort We nominate genes as CPG candidates two of which are known cancer drivers As we have shown Fig a0 somatic mutations in the nondriver validated CPG candidates resulted in a significant negative effect on the patients™ survival rateMaterials and methodsStudy population The UKBB has recruited people from the general population of the UK using National Health Service patient registers with no exclusion criteria39 Participants were between and a0years of age at the time of recruitment between and To avoid biases due to familial relationships we removed samples keeping only one representative of each kinship group of related individuals We derived the kinship group from the familial information provided by the UKBB fam files Additionally samples had mismatching sex between the selfreported and the geneticsderived and samples had only partial genotypingWe divided the remaining participants into two groups ˜Caucasians™”individuals that were both genetically verified as Caucasians and declared themselves as ˜white™ ˜nonCaucasians™”individuals not matching the previous criterion The Caucasian cohort includes individuals of whom had cancer and the nonCaucasian cohort includes individuals had cancer We used the Caucasian cohort for our primary analysis and the nonCaucasian cohort for additional validation purposesVariant filtration pipeline We considered a heterozygous variation as cancerexclusive when there were at least cancer patients exhibiting the variation and no healthy individuals with the variation in the Caucasian cohort We considered a homozygous variation as cancerexclusive when there were at least cancer patients exhibiting the variation ie homozygous to the alternative SNP and no healthy individuals with the homozygous variation in the Caucasian cohort The ensemble Variant effect predictor40 was used to annotate the variantsWe applied two additional filtration steps for the exomesplicingregion variants The first filter was applied using the ˜nonCaucasian™ data set we filtered heterozygous variations with MAF and homozygous variations with homozygous frequency in this set This filtration step is meant to diminish variations which are mostly ethnic artifacts The second filter was applied to assure the variations rarity We applied the same filter heterozygous variations with MAF and homozygous variations with homozygous frequency using gnomAD v21141 The used gnomAD threshold was based on the summation of gnomAD v211 exomes and genomes We also used gnomAD for the TCGAgermline validation by extracting TCGA appearances from the databaseStatistical analysis The UKBB ultrarare variants are enriched with CPGs variants We accounted for this imbalance by calculating the expected number of cancer predisposed genes when gradually removing highlyrepresented genes from the ultrarare variant collection for heterozygotes We calculated pvalues for each datapoint using a twoside binomial testWe downloaded survival data from cBioPortal The data only included survival months We used Cox regression without covariates to calculate Hazard Ratio and confidence intervals The results are listed in Supplementary Table a0S4Rare variants reliability Our CUV collection includes variants that appeared at least twice in the filtered Caucasian cohort thereby evading many SNPgenotyping inaccuracies38 We further ascertain the validity of prominent variants with additional genomic evidenceCancer type definition The UKBB provides an ICD10 code for each diagnosed condition We considered an individual diagnosed with malignant neoplasm ICD10 codes C00C97 as individuals with cancer and otherwise as cancerfree individuals The codes were aggregated to improve data readability using the assembly described in Supplementary Table a0S1Ethical approval All methods were performed in accordance with the relevant guidelines and regulations UKBB approval was obtained as part of the project Ethical approval for this study was obtained from the Scientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0ccommittee for ethics in research involving human subjects for the faculty of medicine The Hebrew University Jerusalem Israel Approval Number UKBB received ethical approval from the NHS National Research Ethics Service North West 11NW0382 UKBB participants provided informed consent forms upon recruitmentData availabilityMost of the data that support the findings of this study are available from the UKBB However restrictions apply to the availability of these data which were used under license for the current study and so are not publicly available Data are available from the authors upon a justified request and with permission of the UKBB Data extracted from gnomAD is available from the authors upon requestReceived February Accepted July a1508 References Rahman N Realizing the promise of cancer predisposition genes Nature 101038natur e1298 Zhang J et al Germline mutations in predisposition genes in pediatric cancer N Engl J Med 101056NEJMo Hanahan D Weinberg R A Hallmarks of cancer the next generation Cell 101016jcell201102013 Vogelstein B Kinzler K W Cancer genes and the pathways they control Nat Med 101038nm108 Bertelsen B et al High frequency of pathogenic germline variants within homologous recombination repair in patients with advanced cancer npj Genom Med 101038s4152 Easton D F How many more breast cancer predisposition genes are there Breast Cancer Res 101186bcr6 Hindorff L A et al Potential etiologic and functional implications of genomewide association loci for human diseases and traits Proc Natl Acad Sci U S A 101073pnas09031 Galvan A Ioannidis J P A Dragani T A Beyond genomewide association studies genetic heterogeneity and individual predisposition to cancer Trends Genet 101016jtig200912008 Baria K Warren C Roberts S A West C M Scott D Chromosomal radiosensitivity as a marker of predisposition to common cancers Br J Cancer 101054bjoc20001701 Hu C et al Association between inherited germline mutations in cancer predisposition genes and risk of pancreatic cancer J Am Med Assoc 101001jama20186228 Verkasalo P K Kaprio J Koskenvuo M Pukkala E Genetic predisposition environment and cancer incidence a nationwide twin study in Finland “ Int J Cancer 101002SICI1097021519991 210836743AIDIJC830CO2Q Frank S A Genetic predisposition to cancer”insights from population genetics Nat Rev Genet 101038nrg14 Law P J et al Association analyses identify new risk loci for colorectal cancer susceptibility Nat Commun 101038s4146 w Czene K Lichtenstein P Hemminki K Environmental and heritable causes of cancer among million individuals in the Swedish FamilyCancer Database Int J Cancer 101002ijc10332 Economopoulou P Dimitriadis G Psyrri A Beyond BRCA new hereditary breast cancer susceptibility genes Cancer Treat Grant R C et al Prevalence of germline mutations in cancer predisposition genes in patients with pancreatic cancer GastroenRev 101016jctrv201410008 terology 101053jgastr o201411042 Petersen G M et al A genomewide association study identifies pancreatic cancer susceptibility loci on chromosomes 13q221 1q321 and 5p1533 Nat Genet 101038ng522 Wolpin B M et al Genomewide association study identifies multiple susceptibility loci for pancreatic cancer Nat Genet Long J et al Genomewide association study in East Asians identifies novel susceptibility loci for breast cancer PLoS Genet 101038ng3052 101371journ alpgen10025 Thomas G et al Multiple loci identified in a genomewide association study of prostate cancer Nat Genet 101038ng91 Mancuso N et al The contribution of rare variation to prostate cancer heritability Nat Genet 101038ng3446 Jiao S et al Estimating the heritability of colorectal cancer Hum Mol Genet 101093hmgddu08 Huang KL et al Pathogenic germline variants in adult cancers Cell 101016jcell201803039 Griffin L L Ali F R Lear J T Nonmelanoma skin cancer Clin Med J R Coll Physicians Lond 107861 Nikolaou V Stratigos A J Tsao H Hereditary nonmelanoma skin cancer Semin Cutan Med Surg 101016jclinm edici ne16162 sder201208005 Roberts M R Asgari M M Toland A E Genomewide association studies and polygenic risk scores for skin cancer clinically useful yet Br J Dermatol 101111bjd17917 Forbes S A et al COSMIC exploring the world™s knowledge of somatic mutations in human cancer Nucleic Acids Res 101093nargku10 cell201802060 Bailey M H et al Comprehensive characterization of cancer driver genes and mutations Cell 101016j Annels N E et al Phase I trial of an ICAM1targeted immunotherapeuticcoxsackievirus A21 CVA21 as an oncolytic agent against non muscleinvasive bladder cancer Clin Cancer Res 10115810780432CCR184022 Hamdi Y et al Family specific ge

Thyroid_Cancer

Genomics Score Based onGenomeWide Network Analysis forPrediction of Survival in GastricCancer A Novel PrognosticSignatureZepang Sun  Hao Chen  Zhen Han  Weicai Huang Yanfeng Hu Mingli Zhao Tian LinJiang Yu Hao Liu Yuming Jiang and Guoxin LiDepartment of General Surgery Nanfang Hospital Southern Medical University Guangzhou ChinaGCPurpose Gastric canceris a product of multiple genetic abnormalitiesincluding genetic and epigenetic modifications This study aimed to integrate variousbiomolecules such as miRNAs mRNA and DNA methylation into a genomewidenetwork and develop a nomogram for predicting the overall survival OS of GCMaterials and Methods A total of GC cases as a training cohort with a random of examples included as a validation cohort were screened from The Cancer GenomeAtlas database A genomewide network was constructed based on a combination ofunivariate Cox regression and least absolute shrinkage and selection operator analysesand a nomogram was established to predict and 5year OS in the trainingcohort The nomogram was then assessed in terms of calibration discriminationand clinical usefulness in the validation cohort Afterward in order to confirm thesuperiority of the whole gene network model and further reduce the biomarkers for theimprovement of clinical usefulness we also constructed eight other models accordingto the different combinations of miRNAs mRNA and DNA methylation sites and madecorresponding comparisons Finally Gene Ontology GO and Kyoto Encyclopedia ofGenes and Genomes KEGG analyses were also performed to describe the function ofthis genomewide networkResults A multivariate analysis revealed a novel prognostic factor a genomics scoreGS comprising seven miRNAs eight mRNA and DNA methylation sites In thevalidation cohort comparing to patients with low GS highGS patients HR P were significantly associated with increased allcause mortality Furthermoreafter stratification of the TNM stage I II III and IV there were significant differencesrevealed in the survival rates between the highGS and lowGS groups as wellP The and 5year Cindex of whole genomicsbased nomogram were and respectively The other models have comparable or relativelypoor comprehensive performance while they had fewer biomarkers Besides thatDAVID further revealed multiple molecules and pathways related to the genomewidenetwork such as cytomembranes cell cycle and adipocytokine signalingEdited byXin Maizie ZhouStanford University United StatesReviewed byHailin TangSun Yatsen University Cancer CenterSYSUCC ChinaJie TianInstitute of Automation CAS ChinaCorrespondenceYuming Jiangjiangymbest163comGuoxin Ligzliguoxin163com These authors have contributedequally to this workSpecialty sectionThis was submitted toComputational Genomicsa section of the journalFrontiers in GeneticsReceived April Accepted July Published August CitationSun Z Chen H Han Z Huang WHu Y Zhao M Lin T Yu J Liu HJiang Y and Li G GenomicsScore Based on GenomeWideNetwork Analysis for Predictionof Survival in Gastric Cancer A NovelPrognostic SignatureFront Genet 103389fgene202000835Frontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreConclusion We successfully developed a GS based on genomewide network whichmay represent a novel prognostic factor for GC A combination of GS and TNMstaging provides additional precision in stratifying patients with different OS prognosesconstituting a more comprehensive subtyping system This could potentially play animportant role in future clinical practiceKeywords gastric cancer genomewide network miRNA mRNA DNA methylation nomogramBACKGROUNDGastric cancer GC is one of the most common malignanthuman tumors and the third leading cause of cancerrelatedmortalities worldwide Reports estimate that nearly one millionnew cases and deaths occur each year across the worldTorre Despite the rapid research advancement GCrelated impacts on human life remain high around the globeAccording to the global cancer burden data hundreds of billionsof dollars in economic losses are incurred each year due to GCAt the same time stomach cancer has been reported to cause million disabilityadjusted life years with of these resultingfrom years of life lost and from years lived with disabilityGlobal Burden of Disease Cancer Collaboration Despite major breakthroughs in GC prevention diagnosisand treatment therapies reported over the past decade prognosisremains a challenge at diï¬erent TNM stages Jiang 2018aSun 2019ab Notably patients with similar clinicalfeatures and at the same tumor stage who receive uniformtreatment have exhibited varying clinical outcomes Bang Jiang 2018b Such evidence indicates the existingchallenges to traditional TNM staging Serra possiblydue to a lack of molecular tools for eï¬ectively predicting theprognosis and the therapeutic eï¬ect of GC patients Thereforemore rigorous and reliable systems that accurately reflect theheterogeneity of diï¬erent patients and guide the development oftreatment approaches are urgently needed Duarte Serra Tumors are a product of multiple genetic mutations includinggenetic gene expression and epigenetic DNA methylation andhistone modification modifications as well as deregulationsof tumorsuppressor genes and protooncogenes Anna Choi In addition changes in a set of geneticmaterials have been closely associated with cancer outcomesAnna Choi Therefore to eï¬ectivelypredict the prognosis of tumors such as GC a single biomarkeris insufficient necessitating the need for a gene networkA variety of mRNAs have been associated with GC prognosisCamargo with microRNAsmiRNAs alsoimplicated in tumor prediction in the recent years Li Ueda Camargo These smallnoncoding RNAs comprising nucleotides primarily functionto regulate protein translation by inhibiting the expressionoffrom geneticsepigenetics is currently receiving considerable research attentionDNA methylation isthe most common epigenetic eventassociated with cancer development and progression Anna Consequently numerous studies have implicated DNAtarget messenger RNAs mRNAs Apartmethylation in the diagnosis and the prognosis of various tumorsincluding GC Camargo Choi Althoughthese studies have revealed several biomarkers that have proved tobe prognostic predictors in GC only a handful have been adoptedin clinical therapies or are used to build predictive models forthe disease Anna Duarte Camargo Choi Serra Previous studies have identified and recommended numerousbiomarkers for GC However since malignant tumors ofteninvolve multiple layers and diï¬erent levels of genetic changesincluding the genome transcriptome and proteome or evenepigenetic content selecting reasonable candidate factors fromtens of thousands of biomarkers and comprehensively analyzingthem as an independent feature is imperative to eï¬ectivelydevelop a suitable prognostic target Therefore genetic networkscontaining a panel of abnormal factors from diï¬erent regulatorylevels represent the best chance for achieving prognostic valueThe whole genomewide network analysis is reported inseveral other cancers such as colorectal cancer breast cancer andlung cancer Hou Zhang and it showsgreat value in diï¬erentiating the prognosis of these patientsTherefore it is feasible and advantageous to apply genomewidenetwork analysis to GCIn the current study we performed a series of sophisticatedstatistical analyses and identified genetic molecules that werehighly correlated with the prognosis of GC Specifically wescreened The Cancer Genome Atlas TCGA a genome projectwith types of cancer including gene expression and DNAmethylation as well as other biological information Furthermorewe extended these independent prognostic factors to theœomics concept Then a genomewide network was constructedInterestingly the genomics score GS obtained herein couldsupplement TNM staging and enhance the prognostic value ofdiï¬erent patients Moreover we developed multiple prognosticmodels then validated and compared them to ascertain theirstrengths and weaknesses Finally we performed pathwayenrichment and gene oncology annotation analyses to elucidatethe function of this gene networkMATERIALS AND METHODSData Acquisition and PreprocessingLevel data were downloaded from the TCGA databaseusing TCGAAssembler Module Ain January whichwas then pretreated with Module B The dataset comprised ofclinical variables from patients including age sex stageFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scoreprimary site grade treatment and survival as well as associatedgenomewide data In addition the expression levels of miRNAs mRNA and DNA methylation sitesIllumina methylation were obtained from and patients respectively Afterward an intersection with a totalof samples was eventually retained Furthermore patientswith missing active followup data were excluded from theanalysis leaving patients in the final cohort Figure Moreover genomewide level data whose expression levelsfor miRNAs mRNA and DNA methylation sites were missingin more than of all samples were excluded from the finalanalysis Finally GC patients with miRNAs mRNA and DNA methylation sites were chosen forfurther analysisGenomeWide Network AnalysisGene expression and DNA methylation data were normalizedusing R package before subsequent processing Univariate andleast absolute shrinkage and selection operator LASSO Coxregression models were combined and used to identify themost useful prognostic factors in miRNAs mRNA and DNAmethylation sites associated with survival Firstly univariate Coxregression was performed on each candidate miRNA mRNA andDNA methylation site to elucidate its role in patient survivalthen signatures with P value less than were retained forsubsequent analysis Thereafter the LASSO Cox regression modelwas applied to select and shrink the data SupplementaryFigure S3 Tibshirani Finally a GS based on a genomewide network comprising seven miRNAs eight mRNAs and DNA methylation sites was constructed for predicting survivalA summary of the whole screening process is displayed inSupplementary Figure S1Development and Comparison ofIndividualized Prediction ModelsThe TCGA cohort with cases was used as the trainingset with a random cases from the total cohort includedas a validation group The random number is Firstlywe developed the original GS based on biomarkers sevenmiRNAs eight mRNAs and DNA methylation sites Thenconsidering the complexity of the original GS and difficultclinical applicationin order to obtain a more concise andFIGURE A Venn diagram displays the patients™ screening processFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scoreeï¬ective GS we also constructed eight other models accordingto the diï¬erent combinations of miRNAs mRNA and DNAmethylation and made corresponding comparisons Finally atotal of nine GS models based on the genomewide networkfrom LASSO were adopted to screen for the most appropriatemarkers These included the following models genomics sevenmiRNAs eight mRNA and DNA methylation sites miRNAsseven miRNAs mRNA eight mRNA methylation DNAmethylation sites miRNAs methylation seven miRNAsand DNA methylation sites miRNAs mRNA sevenmiRNAs and eight mRNA mRNA methylation eightmRNA and DNA methylation sites Coxmodel twomiRNAs six mRNA and nine DNA methylation sites andCoxmodel one miRNA one mRNA and seven DNAmethylation sites Among them markers from Coxmodel were separately detected from miRNAs mRNA or DNAmethylation sites using multivariate Cox regression analysis afterLASSO Supplementary Tables S2“S4 On the other handmarkers from Coxmodel depended on signatures from amultivariate Cox regression analysis combining the genomewide network and the clinical characteristics SupplementaryTable S5 Thereafter we constructed several nomograms byincorporating significant P GS variables and otherclinical features following multivariate Cox regression Iasonos and a clinical nomogram was built as a blank controlThe equations used for calculating the GS of these models arelisted in Supplementary Table S6To calculate the discrimination and the stability of diï¬erentCox regression models we applied Cstatistics and calibrationAdditionally we performed an analysis oftimedependentreceiver operator characteristics ROC based on the and 5year survival endpoints to assess the prognostic accuracyof the diï¬erent nomograms Furthermore we evaluated thepotential net benefit of diï¬erent predictive models using decisioncurve analysis DCA DCA compares the clinical usefulness ofdiï¬erent indicators by calculating the potential net benefit of eachdecision strategy at each threshold probability Thus DCA wasa significant novel approach for comparing the old and the newmodels Vickers and Elkin Screening for Potential miRNA TargetGenesWe predicted the potential target genes of the seven miRNAsfrom LASSO by screening the miRTarBase miRDB andTargetScan databases Common genes from each miRNA acrossthe three databases were then used for subsequent studies Morethan of the miRNAs showed negative regulation to targetgenes Consequently the expression data from TCGA were usedto perform a batch of correlation analysis of each miRNAwith corresponding target genes and the three genes with thelargest absolute negative correlation were retained as the mostlikely targets Additionally at least three potential target genesfrom miRTarBase which is coexpressed with miRNAs wereconsidered as equally important and were subjected to Cytoscapeversion for identification of miRNA“target genes coexpression network analysis Supplementary Figure S2Functional Enrichment AnalysisThe potential target genes that were negatively correlated withmiRNAs in TCGA as well as the coding sequences for mRNAand DNA methylation sites were used for functional enrichmentanalysis using the Kyoto Encyclopedia of Genes and GenomesKEGG pathway and Gene Ontology GO using DAVID Supplementary Figure S2 Functional enrichment analysisindicates why the gene network produces images on the survivalof GC from a molecular mechanism Visualization was then doneusing the œggplot2 package implemented in RStatistical AnalysisThe patients were divided into lowrisk and highrisk groupsby the median GS as the cutoï¬ point Survival estimates wereobtained according to the Kaplan“Meier method and comparedusing the logrank test Variables that reached significance withP were entered into the multivariable analyses usingthe Cox proportional hazards model with an entry stepwiseapproach to identify covariates associated with increased allcause mortality and then hazard ratio with confidenceintervals CIs of each variable was achieved All the statisticalsignificance values were set as twosided P LASSOCox regression was performed through the œglmnet packageTimedependent ROC analysis at diï¬erent followup times wasimplemented using the œtimeROC package of R project in orderto further expound the performance of diï¬erent GS modelsand DCA was used to compare their clinical use by œrmdapackage Finally nomogram based on the Cox regression modelwas constructed using the œrms package Cindex and calibrationto calculate the discrimination and the stability of these modelswere performed using cstatistics and Bootstrap sample Harrell™sconcordance index Cindex indicated a better prognostic modelif its value was closer to and the calibration diagram showedthat the better the prediction if the closer the correction line wasto the diagonal All statistical methods are applied to both thetraining group and the validation group Statistical analyses wereperformed using SPSS statistical software version and Rsoftware version RESULTSPatient CharacteristicsAmong the GC patients analyzed in this study were male whereas were female The average ageof the entire study population was ± years In termsof pathological stage cases were identified as stageI were stage II were stage III and were at stage IV With regards to treatment patients received surgery cases of R0 surgery R1 and nineR2 whereas were subjected to fluorouracilbasedchemotherapy The genomic nomogram classified samplesinto low GS GS ‰¤ and into high GS GS groups based on the median cutoï¬ Figure A detaileddescription of tumor location pathology grade and Laurenclassification is outlined in Supplementary Table S1 while aheat map of the genomic scores layered by clinicopathologicalFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE Distribution of patient cases and density based on genomics score GS in the total The Cancer Genome Atlas cohort AB Scatter plots of genomicsscores regarding the classification of low and high GS CD and the bold line represents the medianfactors is illustrated in Supplementary Figure S4 The medianmean CI survival time for OS was “days in the total cohort “ days in the highGS group and mean “ days in the lowGSgroup Supplementary Figure S5 Toward the last followupa total of deaths and censoring were recorded Theestimated cumulative and 5year OS in the total cohortwere and respectively although these ratesincrease to and respectivelyin the lowGS group Conversely the and 5year OS decreased to and respectively in the highGS group Thebaseline information of the validation cohort is also listed inSupplementary Table S1 and Supplementary Figure S6Survival AnalysisWe identified a basic genomewide network comprisingseven miRNAs eight mRNAs and DNA methylationsites as the prognostic factor for OSfrom hundreds ofthousands of univariate Cox regression and LASSO analysesThis network was then classified as other models in thetraining and the validation groups Among the featuresidentified poor prognosis was significantly associated witha high expression of seven miRNAs hsamir100 hsamir hsamir136 hsamir193b hsamir22 hsamir653and hsamir6808 six mRNAs NRP18829 RNF144A9781ZNF227570 DUSP11843 CPNE8144402 MAGED19500and LOC9145091450 and seven DNA methylation sitesFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scorecg22813794cg26014401cg25361506cg07020967 cg08859156 cg12485556 cg15861578 cg15861578cg25161386 and cg22740006 Conversely poor prognosis wasstrongly associated with a low expression of SOX148403 and DNA methylation sites including cg02223323 cg00481239cg14791193 cg15486740 cg20100408 cg20350671 cg22395807cg24361571andcg26856948 Table Univariate analysis performed onclinical characteristics revealed a significant association betweenage pathological stage TNM and surgery with OS Table On the other hand results from multivariable Cox regressionshowed that age pathological stage and GS were significantlyassociated with allcause mortality in GC Table and Figure Furthermore stratification of the pathological stage I II IIIand IV revealed significant diï¬erences in survival rates betweenthe highGS and the lowGS groups Figure A similarresult was found when the data were stratified by demographicvariables sex and age clinical characteristics primary sitegrade and Lauren classification as well as treatments surgeryand chemotherapy Supplementary Figures S7 S8 Onthe other hand categorizing GS into high or low groupsusing the median value across diï¬erent modelsindicatedthe genomics nomogram had the highest HR valuethatInterestingly HR was almost equal to miRNAs methylationmRNA methylation Coxmodel and Coxmodel nomograms which contained fewer gene features Moreoverthe HR value showed a marked decrease in miRNAs mRNAmethylation and miRNAs mRNA nomograms which includedthe least characteristics Table Nomograms Based on GenomeWideNetworkA genomics nomogram was first constructed based on thegenomewide network comprising gene features Figure To obtain a more concise and eï¬ective nomogram we also builta Coxmodel gene features and Coxmodel nine genefeatures nomograms Supplementary Figures S9 S10 Nexta clinical nomogram based on stage and age was built as acontrol Supplementary Figure S11 Thereafter we performedinternal and external validation to evaluate the feasibility of allnomograms using a threegrouped random bootstrap samplingFigure and Supplementary Figures S9“S11 We observedgood predictive performance in the first three nomograms butnot in the simple clinical modelValidation of the Nomograms Using ROCand DCATo ensure a good comparison across diï¬erent GS nomogramswe performed a timedependent ROC at and years offollowup as well as DCA In the validation group genomicsnomogram revealed the best comprehensive performance with and 5year area under the curve AUC values of and respectively Table and Coxmodel miRNAs methylation and mRNA methylation nomogramshad a comparable performance with and 5year AUCvalues of “ “ and “ respectivelybut it had fewer biomarkers Table Although the Coxmodel nomograms had the least biomarkers including miRNA mRNAand DNA methylation sites it had a relatively poor performancewith and 5year AUC values of and respectively Besides that the miRNA mRNA methylationmiRNAs methylation and miRNAs mRNA nomogramsrecorded and 5year AUC values of “ “ and “ respectively Finally we found thatcompared to miRNA and and mRNAnomogram and methylation nomogramhad higher and 5year AUC values of and Nevertheless all of them showed better performance thanthe clinical nomogram which recorded and 5year AUCvalues of and respectively Figures 6AB andSupplementary Figure S12 The Cindex based on diï¬erentnomograms exhibited a similar eï¬ect Supplementary Table S8Additionally DCA showed that the genomics Coxmodel mRNA methylation and methylation nomograms had asignificant net benefit compared to other GS models and theclinical nomogram Figures 6CDPotential miRNA Target GenesA total of hsamir22 hsamir100 hsamir136 hsamir193b hsamir653 hsamir1304 and hsamir6808 potential target genes were identified from themiRTarBase miRDB and TargetScan databases SupplementaryFigure S14 We then performed a correlation analysis betweeneach target gene and miRNAs and finally generated a miRNA“potential target gene plot Supplementary Figure S15A as wellas a miRNA“target gene coexpression network SupplementaryFigure S15B using CytoscapeFunctional Analysis of GenomeWideNetworkWe imported the potentialtarget genes mRNA andDNA methylation sitecoding sequences identified above intoDAVID for KEGG and GO analyses and identified biologicalprocesses molecular functions as well as cellular componentsFigures 7A“C Their corresponding KEGG pathways were alsoplotted Figure 7DDISCUSSIONGC can be divided into two types or four main categoriesaccording to the Lauren and World Health anizationWHO classifications Lauren Nagtegaal although neither of these classifications is based on molecularmarkers In the last decade however three novel molecularbased classification systems have been suggested for GC TheSingaporeDuke Group was the first to describe a classificationwith two intrinsic genomic subtypes GINT and GDIF whichhad diï¬erent gene expression Tan Serra The subtypes have diï¬erent levels of resistance to variouschemotherapy drugs and show limited prognostic value LaterTCGA used molecular evaluation to propose a new classificationwith four subtypes EBV MSI GS and CIN The identification ofthese subtypes has provided a roadmap for patient stratificationFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE Univariable and multivariable analyses of the genomics score and the clinicopathological characteristics for overall survival in the training group and thevalidation groupVariablesTraining group n Validation group n Univariable analysisMultivariable analysisUnivariable analysisMultivariable analysisHR CIP valueHR CIP valueHR CIP valueHR CIP valueAge at diagnosis tears‰¥Pathological stageIIIIIIIVSurgeryR0R1R2UnknownGenomics scoreaLowHighT stagingT1T2T3T4N stagingN0N1N2N3M stagingM0M1SexFemaleMalePrimary siteCardiaFundusbodyAntrumUnknownPathology gradeI“IIIII“IVUnknownLauren classificationIntestinal typeDiffused typeUnknownChemotherapyYesNo“NANA““NA“NA“““ NA““ “““““““NANANA“““ NA““ “““““““NANANA“““ “NANANANA““““““NANANA““““““““““NANANANANA““““““NANANA“ “““““““““NANANANANAaBased on biomarkers seven miRNAs eight mRNAs and DNA methylation sitesFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE miRNAs mRNA and DNA methylation whose expression levels showed a significant association with overall survival in least absolute shrinkage andselection operatorMolecular probeID reference geneCoefficientmiRNAsmRNAcg02223323cg00481239cg07020967cg08859156cg12485556cg14791193cg15861578cg15486740cg20100408cg20350671cg22395807cg24361571cg25361506cg25622155cg25161386cg22740006cg22813794cg26014401cg26856948hsamir100hsamir1304hsamir136hsamir193bhsamir22hsamir653hsamir6808NRP18829RNF144A9781ZNF227570SOX148403DUSP11843CPNE8144402MAGED19500LOC9145091450MAP7D2SHC4EID1TMEM117RPS4XPREPC1orf144ZC3H10ACOT13TTRAPHLADPB1IL1RAPL1ATXN10MIR3652UnconfirmedUnconfirmedNUFIP2PCLRFN4STYXL1MDH2UnconfirmedGOLGA3ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’HR CI““““““““““““““““““““““““““““““““““SEz valuep valueˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’as well as targeted therapeutic trials Cancer Genome AtlasResearch However initial data on disease outcomes fromthis cohort did not show diï¬erences in survival among thefour groups A series of positive studies on prognosis basedon TCGA classification was also reported Sohn In addition the Asian Cancer Research group divided GCinto four subtypes MSI EMT MSSTP53 and MSS TP53based on gene expression data and found significantly diï¬erentsurvival outcomes across them Cristescu Serra Despite the significant milestones of these studiesthey are all mainly based on the analysis of gene expressionmRNA Besides that a study proposed a fivemiRNAmodel while it had a Cindex of only Zhang In the current study we included methylation data andperformed functional enrichment analysis making our workstronger The aforementioned classifications are also complicatedand need further optimization to increase clinical applicabilityFurthermore they focused on typing and finding new targetswhereas our study reports on prognostic analysisSome of the biomarkers we identified herein including hsamir22 hsamir100 hsamir136 hsamir193b hsamir1304NRP1 DUSP1 and MAP7D2 cg02223323 have previouslybeen reported in GC Grandclement and B Chen Mu Zuo Zheng Chen Kurata and Lin Liu KT Song Teng Liu Pan Wang Others such as CPNE8MAGED1 RNF144A SOX14 ACOT13 cg15486740 EID1cg00481239 RPS4X cg08859156 and TTRAP cg15486740have been identified in various tumors other than GC Kamio Zeng Zhou Kuang Stanisavljevic Liu X Tosic Nagasawa Yang The remainingbiomarkers including hsamir653 hsamir6808 LOC91450Frontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE Kaplan“Meier curve of overall survival in all patients then stratified by genomics score GS pathological stage and age Survival analysis in the lowand highGS groups was further divided based on stages stages I“IVcg20100408 LRFN4cg14791193 GOLGA3ZNF22 C1orf144cg26856948HLADPB1cg22740006 MDH2cg22813794 MIR3652 cg24361571 NUFIP2 cg25161386PREPcg22813794 TMEM117cg07020967 ZC3H10 ZC3H10 IL1RAPL1 cg20350671 PCcg22740006 SHC4 cg00481239 and ATXN10 cg22395807have not been previously reportedcg12485556STYXL1Currently focus has been directed on identifying prognosticmiRNAs for GC Particularly one miRNA can regulate multipletargets while multiple miRNAs can regulate a single mRNATherefore a single miRNA may play an opposite role incancer progression by regulating diï¬erent target genes Forexample Mir22 and Mir100 were found to be tumorsuppressors in various cancers including GC Chen Zuo Similarly a high expression of Mir136 wasfound to promote proliferation and invasion in GC cell linesby inhibiting PTEN expression Chen while acontrasting result was reported when HOXC10 was targetedZheng Similarly Mir193b reportedly inducedGC proliferation or apoptosis by mediating diï¬erent mRNAexpressions Mu Song whereas a highMir1304 expression in GC was reported as a negative predictorfor prognosis of lung and thyroid cancers Kurata and Lin Liu Pan However the function of Mir653and Mir6808 has not been previously reported In the currentstudy we found an association between a high expression of allmiRNAs and poor survival Diï¬erent outcomes may be observedin our study relative to previous reports owing to the hugeFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE Comparison of different genomics score models based on the median value for overall survival in the training group and the validation groupVariablesTraining group n Validation group n Hazard ratio CIP valueHazard ratio CIP valueGenomics nomogramAgePathological stageGenomics scoreaClinical nomogramAgePathological stagemiRNAs nomogramAgePathological stageGenomics scorebmRNA nomogramAgePathological stageGenomics scorecMethylation nomogramAgePathological stageGenomics scoredmiRNAs methylation nomogramAgePathological stageGenomics scoreemiRNAs mRNA nomogramAgePathological stageGenomics scorefmRNA methylation nomogramAgePathological stageGenomics scoregCoxmodel nomogramAgePathological stageGenomics scorehCoxmodel nomogramAgePathological stageGenomics scorei““““““““““““““““““““““““““““““““““““““““““““““““““““““““““aBased on biomarkers seven miRNAs eight mRNA and DNA methylation sites bBased on seven miRNAs cBased on eight mRNA dBased on DNA methylationsites eBased on seven miRNAs DNA methylation sites fBased on seven miRNAs eight mRNA gBased on eight mRNA DNA methylation sites hBased ontwo miRNAs six mRNA nine DNA methylation sites iBased on one miRNAs one mRNA seven DNA methylation sitesnumber of corresponding miRNA target genes herein and lackof evidence on their role in GC developmentMessenger RNAs have been reported to play an essentialrole in GC cancer For example high NRP1 expression andhypermethylation were associated with poor GC prognosisWang whereas another study indicated thatit could be an antitumor target Grandclement and B In addition high DUSP1 expression levels were foundto promote progression drug resistance and poor prognosisof GC Teng On the other hand SOX14showed opposite prognostic values in cervical cancer andleukemia with antitumor and carcinogenic eï¬ects respectivelyStanisavljevic Tosic Studies have alsoimplicated CPNE8 MAGED1 and RNF144A in ovarian andbreast cancers Zeng Nagasawa Yang However LOC91450 and ZNF22 have not beenreported in cancerAccumulating evidence indicates that DNA methylation playsa significant role in cancer progression However only a handfulof studies have described the relationship between levels ofFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE Genomics nomogram to predict the probability of and 5year overall survival OS in the training cohort A and the validation cohort B todetermine how many points for each variable to the probability of OS locate the variable on its axis draw a line straight upward to the point axis repeat this processfor each variable sum up the points achieved for each of the risk factors locate the final sum on the total point axis and draw a line straight down to find thepatient™s probability of OSsinglesite methylation and GC prognosis Particularly highexpressions of MAP7D2 ACOT13 EID1 RPS4X and TTRAPhave been associated with poor prognosis in gastric lung andpancreatic cancers as well as hepatic carcinoma respectivelywhile a high TTRAP expression reportedly inhibits the growthof osteosarcoma Kamio Zhou Kuang Liu KT Liu X Notably therelationship between methylation levels and corresponding geneexpression profiles is unknown necessitating further researchFurthermore the remaining DNA methylation sites and theircorresponding genes have not been reported Lastly no studyhas described the prognostic significance using a genomewide networkLast but not l

Thyroid_Cancer

"Tumor microenvironment TME plays an important role in malignant tumors Our study aimed toinvestigate the effect of the TME and related genes in osteosarcoma patientsMethods Gene expression profiles and clinical data of osteosarcoma patients were downloaded from the TARGETdataset ESTIMATE algorithm was used to quantify the immune score Then the association between immune scoreand prognosis was studied Afterward a differential analysis was performed based on the high and lowimmunescores to determine TMErelated genes Additionally Cox analyses were performed to construct two prognosticsignatures for overall survival OS and diseasefree survival DFS respectively Two datasets obtained from the GEOdatabase were used to validate signaturesResults Eightyfive patients were included in our research The survival analysis indicated that patients with higherimmune score have a favorable OS and DFS Moreover genes were determined as TMErelated genes Theunsupervised clustering analysis revealed two clusters were significantly related to immune score and T cells CD4memory fraction In addition two signatures were generated based on three and two TMErelated genesrespectively Both two signatures can significantly divide patients into low and highrisk groups and were validatedin two GEO datasets Afterward the risk score and metastatic status were identified as independent prognosticfactors for both OS and DFS and two nomograms were generated The Cindexes of OS nomogram and DFSnomogram were and respectivelyConclusion TME was associated with the prognosis of osteosarcoma patients Prognostic models based on TMErelated genes can effectively predict OS and DFS of osteosarcoma patientsKeywords Tumor microenvironment Osteosarcoma Prognosis Immune features NomogramBackgroundOsteosarcoma is the most common bone tumor especiallyin children and adolescents [] It was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group [] Currently surgery and Correspondence 407404159qqcom4Wenzhou Medical University Wenzhou ChinaFull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years However due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable[] Previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively [] Thereforeit is necessary to 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 0cHu BMC Cancer Page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsRecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified For example Chen [] reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients Moreover Huang [] discovered that dysregulated circRNAs serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microRNA In addition lncRNA [] microRNA [] and many clinical data [] were also identified asprognostic biomarkers for osteosarcoma patients However osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humansTherefore it is necessary to find accurate biomarkers forosteosarcomaIn recent years researchers have paid more and moreattention to the role of the tumor microenvironmentTME in malignant tumors The function of TME inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] More importantly Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data ESTIMATE an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in [] Based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [“] Generally the above research indicatedthat TME can serve as the prognostic biomarker in tumorsand many TMErelated genes were determined as the prognostic genes However the role of TME and TMErelatedgenes in osteosarcoma patients remains unclearIn the present study gene expression data and corresponding clinicopathologic data were obtained from TheTherapeutically Applicable Research to Generate EffectiveTreatments TARGET dataset Then the ESTIMATEalgorithm was performed to quantify the immune score ofosteosarcoma and the TMErelated genes were identifiedby the differential expression analysis Subsequently theprognostic value of TME and TMErelated genes weredetermined by a series of bioinformatics methodsMethodsGene expression datasetsLevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom TARGET dataset ocgcancergovprogramstarget accessed on Oct The correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus After data were extracted from the public domainthe ESTIMATE an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in R software version [] Meanwhile the messenger RNAmRNA expressionprofiles and clinical data ofincludingGSE21257 [] and GSE39055 [] were obtained fromthe Gene Expression Omnibus as external validationcohortstwo cohortsSurvival analysis and correlation analysisAfter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score The KaplanMeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival OS and diseasefreesurvival DFS between high and lowscore cohorts Inaddition the association between clinicopathologic dataand TME score was also studied MannWhitney signedrank test was performed to compare the differences ofimmune score between each clinical group All statisticalanalyses in the present study were performed using Rsoftware Except for the special instructions p value twoside was identified as statistically significantin the present studyDEGexpressed geneDifferentially expressed gene analysisDifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group The limma package in Rsoftware was used to perform the differential analysisand genes with log FC and adjusted pvalue qvalue were identified as DEGs []To further understand the function of DEGs identifiedin the present study Gene Ontology GOincludingbiological processes BP molecular functions MF andcellular componentsCC and Kyoto Encyclopedia ofGenes and Genomes KEGG analysis were performedby clusterProfiler package in R software []Evaluation of association with immune cellsTo further investigate the association between DEGs andimmune cells the CIBERSORT package was used toestimate the relative proportions of types of immunecells [] Meanwhile the œConsensusClusterPlus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping DEGs [] Cumulative distribution function CDF and relative change inarea under the CDF curve were used to determine theoptimal number of clusters k Then MannWhitney 0cHu BMC Cancer Page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters []Survival analysis of DEGsBased on the DEGs the univariate COX analysis was performed to determine the prognostic value of immunerelated genes Then the OSrelated genes were validatedin the GSE21257 dataset while the DFSrelated geneswere validated in the GSE39055 dataset Only genes successfully validated were selected for further analysis Afterward based on the validated genes the multivariate COXanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsThe risk score for each patient was calculated based onthe coefficient from the multivariate COX analysis and thecorresponding gene expression Meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score The survival receiver operating characteristic ROC curve was used to show the discrimination of signatures and the KaplanMeier survivalcurve with the logrank test was generated to show thedifferences of OS and DFS between high and lowriskgroups In addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature The KaplanMeier survivalcurve and survival ROC curve were generated to show thepredictive ability of the signature in the validation cohortDevelopment of a nomogram for osteosarcoma patientsNomogram is a tool to visualize the predictive model andconvenient for clinical practice Therefore we attemptedto develop a nomogram based on the TMErelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients Firstlythe univariateCOX analysis was performed to filter prognostic variableswhich will be further included in the multivariate COXanalysis Secondly based on independent prognostic variables two nomograms were established for predicting theOS and DFS respectively The Cindex was used to assessthe discriminatory performance of the nomogram whichrange from to [] A Cindex of means agreement by chance and a Cindex of represents perfectdiscriminatory performance The higher value of the Cindex the better performance of the nomogram is Furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsResultsImmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females The immunescore of the cohort range from ˆ’ to Tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group The survival analysis indicated thatpatients with higher immune score had a favorable OSand DFS Fig 1a and b After adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both OS and DFSFig 1a and b Inaddition the relationship between immune score and clinical features was also investigated However there was nosignificant relationship between immune score and clinicalvariables Supplementary Figure 1A1CDifferential expression analysisAccording to the median of the immune score patients were divided into highscore n and lowFig Association between immune score and prognosis in osteosarcoma patients a KaplanMeier survival analysis of overall survival for patientswith high vs low immune score b KaplanMeier survival analysis of diseasefree survival for patients with high vs low immune score 0cHu BMC Cancer Page of score group n There were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesFig 2a b and Supplementary Table To furtherunderstand the function of DEGs GO analysisand KEGG analysis were performed The top significant results of GO analysis among three types wereillustrated in Fig 2c Interestingly we can find that theresults of GO analysis are mostly associated with immunity which further verify that the immunerelated DEGsare associated with immune features In addition the results of KEGG also confirmed it Such as œPhagosomeœAutoimmune thyroid disease œAntigen processing andpresentation œB cell receptor signaling pathway œIntestinal immune network for IgA production œInflammatorybowel disease œPrimary immunodeficiency œTh1 andTh2 cell differentiation œTh17 cell differentiation œNatural killer cell mediated cytotoxicity and œNFˆ’kappa Bsignaling pathway Fig 2dconsensusunsupervisedEvaluation of DEGs and immune cellsTo further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns Based on the consensus matrix heat map patientswere clearly divided into two clustersFig 3a In additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined Fig 3bc The immune score betweentwo clusters was significantly different Fig 3d In additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot Fig 3e Interestinglywe can see that the T cells CD4 memory activated ofcluster is significantly higher than cluster Fig 5fPrognostic value of TMErelated genesPrevious studies indicated that TMErelated genes canserve as the prognostic biomarker for tumor patientsFig Differentially expressed genes with the immune score in osteosarcoma patients a Heatmap of significantly differentially expressed genesbased on immune score b The volcano figure to show the upregulated and downregulated genes c GO analysis of differentially expressedgenes d KEGG of differentially expressed genes GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes 0cHu BMC Cancer Page of Fig The immune landscape of the tumor microenvironment ac Unsupervised clustering of all samples based on the overlapping DEGs dComparison of immune score between two clusters e The distribution of types of immune cells in osteosarcoma patients f The comparisonof types of immune cells between clusters DEG Differentially expressed geneHence we performed the univariate COX analysis toidentify prognostic DEGs The results showed that and genes were identified as OS and DFSrelatedDEGs respectively Supplementary Table and Afterward five OSrelated genes were successfully validated inthe GSE21257 data set and five DFSrelated genes were successfully validated in the GSE39055 cohort Furthermoremultivariate COX analysis was performed and two prognostic signatures were generated for predicting the OS andDFS respectively The risk score for predicting the OS wasasrisk score FCGR2B0766 GFAP0702 MPP70387 In addition the risk score for predicting theDFS was as follows risk score CYP2S10574 ICAM3 The AUC values of OSrelated signature were follows 0cHu BMC Cancer Page of and in and 3year respectively Fig 4aand the AUC values of DFSrelated signature were and in and 3year respectively Fig 5aMoreover survival curves showed that patients in the highrisk group had worse OS and DFS compared with the lowrisk patients Figs 4b and 5b Heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients Figs 4ceand 5ce Then both signatures were validated in independent cohorts For OS signature the AUC values ofvalidation cohort were and at and3year Fig 4f For DFS signature the AUC values ofvalidation cohort were and at and3year Fig 5f Additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients Figs 4g and 5gHeat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsFigs 4hj and f 5hjDevelopment of a nomogram for osteosarcoma patientsTo generate a nomogram for clinical use the COX analysiswas performed to select the clinical prognostic variables InFig Establishment and validation of the prognostic model for overall survival based on significant DEGs a Receiver operating characteristiccurves of prognostic signature in the training cohort b The survival curve showed the different overall survival status between high and lowriskpatients c The heat map showed the expression of prognostic genes in the training cohort d The risk curve of each sample reordered by riskscore e The scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f Receiver operating characteristiccurves of prognostic signature in validation cohort g The survival curve showed the different overall survival status between high and lowriskpatients h The heat map showed the expression of prognostic genes in the validation cohort i The risk curve of each sample reordered by riskscore j The scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0cHu BMC Cancer Page of Fig Establishment and validation of the prognostic model for diseasefree survival based on significant DEGs a Receiver operatingcharacteristic curves of prognostic signature in the training cohort b The survival curve showed the different diseasefree status between highand lowrisk patients c The heat map showed the expression of prognostic genes in the training cohort d The risk curve of each samplereordered by risk score e The scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f Receiver operatingcharacteristic curves of prognostic signature in validation cohort g The survival curve showed the different diseasefree status between high andlowrisk patients h The heat map showed the expression of prognostic genes in the validation cohort i The risk curve of each sample reorderedby risk score j The scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate COX analysis risk score and metastatic status were identified as both OS and DFSrelated variablesFig 6a and e Afterward risk score and metastatic statuswere determined as both independent OS and DFSrelated variables in the multivariate COX analysis Fig 6band f Based on independent variables two nomogramswere established for predicting the OS and DFS in osteosarcoma patients respectively Fig 6c and g The Cindexvalues were and in OS nomogram and DFSnomogram respectively The results of Cindex mean thatboth two nomograms have good discrimination Meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve Fig 6dand h which indicated a good calibrationDiscussionThe relationship between TME and tumor have beenwidely studied in recent years In the present study ESTIMATE algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from TARGET database We confirmed that theTME is significantly associated with the prognosis ofosteosarcoma patientsInadditionfunctional enrichment analyses of TMErelated genes indicated that immunerelated processesincluding OS and DFS 0cHu BMC Cancer Page of Fig Nomograms based on the tumor microenvironment related genes for osteosarcoma patients a Univariate COX analysis of overall survivalrelated variables b Multivariate COX analysis of overall survivalrelated variables c Nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curveS of overall survival nomogram e Univariate COX analysis of diseasefree survivalrelated variables fMultivariate COX analysis of diseasefree survivalrelated variables g Nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curveS of diseasefree survival nomogramknown to contribute to tumor progression More importantly DEGs based on the TME were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the OS and DFS of osteosarcoma patientsrespectivelyIn recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the TME and the ESTIMATE algorithm is oneof the most important quantitative tools for this researchfield Based on the ESTIMATE algorithm the association between the prognosis and TME has been initially 0cHu BMC Cancer Page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ “] However previousstudies indicated that TME scores serve as a differentrole in different tumors For example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ “] However for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] Therefore we can find great heterogeneity among different tumors from the perspectiveof TME For osteosarcoma patients the present studyindicated that patients with higher immune score had abetter OS and DFS Hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionIn our research TMErelated genes were identified by comparing the highscore and lowscore osteosarcoma patients The functional enrichment includingGO and KEGG analyses showed that TMErelated geneswere mainly involved in the immune features such asregulation of leukocyte activation MHC protein complex MHC protein and complex binding More importantly the unsupervised cluster analysis based on DEGswas performed and all patients were divided into twoclusters Immune score and T cell CD4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between DEGs and immune featuresDue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantThe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients Emilie etal []performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma Recently the prognostic signature based onTMErelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients [] Compared with the study performedby Zhang [] we think that our research have someadvantages Firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts Secondlythe outcome of DFS was not reported in the previousstudy As reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]Hence the DFS nomogram can improve the managementof osteosarcoma patients Finally two nomograms incorporated TMErelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsIn our research five genes were incorporated into thefinal prognostic signatures FCGR2B GFAP and MPP7were identified and validated as OSrelated biomarkerswhile CYP2S1 and ICAM3 were DFSrelated biomarkersThe role of these genes in tumor prognosis had beenwidely reported in previous studies [“] FCGR2Bhas been confirmed as an immunerelated gene previously [] Although the relationship between FCGR2Band prognosis in sarcoma patients had not been reported the prognostic value of FCGR2B had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] In addition NewM etal [] demonstrated that MPP7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomaCYP2S1 described as Cytochrome P450 Family Subfamily S Member was reported significantly associatedwith colorectal cancer In primary colorectal cancerCYP2S1 was present at a significantly higher level ofintensity compared with normal colon [] More importantly the presence of strong CYP2S1 immunoreactivity was associated with poor prognosis [] The roleof ICAM3 in cancer was also widely reported in published studies and the Akt pathway plays an importantrole in the impact of ICAM3 on tumors YG Kim etal[] reported that ICAM3 can induce the proliferationof cancer cells through the PI3KAkt pathway Additionally JK Park etal showed that the ICAM3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing MMP2 andMMP9 via Akt pathway [] showed that the ICAM3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingMMP2 and MMP9 via Akt pathwayAlthough the role of TME and TMErelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated Firstly the treatmentinformation cannot be obtained from the TARGET database which may influence the prognosis of osteosarcomapatients Secondly two nomograms were generated andshowed good performance in our study However externalvalidation by a large cohort is needed Thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear Finally inthe training cohort and DEGs were identified asOS and DFSrelated DEGs respectively However onlyfive OS and five DFSrelated genes were identified in thevalidation cohort The different age structures smaller 0cHu BMC Cancer Page of sample sizes and the platform covering only part of thegenes may contribute to this resultReceived February Accepted July ConclusionIn conclusion TME plays an important role in osteosarcoma patients and related with the progression of thetumor Moreover TMErelated genes can serve as prognostic biomarkers in osteosarcoma patients Howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studySupplementary informationSupplementary information accompanies this paper at doi101186s12885020072162Additional file Additional file Additional file Additional file AbbreviationsTME Tumor microenvironment DEG Differentially expressed genesOS Overall survival DFS Diseasesfree survival ROC Receiver characteristiccurve ESTIMATE Estimation of STromal and Immune cells in MAlignantTumor tissues using Expression data TARGET Therapeutically ApplicableResearch to Generate Effective Treatments GO Gene Ontology BP Biologicalprocesses MF Molecular functions CC Cellular components KEGG KyotoEncyclopedia of Genes and Genomes CDF Cumulative distribution functionAcknowledgementsNoneAuthors™ contributionsC H L Y Sq T C L and Yh W conceived of and designed the study C H R Sand C L performed literature search R S L Y and B C generated the figuresand tables L Y Hl R X Y and Jy L analyzed the data C H wrote themanuscript and Sq T and L Y critically reviewed the manuscript L Ysupervised the research All authors have read and approved the manuscriptFundingWe received no external funding for this studyAvailability of data and materialsThe data of this study are from TARGET and GEO databaseEthics approval and consent to participateThe research didn™t involve animal experiments and human specimens noethics related issuesConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Joint Surgery the Affiliated Hospital of Qingdao UniversityQingdao China 2Department of Medical Oncology the First Hospital ofChina Medical University Shenyang China 3Department of Nursing Sir RunRun Shaw Hospital Affiliated to Zhejiang University Hangzhou China4Wenzhou Medical University Wenzhou ChinaReferencesJaffe N Bruland OS Bielack S Pediatric and adolescent osteosarcoma vol New York Springer Science Business Media Vander RG Osteosarcoma and its variants Orthopedic Clin North Am “Biermann JS Adkins D Benjamin R Brigman B Chow W Conrad EU 3rdFrassica D Frassica FJ Gee S Healey JH Bone cancer J Natl ComprCancer Netw “Simpson S Dunning MD de Brot S GrauRoma L Mongan NP Rutland CSComparative review of human and canine osteosarcoma morphologyepidemiology prognosis treatment and genetics Acta Vet Scand Chen X Cates JM Du YC Jain A Jung SY Li XN Hicks JM Man TKMislocalized cytoplasmic p27 activates PAK1mediated metastasis and is aprognostic factor in osteosarcoma Mol Oncol “Huang X Yang W Zhang Z Shao Z Dysregulated circRNAs serve as prognosticand diagnostic markers in osteosarcoma by sponging microRNA to regulatethe downstream signaling pathway J Cell Biochem “Liu M Yang P Mao G Deng J Peng G Ning X Yang H Sun H Long noncoding RNA MALAT1 as a valuable biomarker for prognosis in osteosarcoma asystematic review and metaanalysis Int J Surg “Xu K Xiong W Zhao S Wang B MicroRNA106b serves as a prognosticbiomarker and is associated with cell proliferation migration and invasionin osteosarcoma Oncol Lett “Zheng W Huang Y Chen H Wang N Xiao W Liang Y Jiang X Su W WenS Nomogram application to predict overall and cancerspecific survival inosteosarcoma Cancer Manag Res Kahlert C Kalluri R Exosomes in tumor microenvironment influence cancerprogression and metastasis J Mol Med “ Binnewies M Roberts EW Kersten K Chan V Fearon DF Merad M CoussensLM Gabrilovich DI OstrandRosenberg S Hedrick CC Understanding thetumor immune microenvironment TIME for effective therapy Nat Med“ Yoshihara K Shahmoradgoli M Martínez E Vegesna R Kim H TorresGarcia WTreviño V Shen H Laird PW Levine DA Inferring tumour purity and stromaland immune cell admixture from expression data Nat Commun Yang S Liu T Nan H Wang Y Chen H Zhang X Zhang Y Shen B Qian PXu S Comprehensive analysis of prognostic immunerelated genes inthe tumor microenvironment of cutaneous melanoma J Cell Physiol “ Deng Z Wang J Xu B Jin Z Wu G Zeng J Peng M Guo Y Wen Z MiningTCGA database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma Biomed Res Int Zhao K Yang H Kang H Wu A Identification of key genes in thyroid Cancermicroenvironment Med Sci Monit Xu WH Xu Y Wang J Wan FN Wang HK Cao DL Shi GH Qu YYZhang HL Ye DW Prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment AgingAlbany NY Chen B Chen W Jin J Wang X Cao Y He Y Data Mining of PrognosticMicroenvironmentRelated Genes in clear cell renal cell carcinoma a studywith TCGA database Dis Markers Li X Gao Y Xu Z Zhang Z Zheng Y Qi F Identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods Cancer Med “ Pan XB Lu Y Huang JL Long Y Yao DS Prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma Aging Albany NY Wang H Wu X Chen Y Stromalimmune scorebased gene signature aprognosis stratification tool in gastric Cancer F

Thyroid_Cancer

"Cowden™s syndrome is an autosomal dominant disease with variable penetrance involving the tumorsuppressor phosphatase and tension homolog gene located on chromosome 10q2223 responsible for cellproliferation migration and cellular apoptosis Its clinical presentation encompasses mucocutaneous lesions whichare present around of the time macrocephaly and cognitive impairment and it precedes the appearance ofneoplasms such as thyroid carcinoma breast cancer among othersIn addition to these malformations arteriovenous malformations of the brain and spine endocrine abnormalitiesskeletal defects and cardiopulmonary lesions may also be foundThe relevance of the case is due to the fact that through a certain phenotype the patient™s genotype can beinferred and thus followed up closelyCase representation The clinical case concerns a 28yearold Caucasian and Portuguese woman with palmar pitsmacrocephaly and cognitive impairment She was diagnosed with papillary thyroid carcinoma at years of ageand proposed total thyroidectomyAt age a pregnancy was diagnosed with a Breast ImagingReporting and Data System 2rated breast lump Afterthe histological verification it was concluded that it was a high metastatic breast sarcoma opting for palliativemastectomy A genetic evaluation confirmed alteration in the phosphatase and tension homolog gene confirmingCowden™s syndrome The patient died at age due to neoplastic pathologyConclusion This report aims to alert to the clinical signs of this entity and the clinical supervision and followup ofthese patients In order to prevent premature deaths and to improve patient™s quality of life genetic diseases withcancer impact should be diagnosed as early as possibleKeywords Cowden™s syndrome PTEN Papillary carcinoma Breast sarcomaBackgroundCowden™s syndrome CS was first described by Lloydand Dennis in in a 20yearold patient RachelCowden who had multiple deformations such as scrotaltongue syndrome papillomatous papules thyroid adenomas fibrocystic breast disease with malignant degeneration central nervoussystem changes and familymembers with a mild form of the disease [] Correspondence sofiamiguelotehotmailcom1Faculty of Medicine University of Porto Porto PortugalFull list of author information is available at the end of the Only in was the susceptible zone of SC 10q2223mapped [] and the tumor suppressor gene phosphataseand tension homolog PTEN that regulates the PI3KAktmTOR pathway was identified which is responsible forthe proliferation migration and cellular apoptosis []adultLhermitteDuclosPTEN hamartoma tumor syndrome PHTS whichcomprises CSdiseaseLDD BannayanRileyRuvalcaba syndrome BRRSand Proteuslike syndrome represents a spectrumof hamartomatous overgrowth manifestations associated with germline mutations in the PTEN gene[] 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 0cMiguelote World Journal of Surgical Oncology Page of LDD is a rare slowgrowing hamartoma which is usually diagnosed when patients are in their twenties orthirties While it appears to be clearly associated withPTEN mutations the incidence of LDD in patients withCS is unknown Vascular venous or arterial anomaliesare reportedly common in BRRS and CS particularly inpatients with a BRRS diagnosis A number of case reports support the association of arteriovenous malformations in clinically diagnosed BRRS patients []Itis importantto recognize thatin addition toLLD other intracranial findings such as multiple venous anomalies meningiomas greater than expectedwhite matter signal abnormality prominent perivascular spaces and cortical malformations may warrant athorough evaluation for CS in the appropriate clinicalsetting [ ]BRRS is typically diagnosed in childhood and ischaracterized by macrocephaly hamartomas including lipomas or intestinal polyps penile freckling inmales and developmental delaysincluding an increased risk of autism spectrum disorder ASD []Other reported features include developmental delayvascular anomalieslarge birth weight and jointhyperextensibility []CS is an autosomal dominant genodermatosis withvariable expressiveness with over germline mutations in the PTEN gene already described but no significant correlation between mutation type and tumortypes and in of the patients no mutations werefound [] Mucocutaneous lesions including trichilemmoma acral keratosis and oral papillomatosis plusmacrocephaly are the most frequent features describedin more than of cases after the third decade []Multiple extramucocutaneous manifestations can alsooccur in CS The skeletal system may form a higharched palate scoliosis or macrocephaly []The estimated prevalence is with a slightpredominance in females and Caucasians and penetrance of up to in the second decade [] The diagnosis normally is based on clinical criteria periodicallyupdated by the National Comprehensive Cancer Network® NCNN® [] though confirmatory genetic testing is frequently usedCase representation sectionThe case being studied was reported at the S£o Jo£oHospital which is part of the Porto Medical School University of Porto in the North of PortugalPatient female Caucasian born on September having concluded year and unemployed at the timeFamily history mother with oligophrenia and maternalaunt died of thyroid carcinomaPersonal history of childhood hydrocephalus menarche at years with irregular catamenia that have beencorrected after the introduction of oral contraceptive In the patient was directed to psychiatry for suspected cognitive impairmentIn she had severe microcytic and hypochromicanemia and was treated with saccharized ferric oxideGynecological ultrasound without major changes andoccultstool was negative Afterhematological research clinical research von Willebrand™s disease was excludedin thebloodIn she was admitted to endocrinology due tochanges in thyroid analytical parameters TSH and T4and 5cm thyroid nodule in the left lobe An ultrasoundwas performed which identifies a 56cm hypoechogenicnodule and anecogenic areas the largest with cmSubsequently she performed a fineneedle biopsy andwas diagnosed with follicular lesion of undetermined significance FLUS Bethesda category III After a multidisciplinary meetingit was decided to intervene thepatient with total thyroidectomyIn September she underwent g total thyroidectomy and was diagnosed with papillary carcinomasolidtrabecular variant Fig Concomitantly she performs ablative therapy with MBq mCi of I131and is treated with levothyroxine and calciumIn January research scintigraphy after 24h oralI131 wasadministration of MBq mCi ofFig Solidtrabecular variant of papillary carcinoma Welldefined tumor with trabecular pattern a composed by follicular cells with nuclearfeatures of papillary carcinoma b and expression of TTF1 c 0cMiguelote World Journal of Surgical Oncology Page of Fig a Right breast lump b Radiological image of the breast lesionconducted revealing anterior cervical uptake focus compatible with functioning thyroid tissue and no otherpathological fixation fociAfter year in routine ultrasound microcalcificationswere detected in the jugulocarotid chain possibly relatedto a secondary injury to papillary thyroid carcinomaIn November she was referred to the center ofthe breast by right breast lump in the upper left quadrant At that time she was weeks pregnant and wasbeing followed in obstetrics with a diagnosis of hydramnios fetal hypothyroidism and suspected fetal macrosomia opting for childbirth this weekThe breast ultrasound identified a wellcircumscribed3cm polylobular nodule classified as Breast ImagingReporting and Data System BIRADS2In May the breast ultrasound revealed a 4cmnodular lesion and FNB was performed The result revealed a heterogeneous mass of about cm classifyingas BIRADS4C Mammarycomputed tomographyshowed multilobulated contours with gross calcificationsFig In the histological study of the microbiopsy of thebreast lesion a malignant neoplasm compatible withhighgrade sarcoma with areas of necrosis was observedThe neoplasm consists of spindle cells atypical and withfrequent figures of mitosis The presence of osteosarcoma and rhabdomyosarcoma components is shown inFig It was proposed to the patient to undergo palliativeright total mastectomy and the recommended therapywas doxorubicin and ifosfamide After explaining theprecautions and warnings of the drugs the patient choseto take doxorubicin onlyIn August she performs bone scintigraphy revealing no image suggestive of focal metastatic andortumoral bone pathology with hyperfixation in rightbreast dependence compatible with heterotopic fixationby the breast lesion studied For staging she underwentcomputed tomography where nodules suggestive of metastases were found in the different lobes of both lungfields all with a diameter less than cm Fig Concomitantly she was admitted for a genetic consultation showing clinical macrocephaly cm roundkeratotic palmar pits and wart vulgaris on the face andscalp Fig The genetic evaluation included the genes PTENTP53 BRCA1 BRCA2 ATM CHEK2 and PALB2PTEN gene alteration was found The result coincideswith a change in heterozygote pathogenic variantc405dupA p Cys136Metfs in the PTEN geneFig Highgrade sarcoma of the right breast composed by atypical spindle cells with rhabdomyoblastic a b and osteosarcomac components 0cMiguelote World Journal of Surgical Oncology Page of Fig Computed axial tomography with evidence of metastasis white arrowThis variant leads to the formation of a premature stopcodon and potentially the appearance of a truncatedproteinShe has stepbrothers and stepsister but she has norelationship with them The mother and uncles do notwant to carry out the genetic test and the siblings areunreachable Genetic tests were not performed on thepatient™s limbs because they do not want to knowIn she died due to extensively metastatic breastcancer Table Since the diagnosis of papillary carcinoma at her death only years have passed which demonstrates that the diagnosis made correctly and the timecould have dictated another pathDiscussion and conclusionCS presents itself with multiple hamartomas and in of cases its initial presentation is mucocutaneous lesionsThese precede by a few years the neoplasms most at riskfor this syndrome such as renal cell neoplasia and thyroidand breast carcinoma [] Table The importance of early diagnosis of this entity is related to the increase risk for certain cancersWhen the clinical phenotype raises suspicion a screening test httpswwwlernerccfgmiccscore can becarried out which takes into account the individual™s ageand phenotype In adults a clinical threshold score of ‰¥ leads to a recommendation for referral to a genetics professional to consider PHTS In children macrocephalyand ‰¥ of the following leads to the consideration ofPHTSdevelopmentaldelayautism ordermatologic features including lipomas trichilemmomasoral papillomas or penile freckling vascular featuressuch as arteriovenous malformations or hemangiomasand gastrointestinal polyps []The diagnosis is mostly clinical and the diagnostic criteria created by Eng have been developed and arereviewed annually by the National Comprehensive Cancer Network® NCCN® Eng Table Confirmation ofthe diagnosis of CS is confirmedwhen it presents Pilarsky Three or more major criteria but one must includemacrocephaly LDD or gastrointestinalhamartomas or Two major and three minor criteriaWhen there is a family history of CS or PTEN mutation CS is considered in the individual presentingPilarsky a Two major criteria with or without minor criteria orb One major criterion and two minor criteria orc Three minor criteriaThe patient had several characteristic CS findings thatsatisfied three major criteria macrocephaly breast cancer and mucocutaneous lesions and two minor criteriafollicular variant of papillary thyroid cancer and intellectual disability so she could be clinically diagnosedwith CSFig Mucocutaneous lesion on the face and scalp 0cMiguelote World Journal of Surgical Oncology Page of Table Clinical condition summaryANOIdadeDIAGN“STICOOligophreniaTable Diagnostic criteria for Cowden syndromeCriteria minorCriteria majorColon cancerBreast cancerMicrocytic and hypochromic anemiaFollicular thyroid cancerEsophageal glycogenicacanthoses ‰¥ Lipomas ‰¥ Thyroid cancer papillary orfollicular variant of papillaryRenal cell carcinomaThyroid structural lesions egadenoma nodules goiterIntellectual disability QI ‰¤ Autism spectrum disorderTesticular lipomatosisVascular anomalies includingmultiple intracranialdevelopmental venousanomaliesEndometrial cancerMultiple gastrointestinal hamartomasLhermitteDuclos disease adultMacrocephaly P ‰¥ Macular pigmentation of glans penisMucocutaneous lesions— Trichilemmoma ‰¥ biopsy proven— Acral keratoses ‰¥ palmoplantarkeratotic pits andor acralhyperkeratotic papules— Oral papillomas particularlyon tongue and gingiva multiple‰¥ or biopsyproven ordermatologistdiagnosedmucocutaneous neuromas‰¥ Papillary thyroid carcinomaBreast sarcomaCowden™s syndrome diagnoseSarcoma metastasisDeathNCCN recommends for woman an annual mammography of tomosynthesis starting at age “ or “years before the earliest known breast cancer in the family Given the risk of developing breast cancer thepossibility of total mastectomy can be considered Counseling should include a discussion regarding the degreeof protection reconstruction options and risks Becauseendometrial cancer can often be detected early based onsymptoms women should be educated regarding the importance of prompt reporting and evaluation of any abnormal uterine bleeding or postmenopausal bleedingThe evaluation of these symptoms should include endometrial biopsy every “ yearsFor both women and men NCCN advocates annualthyroid ultrasound starting at the time of CSPHTS diagnosis including childhood and colonoscopy starting at age years unless symptomatic or if the close relative withcolon cancer before age then start “ years beforethe earliest known colon cancer in the family Colonoscopyshould be done every years or more frequently if the patient is symptomatic or polyps are found Renal ultrasoundshould be considered starting at age and then every “ years Dermatological evaluation and treatment may benecessary for some patients Children should have a thorough psychomotor assessment and brain symptomsshould be assessed with magnetic resonance imagingThe differential diagnosis of entities related to PTENhamartoma must be considered Some characteristics ofCS are similar to BRRS such as macrocephaly gastrointestinal hamartomas cognitive impairment and pigmentedmacules on the penis The mutational prevalence in PTENAdapted from httpswwwnccn Copyright2019is around however specific diagnostic criteria for thisentity have not yet been crated Regarding LDD in whichthe prevalence of the affected PTEN is around theclinical findings are fundamentally based on slowly growing hamartoma of the cerebellum and usually diagnosedwhen patients are in their twenties or thirties []When the trio of thyroid breast and renal cell carcinoma arises not only the PTEN gene but also the SDHBCD gene should be screened []The clinical evaluation and followup of these patientsshould be thorough so that the evolution of certain malignancies is detected in a timely manner and their diagnosis and treatment as appropriate as possibleWhen diagnosed patients should be instructed to beaware of the signs and symptoms of cancers with ahigher incidence in this disease Sirolimus is in phase IIfor PHTS and other drugs are being studied for solidneoplasms in individuals with CS that act on the pathway PI3KaktmTOR BGT226 and BEZ235 in phase IITable Neoplasic risk and age of onsetRisk to life TumorBreastBeginning ageAbbreviationsCS Cowden™s syndrome PTEN Phosphatase and tension homologLLD LhermitteDuclos disease BRRS BannayanRileyRuvalcaba syndromeBIRADS Breast ImagingReporting and Data SystemThyroidKidney cellsEndometriumGastrointestinalMelanomaAdaptedfrom httpsrarediseasesrarediseasesptenhamartomatumorsyndromeCopyright2019AcknowledgementsNot applicableAuthors™ contributionsSM was the main contributor to the writing of the manuscript RHperformed the histological examination of the thyroid and breast LFprovided some patient data and images LE analyzed and interpreted thepatient data JAT is the first author™s advisor for the elaboration of themaster™s thesis in medicine The authors read and approved the finalmanuscript 0cMiguelote World Journal of Surgical Oncology Page of FundingNo financial sourcesAvailability of data and materialsThe datasets generated and analyzed during the current study are not publiclyavailable due to belonging to the clinical entity S£o Jo£o University HospitalCenter but are available from the corresponding author on reasonable requestEthics approval and consent to participateAll clinical information whether imaging or histological was provided by thebioethics department of the University Hospital Center of S Jo£o for theelaboration of the master™s thesis in medicine The respective authorizationsof the hospital ethics committee are in the supplementConsent for publicationInformed consent was provided by the patient™s hospitalCompeting interestsThe authors declare that they have no competing interestAuthor details1Faculty of Medicine University of Porto Porto Portugal 2University HospitalCenter of S Jo£o Porto Portugal 3Pathological Anatomy DepartmentUniversity Hospital Center of S Jo£o Porto Portugal 4Breast CenterUniversity Hospital Center of S Jo£o Porto Portugal 5Surgery DepartmentUniversity Hospital Center of S Jo£o Porto PortugalReceived March Accepted July ReferencesYehia L Eng One gene many endocrine and metabolic syndromes PTENopathies and precision medicine Endocrine Related Cancer Nelen MR Padberg GW Peeters EA Lin AY van den Helm B Frants RR CoulonV Goldstein AM Van Reen MM Easton DF Localization of the gene forCowden disease to chromosome 10q2223 Nature Genetics “Shenandoah R Alan RC Cowden disease and LhermitteDuclos disease anupdate Case report and review of the literature Neurosurg Focus 2016201E6Ueno Y Enokizono H Ohto T Imagawa K Tanaka M Sakai A Suzuki HUehara T Takenouchi T Kosaki K Takada H A novel missense PTENmutation identified in a patient with macrocephaly and developmentaldelay Human Genome Variation Pilarski R PTEN hamartoma tumor syndrome a clinical overview Division ofHuman Genetics Department of Internal Medicine and ComprehensiveCancer Center Ohio State University Columbus OH USA Dhamija R Weindling SM Porter AB Hu LS Wood CP HoxworthNeuroimaging abnormalities in patients with Cowden syndromeretrospective singlecenter study Neurol Clin Pract Kimura F Ueda A Sato E Akimoto J Kaise H Yamada K Hosonaga M KawaiY Teraoka S Okazaki M Ishikawa T Hereditary breast cancer associated withCowden syndrome “ related PTEN mutation with LhermitteDuclos diseaseSurgical Case Report Porto AC Roider E Ruzicka T Cowden syndrome report of a case and briefreview of literature An Bras Dermatol NovDec Suppl Cavaill© M PonelleChachuat F Uhrhammer N Viala S GayBellile M Privat MBidet Y Bignon Y Early onset multiple primary tumors in atypical presentation ofCowden syndrome identified by wholeexomesequencing Front Genet Garofola C Gross G Cowden disease multiple hamartoma syndrome NCBIBookshelf Farooq A Walker LJ Bowling J Audisio RA Cowden syndromeCancer Treat Rev “ National Comprehensive Cancer Network NCCN Cowden syndromePTENhamartoma tumor syndrome Eng C PTEN hamartoma tumor syndrome PHTS “ GeneReviews Updated Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

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

Glioblastoma GBM is the most aggressive and common form of primary brain cancer Survival is poor and improved treatment options are urgently needed Dual specificity phosphatase6 DUSP6 is actively involved in oncogenesis showing unexpected tumorpromoting properties in human glioblastoma contributing to the development and expression of the full malignant and invasive phenotype The purpose of this study was to assess if DUSP6 activates epithelialtomesenchymal transition EMT in glioblastoma and its connection with the invasive capacityResults We found high levels of transcripts mRNA by qPCR analysis in a panel of primary GBM compared to adult or fetal normal tissues At translational levels these data correlate with high protein expression and long halflife values by cycloheximidechase assay in immunoblot experiments Next we demonstrate that DUSP6 gene is involved in epithelialtomesenchymal transition EMT in GBM by immunoblot characterization of the mesenchymal and epithelial markers Vimentin NCadherin ECadherin and fibronectin were measured with and without DUSP6 overexpression and in response to several stimuli such as chemotherapy treatment In particular the high levels of vimentin were blunted at increasing doses of cisplatin in condition of DUSP6 overexpression while NCadherin contextually increased Finally DUSP6 per se increased invasion capacity of GBM Overall our data unveil the DUSP6 involvement in invasive mesenchymallike properties in GBMKeywords Dualspecificity phosphatase DUSP6 Glioblastoma Epithelialtomesenchymal transition EMTIntroductionDUSP6 plays a prooncogenic role in cancers such as human glioblastoma thyroid carcinoma breast cancer and acute myeloid leukemia [“] Particularly DUSP6 is upregulated in human glioblastoma where its overexpression induces reduction in proliferation rate Cell morphology exhibits a more flattened appearance lower Correspondence samanthamessinauniroma3it samanthamessinauniroma1it Department of Science Roma Tre University Viale Guglielmo Marconi Rome ItalyFull list of author information is available at the end of the levels of cellular detachment after stimulation with EGF and an increased pr sity to form colonies in soft agar Surprisingly mouse xenograft tumors expressing DUSP6 grew significantly faster than controls thus reflecting these changes in cell adhesion and morphology [] Moreover overexpression of DUSP6 has also been identified in a subset of mouse melanoma cell lines where it is associated with enhanced anchorageindependent growth and invasive capacity [] and its overexpression in papillary thyroid carcinoma PTC is associated with increased cell migration and invasion [ ] Finally in acute lymphoblastic leukemia ALL DUSP6 acts as prooncogenic phosphatase in preB cell transformation [] 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 0cZuchegna a0et a0al BMC Res Notes Page of Glioblastoma GBM is the most common and malignant type of primary brain tumor with relevant invasive and resistant properties [] The involvement of epithelialtomesenchymal transition EMT has been extensively investigated in glioblastoma although the real relevance of this program in malignant glioma is still controversial A number of preclinical studies have been launched to target the process considering the critical role played by EMT on GBM dissemination resistance to apoptosis and cancer stemness maintenance [“] Of note DUSP6 is involved in epithelialtomesenchymal transition EMT in epithelial cancers such as breast and endometrial adenocarcinoma [ ] Moreover GBMs mesenchymal subtype are characterized by an elevated invasive potential and of note the most commonly used glioma cell lines ie U87MG and U251 also present a predominant mesenchymal signature [] with elevation in mesenchymal markers [] The aim of this study was to assess the importance of DUSP6 gene in epithelialtomesenchymal transition in GBM in correlation with its invasive capacityMain textMethodsCell culturesAstrocytoma primary WHO grade IV GBM1 GBM10 etc were established from tumor specimens of patients and cultured as described [] NHA Normal Human Astrocytes and NSC Neural Stem Cells were purchased from Cambrex Corporate NJ USA and grown according to the manufacturer™s instructions Normal Human Astrocytes were used as reference because of the presumed similarity between astrocytes and the celloforigin from which glioblastoma develops both adult NHA and foetal Primary Fetal Normal Neural Stem Cells from SVZ U87MG human GBM“astrocytoma cell line was purchased from the bank of biological material Interlab Cell Line Collection Genova Italy Human breast cell cultures MCF7 MCF10A and MDA231 cell lines were purchased from the American Type Culture Collection ATTC LGC Standards srl Italy Cells were cultured at a0°C in CO2 in DMEM with high glucose plus vv fetal bovine serum FBS Euroclone Milan Italy penicillin“streptomycin a0 UmL Euroclone and lglutamine a0 mM Euroclone according to manufacturer™s instructions Primary glioblastomas cell lines WHO grade IV were established from tumor specimens of patients and cultured as described [ ] Cells were plated at of confluence on a0mm dishes and the day after infected with recombinant adenovirus as previously described with the amounts according to the scheme indicated in the figures [] In cycloheximidechase assay experiments the cells were treated with a0µgmL CHX in complete medium and then lysed at the indicated times as described in the figures™ legendsWestern Blot analysisCultured U87MG and U251MG cells were washed with PBS and lysed for a0min in icecold Radioimmunoprecipitation RIPA buffer Triton X100 deoxycholateDOC sodium dodecyl sulphate SDS a0mM Tris pH a0mM NaCl a0mM phenylmethylsulfonyl fluoride PMSF a0mgmL aprotinin leupeptin and pepstatin Cell lysates were clarified at rcf for a0 min at a0 °C and the cytosolic fraction was immediately subjected to protein determination using a Bradford colorimetric assay BioRad Laboratories Inc Hercules CA USA DUSP6 was detected with a ˜homemade™ rabbit polyclonal specific antibody against DUSP6 Lennartson™s lab Monoclonal antiαtubulin as loading control antiphosphoERK and antiFibronectin were purchased by SigmaAldrich St Louis MO USA AntiVimentin was purchased from Millipore and antiERK was purchased from Cell Signaling Technology Anti NCadherin and antiECadherin were purchased from Santa Cruz Biotechnology Goat antiMouse IgG H L Highly CrossAdsorbed Secondary Antibody HRP A16078 e Goat antiRabbit IgG H L Highly CrossAdsorbed Secondary Antibody HRP A16119 Thermofisher Scientific ECL detection kit from Amersham GEHealthcareReal‘time quantitative PCRA quantitative assay for Human DUSP6 mRNA seq ref nePlus„¢ RealTime PCR System Applied Biosystems„¢ NM_0019464 expression was established using StepOusing PowerUp„¢ SYBR„¢ Green Master Mix Applied Biosystems„¢ using the following program a0°C10 a0min — cycle a0°C15 a0s a0°C90 a0s — cycles a0°C15 a0s a0 °C90 a0 s — cycles All reactions were normalized with the housekeeping gene for 18S PCR oligoprimers were Human DUSP6 forward primer ²CgAggACCgggACCgCTT CAC C3² and reverse primer ²CCgAgATggggATTTgCTTgTATT3² generating a a0 bp fragment Human 18S forward primer ²gACCgATgTATATgCTTgCAgAgT3² and reverse primer ²ggATCTggAgTTA AAC TggTCCAg3² The two transcripts of mRNA DUSP6 were detected by PCR using the following primers Forward primer ²CgAggACCgggACCgCTT CAC C3² Reverse primer ²AgTTAggggATATgTTggATTTT3² The expected size of the fragment was a0 bp for the transcript variant NM_0019464 and a0bp for the transcript variant NM_0226524 0cZuchegna a0et a0al BMC Res Notes Page of Cell invasion assayTranswell inserts Corning® FluoroBlok„¢ Plate Permeable Support with a0µm Colored PET Membrane for trex„¢ Matrix Gibco by Life Technologies U87MG cells 24well plates were used Inserts were coated with Gelwere transduced with TRK or DUSP6 expressing adenoviral vectors and — cells were seeded in serumfree Dulbecco™s modified Eagle™s medium in the upper chamber on the top of the matrigel Dulbecco™s modified Eagle™s medium FBS a0μL was dispensed in the lower chamber as a chemoattractant After a0h the apparatus was washed with PBS and cells that did not migrate were removed with a cotton swab then inserts were fixed paraformaldehyde for a0min at room temperature The results were quantified by counting all the cells of the inserts in duplicate from two independent experiments using the — objective Data were tested for normal distribution of variables using the Shapiro“Wilks test and statistical significance between groups was determined using Student™s ttestResultsQuantitative transcriptional analysis of DUSP6 was assessed by RTqPCR measuring high mRNA levels in a panel of twenty primary glioblastomas GBM WHO grade Fig a01b by quantifying the mRNA foldinduction over Normal Human Astrocytes NHA and Neural Stem Cells NSC which specify distinct glioblastoma subtype [“] We measured increased expression in primary samples approximatively sevenfold enrichment in GBM15 GBM53 and GBM176 compared to controls In addition human longterm cultures U87MG U251MG and T98G displayed high mRNA levels compared to primary samples of GBM their values differ by several orders of magnitude Fig a0 1c Furthermore mRNA levels in breast cancers lines MCF7 MCF10 were undetectable but not in MDAMB231 a model for triplenegative breast cancer expressing aberrantly high levels and 6000fold enrichment Fig a01c Interestingly the gene contains three distinct introns Fig a0 1a producing short and longPCR products sized a0bp and a0 bp respectively with a marked prevalence of the longtranscript in primary glioblastomas personal observation ie samples GBM11 and GBM15 Fig a01eFurther we used cycloheximidechase assay after singletime point western blotting as a measurement of halflife endogenous DUSP6 protein Fig a0 Unstimulated primary GBM Panel a and longterm cultures Panel b show high protein levels in both primary and longterm glioblastoma as assayed by semiquantitative Western blot analysis Fig a0 2a b The ERKMAPK cascade activation was sustained in both primary and longterm cultures U87MG U251 and T98G while overexpression adenovirusmediated CTRL completely abrogate pERK and ERK ½ signals Fig a0 2a De novo protein synthesis was measured by timecourse cycloheximide CHX experiment in both cell lines Fig a0 2c d We found that acutely inhibiting protein synthesis diminished DUSP6 diminished expression in both U87MG and U251MG but did so with different kinetics In contrast to previously published data [] we report long halflife in both cell lines U87MG and U251 respectively more than a0h and up to a0h These results agree with the stable endogenous protein exerting oncogenic properties in cancersThen we examined the expression of mesenchymal markers associated with EMT in glioblastoma cultures in na¯ve condition and in DUSP6overexpression Firstly we assayed protein endogenous levels of Vimentin NCadherin ECadherin Fibronectin in U87MG upon several stimuli serum addition serum deprivation EGF and cisplatin by Western Blot with specific antibodies Fig a03a Immunoblot analysis for pERKERK ratio shows different kinetics of ERKMAPK kinases phosphorylation upon EGF and cisplatin CDDP treatmentsInterestingly cisplatin treatment reduces the amount of NCadherin ECadherin and Fibronectin but not Vimentin Fig a0 3a NCadherin and Fibronectin were slightly upregulated in adenovirusmediated DUSP6 overexpression Fig a03b Otherwise the epithelial marker ECadherin was almost absent in both na¯ve and adenoviralexpressing cells Next in DUSP6overexpressing U87MG cells increasing doses of cisplatin ranging from to a0 µLmL affected the EMT markers in opposite fashion Vimentin protein expression is completely blunted upon treatment whereas high levels are shown in adenoviralexpressing control vector CTRL and in adenoviralexpressing DUSP6 untreated lane Fig a0 3b Finally we assayed invasive ability of both na¯ve glioblastoma cells compared to DUSP6adenoviralexpressing U87MG cells by Transwell invasion assay see œMethods We here report that DUSP6 increased the invasion capacity of the glioma U87MG cells Fig a03c compared to MOCK cells na¯ve U87MG and adenoviral empty vector as negative control TRKin epithelialtomesenchymal DiscussionThis study was designed to explore DUSP6 involvement in glioblastoma We previously showed that DUSP6 is upregulated in human glioblastoma and in a0vitro adenovirusmediated overexpression results in a transformed phenotype [] Here we extended our transcriptional analysis to include a new set of primary cell cultures transition 0cZuchegna a0et a0al BMC Res Notes Page of Fig DUSP6 mRNA is overexpressed in human glioblastomas a Schematic grey boxes denote protein coding region of DUSP6 gene and colored arrows represent the approximate locations of the primers™ annealing sites in DUSP6 gene b Bar diagram shows relative quantification of total DUSP6 mRNA across a panel of human glioblastoma samples grade IV astrocytoma Normal Human Astrocytes NHA and Neural Stem Cells NSC were used as controls because of the presumed similarity between astrocytes and the celloforigin from which glioblastoma develops both adult NHA and foetal NSC Relative expression was normalized to housekeeping gene 18S expression c Upper panel relative DUSP6 mRNA expression quantified by qPCR on human glioblastoma cultures U251MG U87MG and T98G Lower panel human breast cell cultures MCF7 MCF10A and MDA231 were assayed as positive controls d Qualitative PCR for DUSP6 mRNA in primary glioblastomas Expression of differentsize transcripts was detected using primers shown in the upper panel Schematic denotes blackgrey boxes exons and the relative location of the forward and reverse primer annealing sites in DUSP6 mRNA gene Ethidium stained agarose gel of endpoint products from the different amplicons DUSP6 using Normal Human astrocytes NHA Neural Stem Cells NCS and two samples of primary glioblastoma mRNA The two alternative transcripts of DUSP6 mRNA are shown transcript variant NM_0019464 and transcript variant NM_0226524of glioblastoma and longterm cultures By using qRTPCR we found high levels of mRNA DUSP6 across the glioblastoma samples Moreover we assayed breast cancer cell lines and found that the mesenchymal MDAMB231 showed the highest levels of mRNA expression compared to normal epithelial MCF10 and MCF7 cell lines Interestingly DUSP6 is involved in maintaining the mesenchymal state in breast cancer [] Moreover here we report high protein expression and long halflife values by cycloheximidechase assay in longterm cultures U87MG and U251 in line with previous data on fibroblasts [] We demonstrate that DUSP6 gene is involved in epithelialtomesenchymal transition EMT in GBM Whereas we clearly show that DUSP6 per se increases invasion capacity of GBM the evidence on epithelialtomesenchymal transition in GBM does not lead to a firm conclusion We report immunoblot characterization of the mesenchymal and epithelial markers with and without DUSP6 overexpression Vimentin is clearly downregulated 0cZuchegna a0et a0al BMC Res Notes Page of Fig DUSP6 protein is overexpressed in human glioblastomas a Western Blot analysis of DUSP6 pERK and ERK in primary GBM samples In both panels CTRL indicates positive immunereactive control obtained with U87MG infected with the adenovirus DUSP6 b Western Blot analysis of DUSP6 pERK and ERK in U87MG U251MG T98G and MCF7 cell lines The corresponding bar graphs show relative expression of proteins normalized to αtubulin c d Posttranslational regulation of DUSP6 protein levels U87MG and U251MG cell lines were treated with cycloheximide CHX µgmL for the time indicated and the decay of the target proteins over time was determined by Western Blot with specific antibodies The corresponding bar graphs show relative expression of proteins normalized to αtubulin 0cZuchegna a0et a0al BMC Res Notes Page of Fig Differential regulation of epithelial and mesenchymal markers by DUSP6 a Immunoblot analysis of proteins DUSP6 pERK ERK vimentin NCadherin ECadherin fibronectin in longterm cultures U87MG treated with different stimuli serum starvation EGF ngmL cisplatin CDDP µgmL for h left lane or h right lane b Immunoblot analysis of mesenchymal and epithelial markers in U87MG adenovirusexpressing DUSP6 compared to TRK empty vector The cells were treated with increasing doses of cisplatin CDDP at different concentrations respectively μgmL and protein expression assayed by western blot on total lysates at h c Invasion assay Bar graph reports the mean of total number of invaded cells per field compared to control MOCK ie U87MG not infected U87MG cells were transduced with adenovirusexpressing DUSP6 or control vector TRK ie U87MG infected with empty vector and seeded on matrigelcoated transwell inserts The experiment was performed for h and the invading cells were counted — objective in nine randomly chosen microscopic fields per transwell Data are presented as mean ± SD from two independent experiments performed in duplicatein cisplatintreated overexpressing DUSP6 cells but not in absence of cisplatin NCadherin is upregulated in cisplatintreated overexpressing DUSP6 whereas it was downregulated in na¯ve cells In addition our data on cadherin switch are in line with the inconsistency of the literature with some reports showing that GBM do not express ECadherin but others showing the occurrence of an E to NCadherin switch More importantly our data show that cisplatin treatment clearly downregulates Fibronectin and both Cadherins while Vimentin underwent no change in na¯ve cells in absence of DUSP6 overexpression Notably classical cadherin switch which is widely accepted as an EMT hallmark in carcinomas is a controversial matter in GBM [ ]Of note it was found that Ncadherin expression is inversely correlated with the invasive behavior of GBM and its ectopical expression reduces cell migration and restores polarity in GBM cells [ ] Conversely lower expression of NCadherin was recognized in a panel of GBM primary samples at mRNA and protein levels [] 0cZuchegna a0et a0al BMC Res Notes Page of Recently association of the EMT transition with chemoresistance has been reported [] Our results clearly show that mesenchymal markers are downregulated by cisplatin in na¯ve cells except for vimentin This is in line with vimentin increased levels in resistant GBM cultures compared to parental ones [] Moreover conflicting results are reported on DUSP6™ role in chemotherapyresistance in epithelial cancers [ ] Here we report the first observation of EMT markers in response to a drug used in chemotherapy during DUSP6 overexpression Particularly the high levels of vimentin were blunted at increasing doses of cisplatin in a condition of DUSP6 overexpression while NCadherin contextually increased These data have implications on chemotherapy response in glioblastoma treatmentLimitationsLimitations of this study include i limited number of lowpassage serumfree cell lines cultured from patient tumor tissue GBM in the text ii the poorlyrepresentative cell line models U87MG and U251MG which are not exhaustive model for glioblastoma multiforme molecular subtypes classical proneural and mesenchymal [] Further this study reports only an in a0vitro characterization based on western blot analysis of the classical mesenchymal markers Additional file a0 and from the pointofview of phenotypic characterization the invasion assay does not exhaustively demonstrate that DUSP6 activates EMT in GBM by enhancing its invasive propertiesSupplementary informationSupplementary information accompanies this paper at https doi101186s1310 yAdditional file a0 Figure S1 Uncropped versions of the western blot used in this manuscript corresponding to Fig Original gels of Western Blot analysis of Fig panels A and B immunereactive bands corresponding to specific antibodies against DUSP6 pERK and ERK and αtubulin as specified in Methods section Original gels of Western Blot analysis of Fig panels C and D immunereactive bands corresponding to specific antibodies against DUSP6 pERK and ERK and αtubulin as specified in Methods section Figure S2 Uncropped versions of the western blot used in this manuscript corresponding to Fig Original gels of Western Blot analysis of Fig panels A and B immunereactive bands corresponding to specific antibodies against DUSP6 pERK ERK Vimentin NCadherin ECadherin Fibronectin as specified in Methods sectionAbbreviationsDUSP6 Dualspecificity phosphatase CHX Cycloheximide ERK12 Extracellular regulated kinases EMT Epithelialtomesenchymal transition ALL Acute lymphoblastic leukaemia GBM GlioblastomaAcknowledgementsWe are grateful to Prof Antonio Porcellini and Dr Luca Persano for their cheerful assistance with preliminary data of this workAuthors™ contributionsSM and BM conceived the project SM CZ and EDZ designed and performed the experiments CZ and EDZ analyzed the experiments and cowrote the paper All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsAll data presented or analyzed in this study are included in this Ethics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Biology Federico II University of Naples Naples Italy Department of Medicine and Health Sciences œV Tiberio University of Molise Campobasso Italy Department of Science Roma Tre University Viale Guglielmo Marconi Rome Italy Received October Accepted July References Messina S Frati L Leonetti C Zuchegna C Di Zazzo E Calogero A Porcellini A Dualspecificity phosphatase DUSP6 has tumorpromoting properties in human glioblastomas Oncogene “ Degl™Innocenti D Romeo P Tarantino E Sensi M Cassinelli G Catalano V Lanzi C Perrone F Pilotti S Seregni E et al DUSP6MKP3 is overexpressed in papillary and poorly differentiated thyroid carcinoma and contributes to neoplastic properties of thyroid cancer cells Endocr Relat Cancer “Song HM Wu CY Wei CK Li DF Hua KY Song JL et al Silencing of DUSP6 gene by RNAimediation inhibits proliferation and growth in MDAMB231 breast cancer cells an in vitro study Int J Clin Exp Med “ Arora D K¶the S van den Eijnden M van Huijsduijnen RH Heidel F Fischer T et al Expression of proteintyrosine phosphatases in Acute Myeloid Leukemia cells FLT3 ITD sustains high levels of DUSP6 expression Cell Commun Signal Li W Song L Ritchie AM Melton DW Increased levels of DUSP6 phosphatase stimulate tumorigenesis in a molecularly distinct melanoma subtype Pigment Cell Melanoma Res “Lee JU Huang S Lee MH Lee SE Ryu MJ Kim SJ Kim YK Kim SY Joung KH Kim JM et al Dual specificity phosphatase as a predictor of invasiveness in papillary thyroid cancer Eur J Endocrinol “Shojaee S Caeser R Buchner M Park E Swaminathan S Hurtz C Geng H Chan LN Klemm L Hofmann WK et al Erk negative feedback control enables PreB cell transformation and represents a therapeutic target in acute lymphoblastic leukemia Cancer Cell “ Kahlert UD Joseph JV Kruyt FAE EMT and METrelated processes in nonepithelial tumors importance for disease progression prognosis and therapeutic opportunities Mol Oncol “ https doi1010021878026112085 Iser IC Pereira MB Lenz G Wink MR The epithelialtomesenchymal transitionlike process in glioblastoma an updated systematic review and in silico investigation Med Res Rev “ Mehta S Lo Cascio C Developmentally regulated signaling pathways in glioma invasion Cell Mol Life Sci “ Kubelt C Hattermann K Sebens S Mehdorn HM Heldfeindt J Epithelialtomesenchymal transition in paired human primary and recurrent glioblastomas Int J Oncol “ 0cZuchegna a0et a0al BMC Res Notes Page of Boulding T Wu F McCuaig R Dunn J Sutton CR Hardy K Tu W Bullman LewisTuffin LJ Rodriguez F Giannini C Scheithauer B Necela BM A Yip D Dahlstrom JE et al Differential roles for DUSP family members in epithelialtomesenchymal transition and cancer stem cell regulation in breast cancer PLoS ONE 201611e0148065 Fan MJ Liang SM He PJ Zhao XB Li MJ Geng F Dusp6 inhibits epithelialmesenchymal transition in endometrial adenocarcinoma via ERK signaling pathway Radiol Oncol “ https doi102478raon20190034 Behnan J Finocchiaro G Hanna G The landscape of the mesenchymal signature in brain tumours Brain “ https doi101093brain awz04 Lu KV Chang JP Parachoniak CA Pandika MM Aghi MK Meyronet D Isachenko N Fouse SD Phillips JJ Cheresh DA Park M Bergers G VEGF inhibits tumor cell invasion and mesenchymal transition through a METVEGFR2 complex Cancer Cell “ https doi101016jccr201205037 Messina S Leonetti C De Gregorio G Affatigato V Ragona G Frati L Zupi G Santoni A Porcellini A Ras inhibition amplifies cisplatin sensitivity of human glioblastoma Biochem Biophys Res Commun “ Lindberg N Kastemar M Olofsson T Smits A Uhrbom L Oligodendrocyte progenitor cells can act as cell of origin for experimental glioma Oncogene “ Liu C Sage JC Miller MR Verhaak RGW Vogel H Foreman O Bronson RT Nishiyama A Mosaic analysis with double markers MADM reveals tumor celloforigin in glioma Cell “ Alcantara Llaguno SR Wang Z Sun D Chen J Xu J Kim E Hatanpaa KJ Raisanen JM Burns DK Johnson J et al Adult lineage restricted CNS progenitors specify distinct glioblastoma subtypes Cancer Cell “ Alcantara L Chen J Kwon C Jackson EL Li Y Burns DK Alvarezbuylla A Parada LF Malignant astrocytomas originate from neural stemprogenitor cells in a somatic tumor suppressor mouse model Cancer Cell “ Marchetti S Gimond C Chambard JC Touboul T Roux D Pouyss©gur J Pag¨s G Extracellular signalregulated kinases phosphorylate mitogenactivated protein kinase phosphatase 3DUSP6 at serines and two sites critical for its proteasomal degradation Mol Cell Biol “ Siebzehnrubl FA Silver DJ Tugertimur B Deleyrolle LP Siebzehnrubl D Sarkisian MR Devers KG Yachnis AT Kupper MD Neal D et al The ZEB1 pathway links glioblastoma initiation invasion and chemoresistance EMBO Mol Med “Misregulated Ecadherin expression associated with an aggressive brain tumor phenotype PLoS ONE 20105e13665 Asano K Duntsch CD Zhou Q Weimar JD Bordelon D Robertson JH Pourmotabbed T Correlation of Ncadherin expression in high grade gliomas with tissue invasion J Neurooncol “ Camand E Peglion F Osmani N Sanson M EtienneManneville S Ncadherin expression level modulates integrinmediated polarity and strongly impacts on the speed and directionality of glial cell migration J Cell Sci “ Musumeci G Magro G Cardile V Coco M Marzagalli R Castrogiovanni P Imbesi R Graziano AC Barone F Di Rosa M Castorina S Castorina A Characterization of matrix metalloproteinase2 and ADAM10 and Ncadherin expression in human glioblastoma multiforme Cell Tissue Res “ https doi101007s0044 Ashrafizadeh M Zarrabi A Hushmandi K Kalantari M Mohammadinejad R Javaheri T Sethi G Association of the epithelialmesenchymal transition EMT with cisplatin resistance Int J Mol Sci https doi103390ijms2 Liao H Bai Y Qiu S Zheng L Huang L Liu T Wang X Liu Y Xu N Yan X Guo H MiR203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelialmesenchymal transition via SNAI2 Oncotarget “ https doi1018632 oncot arget Gao Y Li H Han Q Li Y Wang T Huang C Mao Y Wang X Zhang Q Tian J Irwin DM Tan H Guo H Overexpression of DUSP6 enhances chemotherapyresistance of ovarian epithelial cancer by regulating the ERK signaling pathway J Cancer “ https doi107150jca37267 James NE Beffa L Oliver MT Bstadt AD Emerson JB Chichester CO Yano N Freiman RN DiSilvestro PA Ribeiro JR Inhibition of DUSP6 sensitizes ovarian cancer cells to chemotherapeutic agents via regulation of ERK signaling response genes Oncotarget “ Clark MJ Homer N O™Connor BD Chen Z Eskin A Lee H Merriman B Nelson SF U87MG decoded the genomic sequence of a cytogenetically aberrant human cancer cell line PLoS Genet 201061e1000832 https doi101371journ alpgen10008 Publisher™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

This study was performed assess the clinical outcomes of elderly patients withosteoporotic femoral neck fractures FNFs AOOTA 31BC treated by initial uncementedtotal hip arthroplasty UTA or cemented total hip arthroplasty CTAMethods This study involved consecutive elderly patients with osteoporotic FNFs AOOTA31BC treated by initial UTA or CTA in our medical centre from to The primaryoutcomes were the Harris hip score HHS and the rates of revision loosening periprostheticfracture and dislocationResults In total patients were included in the final analysis UTA n¼ CTA n¼ The mean followup duration was months range “ months The mean HHS was1Department of Microsurgery Trauma and Hand SurgeryThe First Affiliated Hospital Sun Yatsen UniversityGuangzhou China2Department of Pediatrics The First Affiliated HospitalSun Yatsen University Guangzhou China3Department of Orthopaedics The First AffiliatedHospital Sun Yatsen University Guangzhou China4Department of Orthopaedics The Third People™sHospital of Wuxi Jiangsu Province The Affiliated Hospitalof Jiangnan University Wuxi China5Department of Urology The First Affiliated Hospital SunYatsen University Guangzhou ChinaThese authors contributed equally to this workCorresponding authorsJunxing Ye Department of Orthopeadics The ThirdPeople™s Hospital of Wuxi Jiangsu Province The AffiliatedHospital of Jiangnan University No Xingyuan NorthRoad Liangxi District Wuxi Jiangsu ChinaEmail yejunxing0514163comJintao Zhuang Department of Urology The First AffiliatedHospital Sun Yatsen University No Zhongshan 2ndRoad Yuexiu District Guangzhou ChinaEmail brianzg86163comCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cJournal of International Medical Research 06 for UTA and 06 for CTA Significant dissimilarities were detected in therates of revision loosening and periprosthetic fracture between UTA and CTA vs vs and vs respectively A significant difference was also detected inthe probability of revision between the two groupsConclusion Elderly patients with osteoporotic FNFs AOOTA 31BC treated with CTA showgreater improvements in functional outcomes and key orthopaedic complications than thosetreated with UTAKeywordsFemoral neck fracture arthroplasty outcome complication retrospective osteoporosisDate received December accepted July Introduction31BCAOOTAfemoral neck fracturesManagement ofin elderlyFNFspatients is still undergoing considerableresearch12 Uncemented total hip arthroplasty UTA or cemented total hip arthroplasty CTA for displaced FNFs tendsto be a recognised surgical strategy3“Comparisons between UTA and CTA forelderly individuals with an FNF generallyfavour CTA this is primarily attributed tothe exceptional clinical outcomes of CTA interms of relieving pain and improving dailyactivities as well as the higher rate of majororthopaedic complications ie revisionloosening periprosthetic fracture and dislocation associated with UTA4 Howeverrecent studies of UTA in elderly individualshave demonstrated encouraging shorttermclinical outcomes67 Moreover cementedprosthesis syndrome tends to occur morefrequently in CTA than UTA8 Cementedprosthesis syndrome theoretically poses asignificantlifealthough the specific probability of thisthreat has not been calculated89 Hencewhether to utilise CTA for elderly individuals may present the clinician with a dilemma9 The lack of consensus regarding whichtechnique UTA or CTA is preferable fortreating FNFs AOOTA 31BC in elderlyindividuals is related to the remarkableto the patient™sthreatdistinction in clinical outcomes betweenthe two types of implants610Most previousconcentrated medicalstudies have involvedhighlycentresand several brands of prostheses356Additionally shortterm followup is commonplace in these studies To overcomethese drawbacks of previous studies and tocompare the midterm results of the twoprostheses we assessed the clinical outcomesof elderly patients with osteoporotic FNFsAOOTA 31BC treated with initial UTAor CTA with a mean followup of yearsMaterials and methodsStudy populationtheandrequirementEthical approval was obtained from theFirst Affiliated Hospital of Sun YatsenUniversityforinformed consent was waived by theInvestigational Review Board Consecutiveelderly individuals with the principle diagnosis of an FNF AOOTA 31BC whounderwentinitial UTA or CTA from March to March and forwhom detailed information was availablethroughoutidentifiedfrom the orthopaedics department of theFirst Affiliated Hospital Sun YatsenUniversity The manufacturer details offollowup were 0cMao et alTable Manufacturer details of stems and cupsemployed in the arthroplasty proceduresProcedureStemCupCORAIL1Exeter3Exeter3REFLECTIONUncemented2UTA n¼ CTA n¼ 1DePuy Synthes Warsaw IN USA2Smith Nephew London UK3Stryker Corporation Kalamazoo MI USAUTA uncemented total hip arthroplasty CTA cementedtotal hip arthroplastyclosed FNFsthe stems and cups employed in the arthroplasty are shown in Table The surgicalprocedure and postoperative rehabilitationprotocol were described in our previouslypublished study11 The inclusion criteriawereAOOTA 31BCactive and cognitively intact patients ageof 15 years independently mobile priorto the injury and a bone mineral densityTscore of at the femoral neck Themajor exclusion criteria were multiple fractures or contralateral limb fractures pathological FNFs lower limb dyskinesia priorto surgery cancer planned surgery polytrauma severe comorbidities eg thyroiddisorder with calcium and phosphorusmetabolism disorderwithcomplications drug abuse affecting bonehealing or bone metabolism early interruption of followup months and cognitive impairment Clinical and radiographicassessments were performed at and months after surgery and every monthsthereafter The primary outcomes were theHarris hip score HHS and the rates ofrevision loosening periprosthetic fractureand dislocationdiabetesStatistical analysisRevision was defined as partial or completereplacement of the prosthesis12 Looseningof the acetabulum andor stem componentstomographycompared usingas well as dislocation were defined based ona previous description13 Periprostheticconfirmed by Xray orfracture wascomputedexaminationContinuous data ie age bone mineraldensity body mass index followup timeand HHS wereanindependentsamples t test and categoricalvariables ie sex side [leftright] fracturetype comorbidities mechanism of injuryAmerican Society of Anesthesiologists classification and major orthopaedic complications were compared using the chisquaretest or the Mann“Whitney test A Kaplan“Meier survival curve was used to assess theprobability of revision Hazard ratios werecalculated using a Cox proportional hazards model The significance threshold wasset at p The statistical analysis wasperformed using SPSS IBM CorpArmonk NY USAResultsIn total consecutive patientsarthroplasties with an FNF AOOTA31BC who underwentinitial UTA orCTA met our inclusion criteria and wereincluded for analysis Figure The meanfollowup duration was months range“ months The patients™ mean age was 06 years for UTA and 06 for CTA The mean body mass indexfor UTA andwas 06 kgm2 for CTA The patients™baselinesimilarbetween the two groups Table 06 kgm2characteristics werePrimary outcomesImproved functional outcomes were notedin both groups as indicated by the HHSUTA 06 prior to surgery vs 06 at final analysis p CTA 06 prior to surgery vs 06 at final analysis p At the end of followup the HHS was 0cJournal of International Medical ResearchFigure Flow diagram exhibiting methods for identifying patients with FNFs AOOTA 31BC whounderwent an initial UTA or CTAFNFs femoral neck fractures UTA uncemented total hip arthroplasty CTA cemented total hiparthroplastysignificantly different between the twoUTA 06 vs CTAgroups 06 p¼ and patients whounderwent CTA had higherfunctionalscores than those who underwent UTANo distinct betweengroup differenceswere observed at any time point before months postoperatively Table No early year postoperative complications were detectedincluding revisionloosening periprosthetic fracture or dislocation The rate of key orthopaedic complications wasfor UTAand for CTA p Table In the UTA group patients underwent revision UTA developed prosthesis loosening developed periprosthetic fracturesand developed prosthesis dislocation In the CTA group patientsunderwent revision UTA developed prosthesis loosening developed periprosthetic fractures and developed prosthesis dislocation The average time interval from the initial surgery torevision UTA was months range “months for UTA and months rangefor CTA p¼ “ monthsSignificant differences in revision looseningand periprosthetic fracture were observedbetween the UTA and CTA groups revision vs p¼ loosening 0cMao et alTable Patient demographics and outcomesVariableSex femalemaleAge yearsBMI kgm2BMDSide leftrightFNFs AOOTA type31B31CComorbiditiesDiabetes mellitusHypertensionCerebrovascular diseaseMechanism of FNFsTraffic accidentFallingTamping accidentASA classificationUTA n¼ 06 06 06 06 HHS prior to surgeryData are presented as n n or mean 06 standard deviationpvalueCTA n¼ 06 06 06 06 UTA uncemented total hip arthroplasty CTA cemented total hip arthroplasty HHS Harris hip score ASA AmericanSociety of Anesthesiologists BMI body mass index BMD bone mineral density FNFs femoral neck fracturesTable Comparison of hip functional scoresMonths postoperativelyFinal followupData are presented as mean 06 standard deviationUTA n¼ 06 06 06 06 06 06 06 06 CTA n¼ 06 06 06 06 06 06 06 06 pvalueStatistically significantUTA uncemented total hip arthroplasty CTA cemented total hip arthroplasty vs p¼ and periprosthetic fracture vs p¼ respectively A significant difference in theprobability of revision was also detectedbetween the groups hazard ratio interval confidence“p¼ Figure No significant difference was found in the rate of prosthesis dislocation between the UTA and CTA groups vs respectively 0cJournal of International Medical ResearchTable Rates of key orthopaedic complicationsComplicationsProsthesis revisionProsthesis looseningPeriprosthetic fractureDislocationUTA n¼ CTA n¼ pvalueData are presented as n Statistically significantUTA uncemented total hip arthroplasty CTA cemented total hip arthroplastyFigure Kaplan“Meier curves showing probability of revision after primary surgery HR was calculatedper the Cox proportional hazards model with age sex American Society of Anesthesiologists classificationbody mass index bone mineral density and femoral neck fracture type as covariates and surgery as the timedependent factorHR hazard ratio CI confidence interval UTA uncemented total hip arthroplasty CTA cemented total hiparthroplastysurgeryProbableDiscussionThis review characterised the outcomes of asolitary brand of a total hip arthroplastyimplant during a mean followup of years in elderly patients with osteoporoticFNFs AOOTA 31BC The data demonstrated that patients treated with CTAshowed better improvements in functionaloutcomes and key orthopaedic complications than those treated with UTAThe current findings are consistent withprevious studies361415 Although the betterfunctional outcomes and lower rates ofrevision loosening periprosthetic fractureand dislocation are apt to favour CTA nosignificant betweengroup differences in theHHS were detected during the initial yearsafterexplanationsinclude the timedependent clinical efficacyof the implants and the properties of theprostheses34616 Whether UTA or CTA ispreferable in elderly patients with a discontroversial6917placed FNF remainsA recent retrospective study involving patients with an FNF AOOTA 31B whounderwent primary unilateral UTA or CTAshowed that the mean HHS was 06 for CTA and 06 for UTAp¼ A singleblinded randomisedcontrolled trial CHANCEtrial involving individuals treated with an uncementedor cemented tapered hydroxyapatitecoatedfemoral stem and a cemented cup demonstratedtaperedhydroxyapatitecoated femoral stem andcementedthatthe 0cMao et alcemented cup provided better functionalresultsthan the uncemented taperedhydroxyapatitecoated femoral stem andcemented cup16ratesrevisionthe bone microstructureIn the present study the Kaplan“Meiersurvival curve demonstrated that at the2year analysis neither group showed evidence of a target event and no significantbetweengroup differences were found inofthelooseningfracture or dislocationperiprostheticNeverthelessit would be interesting toexplore whether the prosthesis materialinfluencesthepeak effect and the duration of the effectand if so what mechanisms affect the bonemicrostructure and whether there is a wayto change the outcome by blocking thiseffect during a 2year followup We currently have one option for prevention oravoidance of adverse events and thesechanges in treatment strategies may play akey role in improving the clinical resultsif the effects of the prosthesis materialitself cannot be blocked18 There is still alack of consensus on standards for prosthesis revision in this context19When assessing the impact of CTA onthe target events we did not observe anincreased incidence of severe orthopaediccomplications other than the complicationsmentioned in this study In one systematicreview the authors presumed that CTA wassuperior to UTA with respect to functionalscores and tolerable orthopaedic complications20 We obtained analogous results interms of hiprelated complications andfunctional scores Multicentre hip arthroplasty data indicate that UTA remains ahighrisk factor for late revision looseningand periprosthetic fractures810 The notabledissimilarities in the results of these variousstudies on hiprelated complications may belargely attributed to the design of the prosthesis prosthesis size and material selection and the surgeon™s experience46affected theThis study has several limitations It hada small sample and its retrospective designis association with some inherent disadvantages We did not stratify the patientsaccording to fracture type or sex In addition the potential comorbidities betweengroups were not well exposed and compared The statistical power used to addressdifferences between the groups was insufficient Differences in the patients™ baselinedata may haveresultsFurthermore our analysis did not determine whether the deaths were instigatedby bone cement The risk of hiprelatedcomplications was not analysed The survivalcurve of other prosthesisrelatedcomplications was estimated using theKaplan“Meier method and competitiverisks ie death could have affected thesurvival of the prosthesis Patients whodied lostrevisionHence the revision rate might have beenunderestimated during this long followupwith a fairly high mortality ratethe opportunity forevidenceIn conclusion the findings described inthe current review uphold an increasingbody ofthat CTA provideshigher functional scores and lower rates ofhiprelated complications than does UTAin elderly patients with osteoporotic displaced FNFs AOOTA 31BC In thiscontext we recommend CTA for the treatment of such FNFs Our findings may beconducive to alleviating continuing debateregarding which prosthesis UTA or CTAis more suitable for the elderly populationA future prospective study may be essentialto confirm whether our conclusion continues to be acceptable as the followup timeincreasesDeclaration of conflicting interestThe authors declare that there is no conflict ofinterest 0cFundingThis research received no specific grant from anyfunding agency in the public commercial ornotforprofit sectorsORCID iDsWeiguang YuGuowei HanReferencesorcid00000001orcid00000003 Chammout G Muren O Laurencikas Eet al More complications with uncementedthan cemented femoral stems in total hipreplacement for displaced femoral neck fractures in the elderly a singleblinded randomized controlled trial with patientsActa Orthop “ Gjertsen JE Lie SA Fevang JM et al Totalhip replacement after femoral neck fracturesin elderly patients results of fracturesreported to the Norwegian ArthroplastyRegister Acta Orthop “ Hailer NP Garellick G and Karrholm JUncemented and cemented primary totalhip arthroplastyin the Swedish HipArthroplasty Registerof operations Acta Orthop “evaluation Makela KT Eskelinen A Paavolainen Pet al Cementless total hip arthroplasty forprimary osteoarthritis in patients aged years and older results ofthe mostcommon cementless designs compared tocemented reference implants in the FinnishArthroplasty Register Acta Orthop “ YliKyyny T Sund R Heinanen M et alCemented or uncemented hemiarthroplastyfor the treatment of femoral neck fracturesActa Orthop “ Yang C Han XL Wang J et al Cementedversus uncemented femoral component totalhip arthroplasty in elderly patients with primary osteoporosisretrospective analysiswith 5year followup J Int Med Res “Journal of International Medical Research Solarino G Zagra L Piazzolla A et alceramiconResults of consecutiveceramic cementless hip arthroplastiesinpatients up to years of age a yearsof followup study J Arthroplasty S232“S237 Hanly RJ Whitehouse SL Lorimer MFet al The outcome of cemented acetabularcomponents in total hip arthroplasty forosteoarthritis defines a proficiency thresholdresults of cases from the AustralianOrthopaedic Association NationalJointReplacement Registry J Arthroplasty “ Imam MA Shehata MSA Elsehili A et alContemporary cemented versus uncementedhemiarthroplasty for the treatment of displaced intracapsular hip fractures a metaanalysis offortytwo thousand fortysixhips Int Orthop “ Jameson SS Baker PN Mason J et al Thedesign of the acetabular component and sizeof the femoral head influence the risk of revision following singlebrand cementedhip replacements a retrospective cohortstudy of mediumterm data from a nationaljoint registry J Bone Joint Surg Br 94B “ Zeng XS Zhan K Zhang LL et alConversion to total hip arthroplasty afterfailed proximal femoral nail antirotationsor dynamic hip screw fixations for stableintertrochanteric femur fractures a retrospective study with a minimum followupof years BMC Musculoskelet Disord Johnson RL Abdel MP Frank RD et alImpact of frailty on outcomes after primaryandarthroplastyJ Arthroplasty “64e5revisiontotalhip Chen KH Tsai SW Wu PK et al Partialcomponentretained twostage reconstruction for chronic infection after uncementedtotal hip arthroplasty results of sixteen casesafter five years of followup Int Orthop “ DeAngelis JP Ademi A Staff I et alCemented versus uncemented hemiarthroplasty for displaced femoral neck fracturesa prospective randomized trial with early 0cMao et alfollowup J Orthop Trauma “ Rolfson O Donahue GS Hallsten M et alPatientreported outcomes in cemented anduncemented total hip replacements Hip Int “ Chammout G Muren O Boden H et alCemented compared to uncemented femoralstems in total hip replacement for displacedfemoral neck fractures in the elderly studyprotocol for a singleblinded randomizedCHANCEtrial BMCcontrolled trialMusculoskelet Disord Liu T Hua X Yu W et al Longtermfollowup outcomes for patients undergoingprimary total hip arthroplasty with uncemented versus cemented femoral components a retrospective observational studywith5year minimum followupJ Orthop Surg Res a Engesaeter LB Espehaug B Lie SA et alDoes cement increase the risk of infection inprimary total hip arthroplasty Revisionrates in cemented and uncementedprimary THAs followed for years inthe Norwegian Arthroplasty Register ActaOrthop “ Schmale GA Lachiewicz PF and Kelley SSEarly failure of revision total hip arthroplasty with cemented precoated femoralcomponents Comparison with uncementedcomponents at to years J Arthroplasty “ Azegami S Gurusamy KS and Parker MJCemented versus uncemented hemiarthroplasty for hip fractures a systematic reviewof randomised controlled trials Hip Int “ 0c'

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

"The principal function of iodine acts on thyroid function but in recent years the role of iodinedeficiency in metabolism has also been gradually revealed We aimed to investigate the current status of iodizedsalt consumption and urinary iodine concentration UIC in an urban Chinese population with type diabetes andto further explore whether UIC was associated with diabetic microvascular complicationsMethods Four thousand five hundred fiftynine subjects with diabetes from communities in downtownShanghai were enrolled in the crosssectional Metal Study in UIC was detected using an inductively coupledplasmamass spectrometer Diabetic kidney disease DKD was defined as urinary albumintocreatinine ratioUACR mgg or estimated glomerular filtration rate mLmin173 m2 Diabetic retinopathy DR wasevaluated by highquality fundus photographs and was remotely read by ophthalmologistResults The median UIC of subjects with diabetes was μgL “ in downtown Shanghai Among allthe subjects consumed noniodized salt and were iodine deficient Iodine deficiency UIC μgLwas associated with an increased odds of DKD OR 95CI “ after adjustment for age sex educationcurrent smokers BMI HbA1c duration of diabetes dyslipidemia thyroidstimulating hormone and free thyroxineNo association was observed between UIC and DR after multivariable adjustmentConclusions A concerning number of subjects with diabetes consumed noniodized salt and suffered from iodinedeficiency in coastal regions of China Low UIC might be a risk factor for DKD which should be further confirmedby longitudinal prospective studiesKeywords Iodized salt Type diabetes Diabetic kidney disease Urinary iodine concentration Epidemiology Correspondence wnj486126com luyingli2008126com Chi Chen and Yi Chen contributed equally to this workInstitute and Department of Endocrinology and Metabolism Shanghai NinthPeople™s Hospital Shanghai JiaoTong University School of MedicineShanghai China 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 Nutrition Metabolism Page of IntroductionType Diabetes Mellitus T2DM has become a seriousglobal health care burden causing microvascular complications which are associated with increased disabilityreduced quality of life and life expectancy [ ] It wasestimated that there were million cases of adult diabetes worldwide in and the number was projectedto increase to million by [] leading to highincidence and prevalence of microvascular complications Approximately one third with T2DM will developdiabetic retinopathy DR and one quarter will developdiabetic kidney disease DKD [] Thus it is critical toidentify and control some novel modifiable risk factorscontributing to microvascular complications in patientswith T2DMIodine is an indispensible micronutrient for the synthesis of thyroid hormones Iodine deficiency ID in early lifeimpairs neurodevelopment and also has many adverse effects throughout various life stages [] Over the past years substantial progress has been achieved in the worldwide effort to eliminate iodine deficiency disorders IDDby salt iodization program However this program is vulnerable and requires a longterm commitment from governments In several countries where ID had been onceeliminated salt iodization programs were discontinuedand ID has now reappeared []The principal function of iodine acts on thyroid function but in recent years the role of ID in metabolismhas also been gradually revealed Analysis of data fromthe National Health and Nutrition Survey NHANES“ found that in US adults low urinary iodineconcentration UIC was associated with dyslipidemia[] and coronary artery disease [] Moreover O S AlAttas [] reported that UIC is markedly decreasedin T2DM and urinary iodine was negatively associatedwith insulin resistance in patients with T2DM Most recently Mingyue Jin [] also found that at a lowerUIC μgL the prevalence of diabetes significantlyincreased relative to an UIC of “ μgL Animalstudies also showed that iodine supplementation couldreduce the blood glucose levels and improve the insulinsensitivity in goats [] However the role of iodine nutrition on diabetic microvascular complications has notbeen studiedThe aim of this study was to investigate the currentstatus of iodized salt consumption and UICs in an urbanChinese population with T2DMand additionallywhether UIC is associated with diabetic microvascularcomplications including DKD and DRMethodsStudy populationThe crosssectional METAL study Environmental Pollutant Exposure and Metabolic Diseases in Shanghaiwwwchictrcn ChiCTR1800017573 was launchedto investigate the association between iodine nutritionand microvascular complications in Chinese adults withdiabetes We recruited study participants from sevencommunities in Huangpu and Pudong new districtShanghai China Huangpu district located in downtownShanghai is the administrative economic and culturalcenter of the metropolitan coastal city [] Pudong newdistrict is the symbol of China™s reform and ingup[] We randomly selected half of patients with diabetesfrom the registration platform in each communityhealthcare center Chinese citizens ‰¥ years old whohad lived in their current area for ‰¥ months were included In August a total of subjects withT2DM who were “ years of age received an examination Participants with missing UIC values n were excluded Finally participants were involvedin the present analysisThe study received ethical approval from the EthicsCommittee of Shanghai Ninth People™s Hospital Shanghai Jiao Tong University School of Medicine All procedures followed were abided by the ethical guidelines ofthe Declaration of Helsinki as reflected in a prioriapproval by the appropriate institutional review committee Informed consent was received from all participantsincluded in the study prior to the data collectionMeasurementsinThe same welltrained and experienced personnelSPECTChina study [“] used a questionnaire to collect information on sociodemographic characteristicseducation medical history family history and lifestylerisk factors Weight and height were measured using abalance beam and a vertical ruler in light clothing andwithout shoes Body mass index BMI was calculated asthe ratio of weight in kilograms divided by height in meters squared Current smoking was defined as havingsmoked at least cigarettes in one™s lifetime and currently smoking cigarettes [] Especially œFor the pastthree years which type of salt was used in your familywas applied to collect information about type of saltThree options for this item were provided only iodized salt only noniodized salt bothBlood samples were drawn between am and am after an overnight fast Blood was refrigerated immediately after phlebotomy and in hours it was centrifugated and the serum was aliquoted and frozen in acentrallaboratory Glycated hemoglobin HbA1c wasmeasured by highperformance liquid chromatographyMQ2000PT Medconn Shanghai China Fastingplasma glucose serum creatininetotalcholesterol high HDL and lowdensity lipoproteinLDL were performed with a Beckman Coulter AU Brea USA Serum thyroidstimulating hormone TSHtriglycerides 0cChen Nutrition Metabolism Page of and free thyroxine FT4 were measured by electrochemiluminescence Roche E601 GermanyMorning fasting spot urine samples collected were refrigerated immediately and frozen at ˆ’ °C in hoursUIC was detected using an inductively coupled plasmamass spectrometer ICPMS No 7700x Agilent Technologies Inc USA The concentrations of urine albuminand creatinine were determined with a Beckman CoulterAU Brea USA using a turbidimetric immunoassayand an enzymatic method respectively Urinary albumintocreatinine ratio UACR was calculated as the urinaryalbumin concentrations divided by the urinary creatinineconcentrations and expressed in mggDR screening was evaluated by mydriatic binocular indirect ophthalmoscopy Topcon TRCNW400 NonMydriatic Retinal Camera Oakland USA Fundus photographs were read by an experienced ophthalmologistspecialized in retinaOutcome definitionDyslipidemia was defined as total cholesterol ‰¥mmolL mgdL triglycerides ‰¥ mmolL mgdL LDL ‰¥ mmolL mgdL HDL mmolL mgdL or selfreported previous diagnosisof hyperlipidemia by physicians according to the modified National Cholesterol Education ProgramAdultTreatment Panel IIIThe estimated glomerular filtration rate eGFR was calculated using the Chronic Kidney Disease EpidemiologyCollaboration CKDEPI equation for œAsian origin []As suggested by American Diabetes Association ADAhigh UACR was defined as UACR ‰¥ mgg reducedeGFR as eGFR mlmin173m2 and DKD as UACR mgg or eGFR mLmin173 m2 []The internationally accepted DR classification by theœGlobal Diabetic Retinopathy Project Group in was applied [] The classification was no retinopathynonproliferative DR intraretinal microaneurysmshemorrhages venous beading prominent microvascularabnormalities and proliferative DR neovascularizationor vitreouspreretinal hemorrhagesStatistical analysisStatistical analysis was run with SPSS IBM Corporation Armonk NY USA General characteristics arepresented as median with the interquartile range IQRfor continuous variables or as proportion for categoricalvariables MannWhitney U test and the KruskalWallistest were used for comparison of two or more groups ofnonnormally distributed data Pearson™s χ2 tests wereperformed to compare categorical variablesThe associations of UIC with elevation of UACR reduction of eGFR DKD and DR were analyzed with logistic regression analyses The results are presented as oddsratio OR and confidence intervals CIs Model was unadjusted Model was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4Table General characteristics of study participants by UIC categoriesUrinary iodineAge yrAdequate “ “ “ “ “ “Low “ “ “ “ “ “Women UIC μgLFPG mmolLHbA1c BMI kgm2Duration of diabetes yrCurrent smokers Beyond high school education TSH mIULFT4 pmolLBlood lipidsTotal cholesterol mmolLLDLC mmolLHDLC mmolLTriglycerides mmolLMore than adequate “ “ “ “ “ “ “ “Excessive “ “ “ “ “ “ “ “ “ “ “ “P “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Data are summarized as median interquartile range for continuous variables or as number with proportion for categorical variablesUIC Urinary iodine concentration FPG Fasting plasma glucose HbA1c Glycated hemoglobin BMI Body mass index LDLC Low density lipoprotein cholesterol HDLC High density lipoprotein cholesterol TSH Thyroidstimulating hormone FT4 Free thyroxineUrinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ‰¥ μgL 0cChen Nutrition Metabolism Page of ResultsGeneral characteristics of the study populationThe general characteristics are presented in Table The mean age of the study population was nearlyone half were women About of the studypopulation were overweight or obese BMI ‰¥ kgm2and were current smokers The percentage of aneducational level beyond high school was and theaverage duration of diabetes was yearsCompared to those with adequate iodine nutritionsubjects with ID were slightly older and were more likelyto be women These subjects also had higher TSH lowerBMI and a lower percentage of current smokers Inaddition subjects with more than adequate and excessiodine nutrition were slightly younger and had higherBMI but comparable TSHUrinary iodine concentration and type of salt intake inthe populationThe distribution of UICs in the study population is presented in Fig The median 25th“75th percentile UICof subjects with diabetes was μgL “ indowntown Shanghai which falls within the range of μgL that WHOUNICEFICCIDD categorize asadequate Urinary iodine measurements indicative of IDUIC μgL were present in of the studypopulation Meanwhile and of the populationshowed more than adequate UIC “ μgL andexcess iodine intake UIC ‰¥ μgL respectivelyThe distribution of type of salt intake is presented inFig As high as of the study population consumed noniodized salt consumed iodized saltand consumed both Logistic regression analysisshowed that compared to those who consumed iodizedsalt subjects consumed noniodized salt were morelikely to be women OR 95CI “ and havea higher educational attainment OR 95CI “ but a comparable age OR 95CI “Association of urinary iodine concentration with elevationof UACR reduction of eGFR and DKDThe association of UIC with elevation of UACR reduction of eGFR and DKD is shown in Table Comparedwith those with adequate iodine nutrition subjects withID had an increased risk of elevation of UACR OR 95CI “ reduction of eGFR OR 95CI“ and DKD OR 95CI “ Adjustment for age sex education current smokers BMIHbA1C duration of diabetes dyslipidemia TSH andFT4 did not attenuate the association of ID with UACRand DKD However multivariable adjustment weakenedthe association between ID and reduction of eGFR further such that it was no longer significant Meanwhilesubjects with more than adequate and excess iodine nutrition did not have an increased risk of elevation ofUACR reduction of eGFR and DKD after multivariableadjustmentAssociation of urinary iodine concentration with DRTable presents the association of UIC with nonproliferative and proliferative DR Compared with thosewith adequate iodine nutritionthe ORs of nonproliferative and proliferative DR in subjects with morethan adequate iodine nutrition were 95CI “Fig Distribution of UICs in the study population 0cChen Nutrition Metabolism Page of Fig Distribution of type of salt consumed in the study populationthan adequate and 95CI “ respectively Multivariable adjustment weakened the association betweenmoreiodine nutrition and nonproliferative DR further such that the association was nolonger significant There was no significant associationobserved between DR and ID and excessiodinenutritionDiscussionIn this study among over communitydwellingChinese adults with diabetes we found that of thesubjects consumed noniodized salt and had IDIodine deficiency was significantly associated with ahigher prevalence of elevated UACR and DKDindependently of age sex education current smokers BMIHbA1C duration of diabetes dyslipidemia TSH andFT4 To the best of our knowledge this is the first studyto investigate the current status of iodized salt consumption and iodine nutrition status in a relatively largepopulation with diabetes and further investigate the association between UIC and diabetic microvascularcomplicationsChina was once severely affected by IDD and hence amandatory Universal Salt Iodization USI program wasTable Association of urinary iodine with elevation of UACR and reduction of eGFRUrinary iodineHigh UACRAdequateLowMore than adequateExcessivePrevalenceOdds RatioModel Model Reduced eGFRPrevalence Odds RatioModel Model DKDPrevalence Odds Ratio Ref Ref Ref Ref “ “ “ “ “ “ “ “ “ “ “ “Model Model Ref Ref “ “Data are expressed as odds ratios 95CI Logistic regression analyses were used for the association of urinary iodine with elevation of UACR reduction of eGFRand DKD P Model was unadjustedModel was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4High UACR was defined as UACR ‰¥ mgg reduced eGFR as eGFR mlmin173 m2 and DKD as UACR mgg or eGFR mLmin173 m2Urinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ‰¥ μgL “ “ “ “ 0cChen Nutrition Metabolism Page of Table Association of urinary iodine with DRUrinary iodineNonproliferative DRAdequatePrevalence Odds RatioModel Model Proliferative DRPrevalence Odds RatioModel Model Ref Ref Ref RefLowMore than adequateExcessive “ “ “ “ “ “ “ “ “ “ “ “P Data are expressed as odds ratios 95CI Logistic regression analyses were used for the association of urinary iodine with DRModel was unadjustedModel was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4Urinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ‰¥ μgLintroduced in and was successful in eliminatingIDD However since the prevalence of thyroid diseaseshas markedly increased in recent years some concernsabout the USI have circulated especially among coastalresidents in urban areas [] In the present analysis themedian UIC of residents with diabetes in downtownShanghai has fallen to marginal levels of iodine sufficiency μgL and more surprisingly more thanhalf of the subjects consumed noniodized saltand were iodine deficient Compared with a studyconducted by the Shanghai Municipal Center for DiseaseControl and Prevention CDC in of participants used iodized salt and were iodine deficientat that time [] During “ Zhongyan Shanand her colleagues performed a crosssectional study ineastern and central China and reported that the medianUIC was μgL in schoolaged children and μgLin the total cohort population [] Our previous studyconducted on the general population found that consumed noniodized salt in the urban area of Shanghai in []The present study indicates that an increasing numberof urban residents in downtown Shanghai prefer to usenoniodized salt in recent years and suffer from IDWomen and those with a higher educational level weremore tended to consume noniodized salt WhyChanges in the reported spectrum and growing incidence of thyroid disorders have been linked to the increased iodine intake resulting from USI in the localmedia and international medical literature [] Thosewith high educational attainment were more likely to beconfused by these information and worry about theirthyroid health and call for liberalizing provincial controlof sales of noniodized salt Formerly needed a prescriptionthe sale of noniodized salt has now beenunofficially allowed by some coastal city authorities []In addition T2DM is associated with an increased riskof multiple thyroid disorders such as thyroid nodule[] thyroid cancer [] and autoimmune thyroid diseases [] Since the prevalence of thyroid abnormalitieswere found to be much higher in females than males[] it is reasonable to deduce that these women wouldhave a higher tendency towards consuming noniodizedsalt after diagnosis of thyroid abnormalitiesseafood alone to provide sufficientConsidering that Shanghai is a coastal city local residents believe that they should never suffer from IDDwith high iodineenriched aquatic products consumption However it may not be true to rely on consumption ofiodineActually the environmental levels of iodine in Shanghaiare deficient μgL [] Furthermore based on theresearch initiated by Shanghai Municipal CDC iodizedsalt contributed of the total dietary iodine inShanghai[] Aquatic products which residentsthought to be rich in iodine accounted for only ofthe total dietary iodine []Thus iodized salt is still themain source foriodine supplementation in coastalpopulationsDKD wascommonlydefinedby ADA asœUACR‰¥30mgg or eGFR60 mlmin per m2 []We found that ID was associated with elevated UACRand DKD The mechanism underlying the association ofID with DKD is not yet fully understood Deficiency ofiodine could reduce thyroid hormone production andelevate TSH It has been reported that lower free triiodothyronine and elevated TSH have significant associationwith risk for albuminuria in T2DM [ ] Moreoverinadequate iodine intake is significantly correlated withan increase in oxidative stress [] Recent evidence hasshown thattumorinflammatory cytokinessuch as 0cChen Nutrition Metabolism Page of necrosis factoralpha and interleukin1 play a pivotalrole in the pathogenesis of DKD [] Therefore we suppose the possible mechanism may be via inflammatoryresponseIn view of the evergrowing prevalence of T2DM andDKD all over the world successive intervention in thislarge population can have important impact on publichealth ID unlike most micronutrient deficiencies is notrestricted to people in developing countries with poordiets Since salt iodization is simple effective and inexpensive the best strategy to control ID is addition ofiodine into salt in nearly all countries [] Monitoringiodine situation of people with diabetes is of critical significance and education programs to diabetes especiallywomen with high academic background may also include information of adequate iodine intake in our clinical practice Our study shed light on the possiblebeneficial effect of iodine supplementation in reducingalbuminuria in T2DM which warrants further investigation in welldesigned randomized controlled trialOur study benefited from its welldefined communitybased participants with a relatively large sample sizeSecond regarding the novelty our study is the first toprovide iodine status and linked iodine insufficiency toan increased risk of albuminuria in people with diabetesThird we used ICPMS to detect UIC in the presentanalysis which was considered as the goldstandardmethod [] There were also some limitations weshould acknowledge First no causal relationship couldbe determined due to the crosssectional design of thestudy and thus our findings need to be validated by longitudinal prospective studies Second although UIC isrecommended by the WHOICCIDDUNICEF for evaluation of iodine status at the population level and widelyused in largescale epidemiological studies [ ] thesingle spot urine measurement may not accurately assesslongterm iodine status at the individual level Actuallyinter and intraindividual variability exists in UIC []However the application of a large sample size from to subjects per subgroup may counteract thebias related to the use of only one casual urine sample[] Future followup studies collecting 24h urine specimens twice are needed to replicate the present resultsConclusionA large proportion of diabetic patients in downtownShanghai consumed noniodized salt and had ID IDmay increase the risk of DKD independent of thyroidfunction in diabetic patients Maintaining USI at an appropriate level is indispensable for diabetic patients Cohort and intervention studies as well as basic researchexploring the effect and mechanism of iodine supplementation on renal function are warrantedAbbreviationsT2DM Type Diabetes Mellitus DKD Diabetic kidney disease DR Diabeticretinopathy ID Iodine deficiency UIC Urinary iodine concentrationTSH Thyroidstimulating hormone BMI Body mass index OR Odds ratioCI Confidence interval UACR Urinary albumintocreatinine ratioeGFR Estimated glomerular filtration rateAcknowledgementsThe authors thank Xiaojin Wang and Bingshun Wang from the Departmentof Biostatistics and Shanghai Jiaotong University School of Medicine for dataprocessingAuthors™ contributionsYL and NW designed the study CC YC HZ FX BH WZ YW HWand NW conducted the research CC and YC analyzed the data and wrotethe manuscript CC and YC contributed equally The final manuscript wasread and approved by all authorsFundingThis study was supported by National Natural Science Foundation of China Science and Technology Commission of ShanghaiMunicipality the Fourth Round of ThreeYear Public HealthAction Plan of Shanghai by the Shanghai Municipal Commission of Healthand Family Planning 20164Y0079 Municipal Human Resources DevelopmentProgram for Outstanding Young Talents in Medical and Health Sciences inShanghai 2017YQ053 Fundamental research program funding of NinthPeople™s Hospital affiliated to Shanghai Jiao Tong university School of MedicineJYZZ099 Shanghai Sailing Program 20YF1423500Availability of data and materialsThe datasets during andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateEthical approval was obtained from the Ethics Committee of Shanghai NinthPeople™s Hospital Shanghai Jiao Tong University School of Medicine Writteninformed consent was received from all participantsConsent for publicationNACompeting interestsNo potential conflicts of interest relevant to this were reportedReceived May Accepted August ReferencesKahm K Laxy M Schneider U Rogowski WH Lhachimi SK Holle R Healthcare costs associated with incident complications in patients with type diabetes in Germany Diabetes Care “Chen C Chen Q Nie B Zhang H Zhai H Zhao L Trends in bonemineral density osteoporosis and oste ia among US adults withprediabetes Diabetes Care “Cho NH Shaw JE Karuranga S Huang Y da Rocha Fernandes JD OhlroggeAW IDF diabetes atlas global estimates of diabetes prevalence for and projections for Diabetes Res Clin Pract “Valencia WM Florez H How to prevent the microvascular complications oftype diabetes beyond glucose control Bmj 2017356i6505Zimmermann MB Boelaert K Iodine deficiency and thyroid disordersLancet Diabetes Endocrinol “Zimmermann MB Andersson M Assessment of iodine nutrition inpopulations past present and future Nutr Rev “Lee KW Shin D Song WO Low urinary iodine concentrations associatedwith dyslipidemia in US adults Nutrients Tran HV Erskine NA Kiefe CI Barton BA Lapane KL Do VTH Is lowiodine a risk factor for cardiovascular disease in Americans without thyroiddysfunction Findings from NHANES Nutr Metab Cardiovasc Dis “AlAttas OS AlDaghri NM Alkharfy KM Alokail MS AlJohani NJ AbdAlrahman SH Urinary iodine is associated with insulin resistance in 0cChen Nutrition Metabolism Page of Wu J Li X Tao Y Wang Y Peng Y Free triiodothyronine levels areassociated with diabetic nephropathy in Euthyroid patients with type diabetes Int J Endocrinol Vidal ZE Rufino SC Tlaxcalteco EH Trejo CH Campos RM Meza MN et alOxidative stress increased in pregnant women with iodine deficiency BiolTrace Elem Res “Jha JC Banal C Chow BS Cooper ME JandeleitDahm K Diabetes andkidney disease role of oxidative stress Antioxid Redox Signal “ Pearce EN Andersson M Zimmermann MB Global iodine nutrition wheredo we stand in Thyroid “Ittermann T Johner S Below H Leiterer M Thamm M Remer T et alInterlaboratory variability of urinary iodine measurements Clin Chem LabMed “Jeon MJ Kim WG Kwon H Kim M Park S Oh HS Excessive iodineintake and thyrotropin reference interval data from the Korean NationalHealth and nutrition examination survey Thyroid “Inoue K Leung AM Sugiyama T Tsujimoto T Makita N Nangaku M et alUrinary iodine concentration and mortality among US adults Thyroid“ Chen W Li X Guo X Shen J Tan L Lin L Urinary iodine excretion UIEestimated by iodinecreatinine ratio from spot urine in Chinese schoolagechildren Clin Endocrinol Oxf “ Andersen S Karmisholt J Pedersen KM Laurberg P Reliability of studies ofiodine intake and recommendations for number of samples in groups andin individuals Br J Nutr “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationssubjects with diabetes mellitus type Exp Clin Endocrinol Diabetes “Jin M Zhang Z Li Y Teng D Shi X Ba J Ushaped associationsbetween urinary iodine concentration and the prevalence of metabolicdisorders a crosssectional study Thyroid “ Nudda A Battacone G Bomboi G Floris B Decandia M Pulina G Effect ofdietary iodine on thyroid hormones and energy blood metabolites inlactating goats Animal “ Qu Y Zhuo L Li N Hu Y Chen W Zhou Y Prevalence of poststrokecognitive impairment in China a communitybased crosssectional studyPLoS One 201510e0122864Sun X Li Y Liu S Lou J Ding Y Liang H Enhanced performance ofcommunity health service centers during medical reforms in Pudong NewDistrict of Shanghai China A longitudinal survey PLoS One e0125469 Wang N Wang X Li Q Han B Chen Y Zhu C The famine exposure inearly life and metabolic syndrome in adulthood Clin Nutr “ Zhai H Chen C Wang N Chen Y Nie X Han B Blood lead level isassociated with nonalcoholic fatty liver disease in the Yangtze River Deltaregion of China in the context of rapid urbanization Environ Health Chen C Zhao L Ning Z Li Q Han B Cheng J Famine exposure inearly life is associated with visceral adipose dysfunction in adult females EurJ Nutr “ Chen C Zhai H Cheng J Weng P Chen Y Li Q Causal link betweenvitamin D and Total testosterone in men a Mendelian randomizationanalysis J Clin Endocrinol Metab “Stevens LA Claybon MA Schmid CH Chen J Horio M Imai E et alEvaluation of the chronic kidney disease epidemiology collaborationequation for estimating the glomerular filtration rate in multiple ethnicitiesKidney Int “Tuttle KR Bakris GL Bilous RW Chiang JL de Boer IH GoldsteinFuchs J Diabetic kidney disease a report from an ADA consensus conferenceDiabetes Care “ Wilkinson CP Ferris FL 3rd Klein RE Lee PP Agardh CD Davis M et alProposed international clinical diabetic retinopathy and diabetic macularedema disease severity scales Ophthalmology “ Zou Y Lou X Ding G Mo Z Zhu W Mao G Iodine nutritional status afterthe implementation of the new iodized salt concentration standard inZhejiang Province China BMC Public Health Zou S Wu F Guo C Song J Huang C Zhu Z Iodine nutrition and theprevalence of thyroid disease after salt iodization a crosssectional survey inShanghai a coastal area in China PLoS One 20127e40718Shan Z Chen L Lian X Liu C Shi B Shi L Iodine status and prevalenceof thyroid disorders after introduction of mandatory universal salt iodizationfor years in China a crosssectional study in cities Thyroid “ Chen C Xu H Chen Y Chen Y Li Q Hu J Iodized salt intake and itsassociation with urinary iodine thyroid peroxidase antibodies andthyroglobulin antibodies among urban Chinese Thyroid “Teng X Shan Z Chen Y Lai Y Yu J Shan L More than adequateiodine intake may increase subclinical hypothyroidism and autoimmunethyroiditis a crosssectional study based on two Chinese communities withdifferent iodine intake levels Eur J Endocrinol “ Wu Y Li X Chang S Liu L Zou S Hipgrave DB Variable iodine intakepersists in the context of universal salt iodization in China J Nutr “ Chen Y Zhu C Chen Y Wang N The Association of Thyroid Nodules withmetabolic status A crosssectional SPECTChina study Int J Endocrinol Qi J He P Yao H Song R Ma C Cao M Cancer risk among patientswith type diabetes a realworld study in Shanghai China SarfoKantanka O Sarfo FS Ansah EO Yorke E Akpalu J Nkum BC et alFrequency and determinants of thyroid autoimmunity in Ghanaian type diabetes patients a casecontrol study BMC Endocr Disord M B T G M P A P PC W Gender differences in thyroid system functionrelevance to bipolar disorder and its treatment Bipolar Disord “ Microvascular Complications and Foot Care Standards of medical Carein Diabetes2018 Diabetes Care 201841S105“s118 Yasuda T Kaneto H Kuroda A Yamamoto T Takahara M Naka T et alSubclinical hypothyroidism is independently associated with albuminuria inpeople with type diabetes Diabetes Res Clin Pract 201194e75“ 0c"

Thyroid_Cancer

IFNBased Biotherapeutics toHarness the Host AgainstFootAndMouth DiseaseGisselle N Medina Teresa de los Santos and Fayna DiazSan Segundo Plum Island Animal Disease Center PIADC ARS USDA Orient Point NY United States Kansas State University Collegeof Veterinary Medicine Manhattan KS United StatesFootandmouth disease FMD is a highly contagious vesicular disease of clovenhoofedanimals that severely constrains international trade of livestock and animal productsCurrently disease control measures include broad surveillance enforcement of sanitarypolicy and use of an inactivated vaccine While use of these measures has contributedto eliminating footandmouth disease virus FMDV from a vast area of the worldthe disease remains endemic in three continents and outbreaks occasionally appearin previously declared FMDfree zones causing economic and social devastationAmong others a very fast rate of viral replication and the need for days to achievevaccineinduced protection are the main limitations in controlling the disease Newfastacting antiviral strategies targeted to boost the innate immunity of the host to blockviral replication are needed Here we review the knowledge on the multiple strategiesFMDV has evolved to block the host innate immunity with particularly focus on the pastand current research toward the development of interferon IFNbased biotherapeuticsin relevant livestock speciesKeywords footandmouth disease virus FMDVIFNλ IFNωinterferon IFN antivirals biotherapeutics IFNα IFNÎINTRODUCTIONThe Disease FootAndMouth DiseaseFootandmouth disease FMD is one the most serious livestock diseases that aï¬ects clovenhoofedanimals including cattle swine sheep and goats as well as numerous species of wild species The disease displays high morbidity but is usually not lethal except when it aï¬ects young animalsthat may develop myocarditis Infected animals secrete copious amounts of virus ps beforethe onset of the clinical phase of the disease Typical FMD clinical signs include fever and theappearance of vesicular lesions on the tongue mouth feet and teats Among ruminants thatrecovered from the disease a relatively large number become asymptomatic virus carriers although it is not clear what is the contribution of these carrier animals to disease transmissionin nature The World anization for Animal Health OIE lists FMD as a reportable diseaseand therefore by law participating nations are required to inform the anization about all FMDoutbreaks OIE member nations with reported cases of FMD are forbidden to engage in tradingof FMDsusceptible animals or their products Thus the presence of FMD in a country can havesevere economic consequencesDiï¬erent interventions to control an FMD outbreak include restriction of susceptible animalmovement slaughter of infectedcontact animals decontamination of infected and surroundingEdited byMariano PrezFilgueiraNational Agricultural TechnologyInstitute ArgentinaReviewed byMargarita S¡izSevero Ochoa Molecular BiologyCenter CSICUAM SpainKenneth James GenoveseAgricultural Research ServiceUnited States Department ofAgriculture United StatesCorrespondenceGisselle N MedinagissellemedinausdagovTeresa de los SantosteresadelossantosusdagovFayna DiazSan SegundofaynadiazsansegundousdagovSpecialty sectionThis was submitted toVeterinary Infectious Diseasesa section of the journalFrontiers in Veterinary ScienceReceived April Accepted June Published August CitationMedina GN de los Santos T andDiazSan Segundo F Use ofIFNBased Biotherapeutics to Harnessthe Host Against FootAndMouthDisease Front Vet Sci 103389fvets202000465Frontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVpremises and vaccination Vaccination is an option used mostlyin countries in which FMD is endemic but diseasefree nationsprefer to abstain from such practice In general FMDfreecountries that occasionally opted to vaccinate to better containthe outbreak did slaughter all vaccinated animals to regaincommerce rights faster as occurred in the outbreak inthe UK and the Netherlands The current approvedFMD vaccine consists of purified chemically inactivated virus[binary ethylenimine BEItreated] formulated with oilbased oraluminum adjuvants that induces serotypespecific protection inapproximately days and it is applied with a boosting protocolfor ensuring longterm protection While this vaccine hasbeen successfully used for many decades leading to diseaseeradication of a vast area of our planet challenges remainFMD is endemic in most of Africa and Asia and occasionallyepizootics appear in South America or in nations that havebeen diseasefree for many years as it happened in the UKthe Netherlands South Korea Taiwan and Japan Novelvaccine technologies have been developed but to this end noneof them has fully addressed the limitations of the commerciallyavailable vaccine or is currently approved for massive use Alternatives or additional therapeutics that could complementor in some instances substitute for vaccination protocols includethe use of antivirals and biotherapeutics that act quickly priorto induction of vaccineinduced immunity The development ofsuch molecules requires a thorough understanding of the biologyof the virus and its intricate interactions particularly with theinnate immune molecular and cellular mechanisms evolved bythe hostThe AgentFootandmouth disease virus FMDV is a member of theAphthovirus genus within the Picornaviridae family and it is theetiologic agent of FMD The virus contains a singlestrandedRNA of positive polarity Its genome of ˆ¼ nucleotidesconsists of a long reading frame ORF flanked by a ² anda ²untranslated region UTR The ORF encodes a polyproteinof about amino acids which is processed by virusencodedproteases Processing results in the generation of precursors andmature protein products including four structural [1A VP4 1BVP2 1C VP3 1D VP1] and ten nonstructural NS proteins[Lpro 2A 2B 2C 3A three distinct copies of 3B VPg 3Cproand 3Dpol] Due to high genetic variability FMDV is categorizedin seven distinct serotypes A Asia1 C O and Southern AfricanTerritories “ SAT “ and numerous subtypes or topotypesUpon infection the virus spreads very rapidly usually achieving morbidity Depending on the route of entry less than tissue culture infectious doses are required to infect andcause disease in animals In fact FMDV is one of thefastest replicating RNA viruses in nature taking as little as “ h to induce cytopathic eï¬ects in susceptible tissue culture cellsOne could envisage that during FMDV replication almost everycomponent of the virus must play a role in dampening interferingcellular responses to allow such rapid virus replicationInnate Immunity and Interferon ActivationEarly protection against viralfundamentallymediated by the action of interferons IFNs the pillar moleculesof the innate immune system “ Expression of IFN isinfection istriggered by the recognition of molecular signatures collectivelynamed pathogenassociated molecular patterns PAMPs viacellular receptors pattern recognition receptors PRRs that candistinguish œself from nonself  molecules Figure Bindingof PAMPs to PRRs triggers a series of signal transduction eventsand posttranslational modifications PTMs phosphorylationubiquitination ISGylation etc that ultimately activate latenttranscription factors to induce IFN transcription Subsequentlysecreted IFN proteins bind to specific receptors on the plasmamembrane to activate in an autocrine and paracrine mannerdiscrete and overlapping cellular signal transduction pathwaysDepending on the cell type and aï¬ected tissue over specificIFNstimulated genes ISGs may be induced many of whichdisplay antiviral activity to control the viral infection There are three families of IFNs based on the specific receptorusage types I II and III Table “ Type I IFNsie IFNα and IFN signal through a heterodimeric receptorcomplex formed by IFNAR1IFNAR2 type II IFN IFNÎsignals through the complex IFNÎR1IFNÎR2 and type IIIIFNs bind the receptor complex IL28RαIL10R Despitethe receptor diï¬erencesthe three IFN families transducesignals through the Janus kinase JAK“signal transducer andactivator of transcription STAT pathway and type I and typeIII IFNs induce redundant responses Figure Overall therapid production of IFN helps to limit viral replication whilemodulating other immune functionsFOOTANDMOUTH DISEASE VIRUSIMPAIRS INNATE IMMUNITY MOLECULARINTERACTIONSRecognition of FMDV RNA by the host cell results in theestablishment of a rapid antiviral state to limit and controlinfection This selective pressure has allowed FMDV to evolvemany strategiesto ensure enhanced virulence and rapidinfectivity In general RNA viruses can bypass the IFN responseby blocking i global cellular transcription and translationii IFN induction and iii IFN signaling Similarly to otherRNA viruses FMDV can also target IFNindependent antiviralresponses mostly associated with cellular metabolic functionsie autophagy apoptosis stress granule formation etc thathave been extensively described elsewhere In thissection we will summarize the current literature on studiesconducted in vitro that explain how FMDV counteracts the hostinnate immune response at the molecular level including RNAsensing activation of adaptoreï¬ector proteins and regulation ofsignaling pathways by specific PTMsBlock on Cellular Transcription andTranslationFMDV inhibition of cellular gene expression and proteinsynthesis during infection is mainly driven by the viralencoded proteases Leader Lpro and 3C FMDV Lpro isa papainlike protease PLP thatthetranslation initiation factor eIF4G including eIF4GI and eIF4GII to disable capdependent protein synthesis AlsoFMDV Lpro causes degradation of the transcription factorinduces cleavage ofFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE Antiviral signaling pathways induced during viral infection Cellular detection of microbial molecules known as pathogenassociated molecular patternsPAMPs ie viral RNA is mediated by pattern recognition receptors PRRs including cytosolic RNA sensors ie RIGI MDA5 or LGP2 andor membraneboundTLRs PAMPPRR interaction activates signal transduction cascades black arrows that result in the production of IFN and inflammatory cytokines RIGI and MDA5contain two caspase recruitment domains CARD and an RNA helicase domain In the case of RIGI ubiquitination green circles is required for its effective activationActivated signals from either RIGI or MDA5 are transmitted downstream via the mitochondrial adaptor MAVS resulting in the formation of MAVS filaments At thisstage different PTMs such as ubiquitination or ISGylation black circles can regulate their functions Endosomal RNAs are detected by TLR3 or TLR78 which signalthrough adaptor proteins TRIF and MyD88 respectively MyD88 uses other adaptors IRAK14 to allow for interaction with TRAF proteins In addition to their role asadaptor proteins TRAFs also serve as E3 ubiquitin Ub ligases to regulate signaling TRAFmediated induction of polyUb is sensed by NEMO thus recruitingdownstream effector kinases such as TBK1 or IKK These proteins form different signaling complexes ie NEMOTBK1 and NEMOIKK leading to phosphorylationblue arrows of transcription factors IRF37 to a lesser extent IRF1 and IRF5 are also phosphorylated IRF phosphorylation triggers dimerization and translocationContinuedFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE orange arrows to the nucleus where they bind mainly to IFN promotersenhancers Alongside with this pathway TRAF6E3 ligases can activate MAPK3and other kinases including ERK12 and JNK which phosphorylate the components of the AP1 heterodimer allowing for translocation to the nucleus and binding tothe IFN promoterenhancer to activate transcription Activated IKK also phosphorylates IκB releasing NFκB which then translocates to the nucleus and binds at theIFN promoter AP1 activating protein CARD caspase activation and recruitment domain DUB deubiquitinase ER endoplasmic reticulum IκB inhibitor of KBkinases IKK IκB kinase IL interleukin IRAK interleukin1 receptorassociated kinase IRF IFN regulatory factor LGP2 laboratory of genetics protein MAPKmitogenactivated protein kinase MAVS mitochondrial antiviral signaling protein MDA5 melanoma differentiationassociated gene MyD88 myeloid differentiationprimary response protein 88d NEMO NFκB essential modulator NFκB nuclear factorκB PKR protein kinase R PTM posttranslational modification RIGIretinoic acidinducible gene I TANK TRAF family memberassociated NFκB activator TBK TANK binding kinase TLR Tolllike receptor TRAF TNF receptorassociated factor TRIF TIRdomaincontaining adapterinducing interferonTABLE Use of IFNbased therapies against FMDVTypeReceptSignalSubtypeSpeciesMilestoneType IIFNAR1IFNAR2JAK1 TYK2IFNαPorcinebovine ¢ Recombinant bacterial expressed IFNα is a potent biotherapeutic againstIFNαPorcine¢ Ad5 delivered poIFNα protects swine against different serotypes of FMDVFMDV in vitro IFNIFNδIFNω7IFNαωIFNτType IIIFNÎ R1 IFNÎ R2JAK1 JAK2IFNÎType IIIIFNλR1IL10R2JAK2 TYK2IFNλ1IFNΓ¢ poIFNαprotection correlates with enhanced tissuespecific innate immune cellinfiltration in swine ¢ poIFNα protection correlates with upregulation of essential ISGs in vitro ¢ Ad5 delivered porcine poIFN protects swine against FMDV ¢ Bacterially expressed poIFNδ8 significantly inhibits FMDV replication in vitro ¢ E coli produced poIFNω7 protects cells against FMDV ¢ Bacterially expressed IFNαω added prior to infection resulted in a significantreduction in FMDV replication in vitro ¢ Ovine IFNτ has antiviral effect against FMDV in vitro ¢ Recombinant bovine IFNÎ reduced FMDV replication in BTY cell culture ¢ High dose of Ad5poIFNÎ protects swine against FMD ¢ Replication of FMDV in IBRS2 cells is inhibited by treatment with the purifiedrecombinant poIFNλ1 PorcinePorcinePorcinePorcineOvineBovinePorcinePorcineIFN CombosOtherIFNvaccinecombosIFNλ3Bovine¢ Inoculation with Ad5boIFNλ3 resulted in the induction of several ISGs in tissuesof the upper respiratory tract and protected cattle against challenge withFMDV PorcinePorcineIFNαIFN΢ Ad5poIFNλ3 protects swine against challenge with FMDV ¢ Use of a combination of Ad5poIFNÎ and Ad5poIFNα or Ad5poIFNαΠshowed an enhancement of the antiviral activity against FMDV in swinePoly ICPorcine¢ Double stranded ds RNA poly ICLC in combination with Ad5poIFNαprotected swine against FMDV siRNAPorcine¢ Combination of Ad5poIFNαΠwith Ad3siRNA targeting FMDV NS codingregions blocked replication of all serotypes of FMDV in vitro IRF73Porcine¢ Inoculation with Ad5IRF735D resulted in induction of IFNα and fully protectedmice and swine challenged with FMDV day after treatment IRESPorcine¢ Use of synthetic IRES in combination with adjuvanted typeO FMD improvedimmune response and protection against FMDV challenge IFNαPorcine¢ Use of a combination of Ad5poIFNα and Ad5A24 in swine resulted incomplete protection after challenge IFNαÎPorcine¢ Ad5poIFNαΠcoadministered with Ad5siRNA targeting NS regions of FMDVand a commercial inactivated FMD vaccine partially protected swine IFNλ3Bovine¢ Use of a combination of Ad5bovIFNÎ3 and AdtO1M in cattle resulted incomplete protection after aerosol challenge nuclear factor NFκB and results in blockage of specificdownstream signaling eï¬ectors Studies in porcine cellsdemonstrated that FMDV Lpro can promote its selfbindingto the transcription factor activitydependent neuroprotectiveprotein ADNP and negatively regulate the activity of the IFNα promoter In contrast chromatin changes that favor theupregulation of IFN and ISGs can inhibit FMDV replication Interestingly deletion or mutations in diï¬erent domainsof Lpro result in viral attenuation in vitro and in vivo “ Furthermore these studies have shown a strong type I IFNactivity upon infection with diï¬erent versions of FMDV Lpromutants Frontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE Type I II and III interferon IFNmediated signaling All type I and type III IFN subtypes bind to respective receptors IFNAR1IFNAR2 and IFNLR1IL10R2These interactions trigger the phosphorylation of JAK1 and TYK2 kinases which in turn phosphorylate STAT1 and STAT2 JAK2 mediates type III IFNdependent STATphosphorylation Phosphorylated heterodimers of STAT1STAT2 bind to IRF9 forming the ISGF3G complex which then translocates to the nucleus and binds toIFNresponsive elements ISREs present in the promoters of over ISGs Type II IFN binds to the heterodimeric IFNÎR1IFNÎR2 receptor also inducingphosphorylation of JAK1JAK2 kinases In turn mostly STAT1 is phosphorylated Phosphorylated homodimers of STAT1 translocate to the nucleus and induce theexpression of genes controlled by gammaactivated sequence GASdependent promoter sequences IFNAR12 IFN alpha receptor12 IFNÎR12 IFNgammareceptor12 IFNALR1 IFNlambda receptor IL10R2 IL10 receptor ISGs IFNstimulated genes ISGF3G ISG factor gamma JAK12 Janus kinase STATsignal transducer and activator of transcriptionInterruption of cellular translation during infection can alsobe mediated by FMDV 3Cpro a chymotrypsinlike cysteineprotease that similarly to Lpro targets eIF4G and the capbindingcomplex eIF4A for cleavage although these events occur later inthe infection 3Cpro can also participate in the inhibitionof host“cell transcription by cleaving histone H3 upon FMDVinfection Block on Interferon InductionDuring infection the initial event that leads to the productionof IFN and proinflammatory cytokines is the recognition ofviral RNA Figure Sensing of FMDVRNA is mediated byMDA5 a protein that belongs to a family of helicasesknown as retinoic acidinducible geneI RIGIlike receptorsRLRs Recent studies have shown that the interaction betweenRLRs RIGI and LGP2 and the FMDV proteins Lpro 2Band 3A interferes with the induction of type I IFN “Indeed overexpression of either FMDV 2B or 3A resulted in thedownregulation of RIGI and MDA5 mRNA expression In contrast upregulation of LGP2 transcripts has been observedduring FMDV infection in porcine cells despite a detectablereduction of LGP2 protein levels presumably due to FMDVFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVLproinduced cleavage The apparent inconsistencybetween the levels of LGP2 mRNA and protein during FMDVinfection may be explained by LGP2™s ability to serve as a positiveand negative regulator of RIGI and MDA5 signaling presumablyaï¬ecting multiple steps of the IFN induction pathway Inaddition to RLRs nucleotidebinding oligomerization domainNODlike receptors NLRs NOD1 and NOD2 also participatein the recognition of RNA A study by Liu et al describedthe association of NOD2 with FMDV 2B 2C and 3Cpro to blockinnate immunity activation Protein kinase R PKR is anotherrecognized PRR that acts as an RNA sensor Binding ofRNA to PKR induces a conformational change that leads toautophosphorylation and activation The primary target ofactivated PKR is the eukaryotic initiation factor α subuniteIF2α whose phosphorylation results in the blockage of cellularprotein synthesis a relatively common process during viralinfection Although no direct interaction between FMDVRNA and PKR has been demonstrated it has been reportedthat PKR activity modulates FMDV infectivity In fact in tissueculture experiments depletion of endogenous levels of PKR usingsiRNA resulted in increased FMDV titers Furthermoreit has been recently shown that overexpression of autophagyrelated ATG5ATG12 proteins induces transcription of PKR andsubsequent reduction of FMDV replication These resultssuggest that PKR has a complex role as an RNA sensor but also asan antiviral agent during FMDV infectionIt has been demonstrated that FMDV also targets DExDHbox RNA helicases formally accepted as PRRs and modulatorsof the antiviral signaling pathway In vitro experimentsintending to analyze protein“protein interactions revealed theassociation between the RNA helicase DDX1 and FMDV 3D Interestingly these studies indicated that during FMDVinfection in porcine cells cleavage of DDX1 was detected whileoverexpression of DDX1 resulted in the upregulation of IFNand other ISG mRNAs which correlated with virus inhibition Other DExDHbox RNA helicases such as RNA helicaseH RHA are hijacked during FMDV infection and interact withFMDV ™UTR 2C and 3A to facilitate virus replication Signaling pathways downstream from RNA sensing involvethe activation of diï¬erent adaptor and eï¬ector proteins Oneof the pathways that lead to signal activation requires theformation of specific complexes such as NFκB essentialmodulator NEMO and the kinase IKK which bridges theactivation of NFκB and IFN regulatory factor IRF signalingpathways It has been demonstrated that FMDV 3Cpro interactswith NEMO and induces its cleavage resulting in impairedinnate immune signaling IRFmediated signals driven byIRF3 and IRF7 can also be targeted by FMDV proteinsSpecifically overexpression of Lpro in PK15 cells resulted inthe downregulation of IRF3 and IRF7 protein levels andinactivation of IFN and IFNλ1 promoter Other factors involved in the activation of IFN includeconventional PTMs such as phosphorylation and ubiquitinationwhich ensure eï¬ective regulation of these signaling pathways Also diï¬erent cellular deubiquitinases DUBs can reverseubiquitination to control the intensity of the immune signalingresponse Interestingly it has been shown that FMDV Lprocan remove ubiquitin Ub molecules from several proteinsrequired for IFN mRNA expression and those involved in theactivationrepression of the IFN loop This role became moreevident by the observation that during infection FMDV Lprocan cleave cellular substrates modified with the Ublike moleculeISG15 Furthermore mutation of Lpro thatimpairsdeISGylaseDUB function results in viral attenuation Inthis regard identification of FMDV targets for deubiquitinationand deISGylation may contribute to elucidate the role ofthose factors in counteracting the innate response and developnovel countermeasuresBlock on Interferon SignalingThe ligandmediated association of the specific IFN receptorspromotes a signaling cascade that results in the phosphorylationof the receptor by the action of JAKs These events result inthe generation of docking sites for downstream adaptor andeï¬ector proteins including signaltransducer and activatoroftranscription STAT proteins that associate with otherfactors and translocate to the nucleus inducing transcriptionof a plethora of ISGs described above and in Figure Although blockage of the JAK“STAT signaling pathway hasnot been reported during FMDV infection overexpressionof either FMDV 3Cpro or VP3 can inhibit this response Forinstance IFNtreated HeLa cells overexpressing FMDV 3Cprosuppressed IFNstimulated promoter activities and inducedproteasome and caspaseindependent protein degradationof karyopherin α1 KPNA1 the nuclear localization signalreceptor Thisinteraction inhibited the nuclear translocation of STAT1STAT2impeding maximal ISG promoter activity In another studyin HEK293T cells overexpression of VP3 followed by coimmunoprecipitation revealed the association between VP3 andJAK1 FMDV VP3 also inhibited virustriggered activation of theIFN promoter leading to the decrease in transcription of ISGspresumably due to lysosomalinduced degradation of JAK1 A yeast twohybrid screen identified FMDV 2C in complex withNmyc and STAT interactor Nmi a protein known to augmentimmune function dependent on STATmediated transcriptionInterestingly such interaction resulted in the recruitment ofNmi to vesicular compartments followed by the induction ofapoptosis in BHK21 cells tyrosinephosphorylated STAT1forEvidently FMDV proteins can also target crosstalk pathwaysinduced by JAKSTAT signaling and due to this versatilityunderstanding of these signaling events during FMDV infectionis challengingFOOTANDMOUTH DISEASE VIRUSIMPAIRS INTERFERONMEDIATEDCELLULAR INNATE IMMUNE RESPONSESSimilarly to what happens in vitro FMDV manipulates theearly innate immune response in vivo to ensure a windowof opportunity that favors viral replication and spread beforeFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVthe onset of eï¬ective adaptive immunity required for virusclearance During infection FMDV interacts with a range of hostcells including natural killer NK cells dendritic cells DCsmonocytesMφ and Îδ T cells All these cells play an importantrole in innate immune responses that trigger the productionof large quantities of IFN and other cytokines which serve asautocrine agents “Shortly after FMDV infection in swinethe number ofcirculating NK cells transiently decreases and the remaining NKcells show a dysfunctional lytic activity against target cells and areduction of IFNÎ production In parallel FMDV blocks theability of porcine DCs to mature into conventional DCs cDCs dampening their response against Tolllike receptor TLRligands Another subset of porcine DCs plasmacytoid DCspDCs also referred to as the major professional systemic IFNαproducers are also aï¬ected by FMDV During infectionpartial depletion of pDCs in the peripheral blood has beendetected and the remaining pDCs are less capable of producingIFNα in response to ex vivo stimulation by TLR ligands or virus Similar to pDCs FMDV infection reduces the productionof IFNα on Langerhans cells LCs a distinct subset oftissueresident DCs of the skin It has also been suggestedthat porcine Îδ T cells and Mφ can serve as targets for FMDVinfection in swine although the interplay betweenthese cells and FMDV remains unclearComparably to swine FMDV infection in cattle triggersseveral early events in the innate immune system althoughthe eï¬ects are not exactly the same For instance bovine NKcells originated from FMDVinfected cows have an elevatedcytotoxic function against bovine target cells in vitro In addition some subsets of cDCs are significantly decreasedduring the peak of viremia while the expression of majorhistocompatibility complex MHC class II molecules on allbovine cDCs is reduced and the processing of exogenous antigenis impaired Furthermore during FMDV infection thenumber of systemic mature bovine pDCs characterized bythe expression of CD4 and MHC class II is increasedpresumably to intensify a humoral response and T cell activationwhile levels ofimmature CD4 MHC class IIpDCs aredeclined Examination of bovine Îδ T cells revealed thatthese cells with the surface expression marker WC1 showa transient activated phenotype and increased expression ofIFNÎ FMDV also aï¬ects the innate immune response at the cytokinelevel in the natural host In vivo cytokine profile analysis duringthe clinical phase of disease shows a systemic decrease of proinflammatory cytokines [IL1 IL6 and tumor necrosis factorTNFα] and an increase of the antiinflammatory cytokine IL and IFNα Most likely these changesare related to the early T cell unresponsiveness and lymph iadescribed in swine and cattle during FMDV infection Interestingly a significant induction of inflammatoryand antiviral factors at the local level is detected in cattle in sitesof abundant viral amplification such as the nasaloropharynx orvesicular lesions “ A consistent upregulation of IFNα Î and λ mRNA in distinct microanatomical compartmentsof the nasopharyngeal mucosa concurrent with occurrence ofviremia has also been detected in cattle In contrast studiesin swine demonstrated that IFN expression in infected swineskin is inhibited These diï¬erences may be due to theanalysis of follicleassociated epithelium of the nasopharyngealmucosa in cattle vs skin in swine or to the specific samplingtechnique used in each experiment While in the cattle studylasercapture microscopy was used to focus only in areas of highFMDV replication in the swine study RNA was extracted froma piece of skin without discriminating between microanatomicalcompartments Evidently more studies are needed to elucidatethe intricate interactions between FMDV and the innate immunesystem of specific animal hostsEFFECTIVE USE OF INTERFERONAGAINST FOOTANDMOUTH DISEASEVIRUS IN VITROType I InterferonThe role of IFN in controlling FMDV replication was firstproposed in when Dinter and Philipson demonstrated thatcalf kidney cells exposed to FMDV could become persistentlyinfected and proposed this was a consequence of the inductionof an IFNlike inhibitor present in the supernatant of infectedcells Later studies also suggested that swine leukocytestreated with phytohemagglutinin produced an inhibitor ofFMDV replication with properties similar to IFN It wasnot until that new studies demonstrated that the abilityof FMDV to form plaques in cell culture correlated with thesuppression of type I IFN α protein expression Theseresults were further supported by detection of IFN protein andantiviral activity in the supernatants of primary porcine ovineand bovine kidney cells infected with an attenuated FMDVmutant leaderless as compared to the supernatants of cellsinfected with wildtype WT virus Later studies by the samegroup provided proof of concept on the use of recombinantbacterial expressed IFNα as a potent biotherapeutic againstFMDV This approach was further developed by deliveringrecombinant porcine IFNα using a replicationdefectivehuman Adenovirus vector Ad5poIFNα Infection ofIBRS2 cells with Ad5poIFNα resulted in secreted poIFNα IFN protein detected as early as h postinfection hpiand lasting for at least h Most important expressed IFNprotein displayed strong biological antiviral activity againstFMDV Followup studies by the same group showed that allFMDV serotypes are very sensitive to Ad5delivered poIFNαand sterile protection could be achieved in vivo highlightingthe potential of this approach for the development into abroad biotherapeutic strategy to control FMDV replicationIn the last years advancements is genomics have ledto the characterization of almost all type I IFN subtypes inthe porcine and bovine genome “ which are morenumerous than those identified in primates and mice This hasrevealed diï¬erent functional genes and pseudogenes with diverseexpression profiles and antiviralfunctions against diï¬erentviruses mostly in swine In fact a recent studyFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVdemonstrated that poIFNω7 known for its ability to inducethe highest levels of antiviral activity when compared to otherpoIFNω subtypes elicits an antiviral state against FMDV inIBRS2 cells treated with the recombinant form of poIFNω7produced in Escherichia coli Other subclasses of type IIFN known to be produced in swine and cattle include IFNalphaomega IFNαω also known as IFNµ and IFN delta IFNδ Significant reduction in FMDV replication has been observedupon treatment of porcine cells with bacterially expressed IFNαω or IFNδ8 prior to viral infection Recently another member of type I IFN family IFNτ whichis only produced in ruminants has been evaluated as an antiviralagainst FMDV IFNτ is a paracrine reproductive hormonesecreted constitutively by trophoblasts and endometrial cells toincrease the life span of the corpus luteum however productionis not induced upon viral infection While its secretionis restricted to ruminantsit has a broadspectrum activityagainst various crossspecies viruses Interestingly IFNτ has homology with the amino acids of IFNα which allows forbinding to type I IFN receptors The property of IFNτ thatmakes it an interesting therapeutic candidate for the treatmentof various viral diseases is its significantly lower toxicity ascompared to other type I IFNsType II InterferonIn contrast to type I IFN the type II IFN family is composedof only one member IFNÎ which exerts its actions througha specific receptor IFNGR1IFNGR2 IFNÎ is weakly resistantto heat and acid and it is able to activate leukocytes suchas macrophages and granulocytes also exerting regulatoryfunctions on T and B lymphocytes Indeed productionof IFNÎ is used as a tool to measure cellmediated immuneresponses against FMDV in vaccinated cattle “ andin swine Interestingly IFNÎ responses as measured b

Thyroid_Cancer

"Pancreatic cancer PC is one of the most aggressive cancers and has an extremely poor prognosisworldwide Long noncoding RNA lncRNA has been reported to be a potential prognostic biomarker in theinitiation and prognosis of PC Nevertheless the biological functions and the detailed molecular mechanism ofLINC00514 in PC remain unclearMethods We measured the expression level of LINC00514 in PC tissues and cell lines by quantitative realtime PCRGain and lossoffunction experiments were performed to explore the bioeffects of LINC00514 on PC developmentboth in vitro and in vivo Subcellular fractionation luciferase reporter assay RNA immunoprecipitation assay pulldown assay and western blotting were performed to investigate the oncogenic molecular mechanisms ofLINC00514Results In this study LINC00514 was shown to be upregulated in PC tissues and cell lines Increased LINC00514expression was significantly associated with the clinical progression and prognosis of PC patients In additionsilencing LINC00514 inhibited PC cell proliferation migration and invasion while LINC00514 overexpressionpromoted these processes Moreover LINC00514 knockdown remarkably inhibited PC development and metastasisin vivo Deeper investigations indicated that LINC00514 acted as a sponge for microRNA285p miR285p in PCand that Rap1b was a downstream target of miR285p Furthermore the positive correlation of LINC00514 andRap1b and the negative correlation between miR285p and LINC00514 or Rap1b were revealed Based on therescue assays Rap1b inhibition partially suppressed the oncogenic effect of LINC00514 overexpression on PC cellproliferation migration and invasionConclusions This study is the first to characterize the oncogenic function of the long noncoding RNA LINC00514in pancreatic cancer progression by acting as a competing endogenous RNA ceRNA of miR285p to upregulateRap1b expression Understanding this molecular mechanism might contribute to further discoveries of betterdiagnostic and therapeutic options for pancreatic cancerKeywords LINC00514 Pancreatic cancer Proliferation Invasion miR285p Rap1b Correspondence songzhiwangtongjieducnDepartment of Oncology the First Affiliated Hospital of Nanchang University Yongwaizheng Street Nanchang Jiangxi People™s Republic ofChina 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 0cHan Journal of Experimental Clinical Cancer Research Page of Table Primers involved in the studyGeneLINC00514Rap1bMiR285pGAPDHForward primerGAGGCAGGAGAATCGCTTGAACCACAGCAATGAGGGATTTATACTGGTGTCGTGGGTCGACGCTCTCTGCTCCTCCTGTTCU6Abbreviation LINC00514 long intergenic nonprotein coding RNA GAPDH glyceraldehyde 3phosphate dehydrogenaseCTCGCTTCGGCAGCACAReverse primerGAGGCAGGAGAATCGCTTGAACCTGACCTTGTTCCTTCCCTACCTCGCTTCGGCAGCACAATCCGTTGACTCCGACCTTCACAACGCTTVACGAATTTGCGTFig LINC00514 was upregulated in PC and predicted a poor prognosis a LINC00514 expression was detected in PC tissue and adjacentnormal tissue by qRTPCR bd Associations between LINC00514 expression and tumor size Lymph node metastasis or clinical stage weredetected by qRTPCR e qRTPCR was applied to confirm the expression level of LINC00514 in PC cell lines and normal pancreatic epithelial cellline f KaplanMeier analysis was used to assess the relation between LINC00514 expression level and overall survival in PC patients p05p01 p001 All experiments were repeated at least for three times and mean ± SD was used to represent the final result PC pancreaticcancer qRTPCR quantitative realtime polymerase chain reaction SD standard deviation 0cHan Journal of Experimental Clinical Cancer Research Page of BackgroundAs an extremely aggressive cancer worldwide pancreatic cancer PC has shown an increasing incidencerate in recent years [] Due to its high level of malignancy PC has become the fourth leading cause ofdeath from malignanttumors with poor prognosisand the 5year survival rate is less than [ ]Early diagnosis of PC has been a considerable challenge due to its complicated pathological process andintricate molecular mechanism While some advancesin imaging and clinical treatment have improved diagnosis and therapy [] the outcome of PC patients remains unsatisfactory Hence a better understandingofthe underlying molecular mechanism is essentialfor seeking a novel therapeutic target for PChavestudiesfunctions HoweverLong noncoding RNA lncRNA is a ribonucleotidechain with a coding length of more than nucleotides[] In the past it was thought that since lncRNAs didnot have the ability to encode proteins they lacked biologicalin recent years scientistshave found that lncRNAs execute their biological effectsin epigenetics [] at the histone modification [] tranlevels [] Accumuscriptional and posttranscriptionallatedelucidatedtheextraordinarysignificance of lncRNAs in the progression of a widerange of diseases such as cardiovascular diseases []diabetes [] neurodegenerative diseases [] and human cancers For instancelncRNA SNHG1 whichcan be positively regulated by miR21 activates theAKT pathway to promote sorafenib resistance in hepatocellular carcinoma cells [] lncRNA EPB41L4AAS1 suppresses the Warburg effect and plays a significant role in metabolic reprogramming of cancer[]intestinalstem cells and promote tumorigenesis of colorectalcancer []lncGata6 could maintain stemness ofLINC00514 is a newly identified lncRNA and very fewreports about it are found in the literature Research byLi [] proved that LINC00514 could be an inhibitor of malignant behaviors of papillary thyroid cancer Inaddition another study has also shown a relationship between LINC00514 and neuroendocrine prostate cancer[] In our study we explored the function and mechanism of LINC00514 in PC We discovered thatLINC00514 expression was increased in PC tissue andPC celllines and that the upregulated expression ofLINC00514 was associated with PC cell proliferationmigration and invasion in vitro and tumor growth andmetastasis in vivo Mechanistically LINC00514 accelerated pancreatic cancer progression via the miR285pRap1b axis AllthatLINC00514 might act as a potential prognostic biomarker of PC occurrence and provide a novel target forPC therapythe evidence above suggestsMethodsClinical samplesPC tissue and adjacent normal tissue were collectedfrom the First Affiliated Hospital of Nanchang University with the informed content of the enrolled patientsin this research Patients received neither chemotherapynor radiotherapy before surgery Our study was approved by the Human Research Ethics Committee ofNanchang UniversityQuantitative realtime PCRRNA was extracted by TRIzol reagent Invitrogen fromtissue samples and cells Extracted RNA was later reverse transcribed into complementary DNA cDNA byPrimeScript RT Reagent Takara Japan A SYBR GreenKit Takara Japan was utilized to perform RTPCRGAPDH and actin were used as internal controlsGene expression levels were calculated by the ˆ’ΔΔCtmethod The primer sequences are shown in Table Cell lines and cell cultureThe normal pancreatic epithelial cell line HPDE andPC celllines BxPC3 SW1990 PANC1 AsPC1Capan2 and MIAPaCa2 were purchased from ATCCCells were cultured in Dulbecco™s modified Eagle™sTable Correlation between LINC00514 expression level andclinical featuresCharacteristicsN2P valueAllAge years ‰¥ GenderMaleFemaleTumor size cm ‰¥ DifferentiationPoorModerateWellLymph node metastasisAbsentPresentClinical stage AJCCIIIIIIIVLINC00514 expressionHighLowAbbreviation LINC00514 long intergenic nonprotein coding RNA p was considered statistically significant 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig LINC00514 promoted PC cell proliferation migration and invasion ab Transfection efficiency of LINC00514 overexpression plasmids andshRNA in BxPC3 and SW1990 cells were evaluated by qRTPCR cd CCK8 assay was performed to detect cell BxPC3 SW1990 proliferationability with LINC00514 overexpression and LINC00514 silencing e Colony formation assay was carried out to further detect cell proliferationcapacity fg Transwell migration and invasion assay were carried out to detect cell migration and invasion under LINC00514 overexpression andknockdown h Western blot was conducted to evaluate the impact of LINC00514 on EMT progression p05 p01 p001 All experimentswere repeated at least for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtimepolymerase chain reaction SD standard deviation CCK8 cell counting kit8 EMT epithelialmesenchymal transitionmedium DMEM containing fetal bovine serumFBS at °C with CO2 in humidified airCell transfectionLINC00514 overexpression plasmid and shRNAs againstLINC00514 and Rap1b were purchased from GenePharma Shanghai China with scramble plasmid andshRNA used as negative controls MiR285p mimics andmiR285p inhibitors were acquired from Gene Pharma aswell All of the above reagents were transfected into cellsvia TF3000 Transfection Reagent Invitrogen accordingto the manufacturer™s recommendationsColony formation assayCells × cells per well were seeded in 6well platesand then incubated for days After being washed withPBS three times colonies were stained with hematoxylinand countedViability assayTo evaluate cell viability a CCK8 assay was carried outCells × cells per well were plated in 96well platesfor h h h and h The cell growth rate was analyzed by Cell Counting Kit8 Solarbio China reagentaccording to the manufacturer™s instructions The opticaldensity value was measured by a microplate reader at nmMigration and invasion assaysA transwell chamber Corning Tewksbury MA wasused to detect cell migration and invasion capacitiesCells × were seeded on the upper chamber covered with Matrigel Corning Tewksbury MA whileDMEM with FBS was placed on the lower chamberAfter h of transfection cells that passed from theupper chamber onto the lower chamber were fixed withmethanol stained with crystal violet and imaged under alight microscopeIn vivo analysisFiveweekold female nude mice were purchased fromthe National Laboratory Animal Center Beijing Chinaand maintained under specific pathogenfree conditionsSubsequently the mice were randomly separated intotwoLINC00514groups Cells × oftheoverexpression group and NC group were subcutaneously injected into the right axillary of nude miceTumor volume was measured every days and weightwas measured at the end of the experimentTo further evaluate the effects of LINC00514 we carried out pulmonary metastasis analysis PC cells × were injected into nude mice via the caudal vein After days the mice were euthanized The lungs of micewere removed to observe tumor metastasis All experiments were approved by the Animal Research EthicsCommittee of Nanchang UniversitySubcellular fractionation assayThe PARIS Kit Life Technologies was used to isolatenuclear and cytoplasmic RNAs according to the manufacturer™s protocol Reverse transcription of extractedRNAs and RTPCR were conducted as described beforeLuciferase reporter assayThe online software StarBase30 httpstarbasesysueducnwas used to predict the binding sites of LINC00514to miRNA285p Wildtype LINC00514 and mutantLINC00514 of the putative binding sites were clonedinto a luciferase vector Promega and cotransfected withmiR285p mimics into PC cells via LF3000 transfectionreagent After h cells were harvested for luciferase activity analysisPulldown assayWtmiR285p and NCmiRNA were labeled with biotinand transfected into BxPC3 and SW1990 cells The celllysates were incubated with streptavidin magnetic beads at °C for h After that the beads were rinsed with precooled lysis buffer and salt buffer The pulldown RNAswere extracted to detect LINC00514 levelsRNA immunoprecipitation assayThe Magna RIP RNABinding Protein ImmunoprecipitationKit Millipore MA was used to conduct the RIP assay according to the manufacturer™s protocol The cells were lysedand incubated with Ago2 and IgG Then cell lysates weremixed with antiAgo2 and antiIgG in RIP buffer MilliporePrecipitated RNAs were collected for RTPCR analysis 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig LINC00514 promoted tumor growth and pulmonary metastasis in vivo ab The images of subcutaneous tumors were obtained on Day cf The tumor volumes and weights of shLINC00514 group compared with NC group and LINC00514 overexpression group compared withempty group were quantified Tumor volumes were analyzed by ANOVA gh qRTPCR was used to assess the transfection efficiency ij Theimage of pulmonary metastasis was photographed at the endpoint kl Pulmonary metastasis of LINC00514 silencing and LINC00514overexpression compared with their control groups were evaluated p05 p01 p001 The mean±SD was used to represent the finalresults of experiments repeated at least three times PC pancreatic cancer qRTPCR quantitative realtime polymerase chain reaction SDstandard deviation NC negative control ANOVA analysis of variancePVDF membranesWestern blotProtein was extracted from cells and transferred topolyvinylidene difluorideafter sodium dodecyl sulfate polyacrylamide gel electrophoresis After that membranes were blocked with nonfat milk and incubated overnight at °C withprimary antibodies After rinsing the membranes threetimes with PBS a secondary antibody labeled withhorseradish peroxidase was used to incubate membranesroom temperature Antibodiesagainst ECadherin NCadherin and Vimentin wereall purchased from CST company and actin andRap1b antibodies were purchased from Abcam Thedilution ratio was determined accordingto theinstructionsfor h atStatistics analysisAll data are presented as the mean ± standard deviationAll experiments were repeated at least three times Student™s t test ANOVA Spearman™s rank correlation testand χ2 test were used for statistical analysis A value ofp was considered statistically significantResultsLINC00514 was upregulated in PC and predicted a poorprognosisFirst we analyzed the LINC00514 profile in PC Wefound that LINC00514 was remarkably increased in PCtissues compared with the corresponding normal tissuesFig 1a The upregulated expression of LINC00514 wassignificantly associated with the LINC00514 level andtumor sizelymph node metastasis and clinical stageFig 1bd while no significant correlation was foundbetween LINC00514 expression and age gender ortumor differentiation Table Additionally LINC00514expression was increased in PC cell lines compared withthe normal pancreatic epithelial cell line Fig 1e Furthermore Kaplan“Meier survival curves revealed thathigh LINC00514 expression was related to a lower overallthe lowLINC00514 level group Fig 1f Overall LINC00514was increased in PC and might be associated with clinical progression and a poor prognosis of PC patientsrate compared with that ofsurvivalcellandpromotedLINC00514 promoted cell proliferation migration andinvasionTo investigate whether LINC00514 is involved in cellproliferation migration and invasion we carried outgain and lossoffunction assays The LINC00514 overexpression plasmid and LINC00514 shRNA were stablytransfected into BxPC3 and SW1990 cells with ascramble plasmid and shRNA used as negative controlsFig 2ab According to the results of CCK8 and colony formation assays LINC00514 overexpression significantlySW1990proliferation capacity while suppression of LINC00514remarkably inhibited these processes Fig 2ce Moreovertranswell assays were utilized to prove thatLINC00514 increased cell migration and invasion capabilities Fig 2fg For further confirmation westernblotting was performed to measure the expression ofEMT markers in both BxPC3 and SW1990 cells As expected Ecadherin was observed to be strikingly downregulated by LINC00514 overexpression whereas Ncadherin and Vimentin were obviously upregulated andthe shLINC00514 group showed the opposite resultsFig 2h In summary LINC00514 promoted PC cellproliferative migratory and invasive capacitiesBxPC3LINC00514 knockdown inhibited tumor growth andpulmonary metastasis in vivoTo further identify the bioeffects of LINC00514 ontumor growth we constructed a subcutaneous xenografttumor model BxPC3 cells transfected with LINC00514shRNA compared with NC shRNA or transfected withthe LINC00514 overexpression plasmid and comparedwith the empty plasmid were subcutaneously injectedinto nude mice The tumors were measured every daysafter injection After euthanizing the mice we obtainedimages of the tumors Fig 3ab Compared with thoseof the NC group the volume and weight of tumors inthe LINC00514 shRNA group were significantly reducedwhileresults were observed in theLINC00514 overexpression group Fig 3cf QRTPCRwas used to assess the transfection efficiency Fig 3ghThen we further investigated the role of LINC00514 inPC metastasis in vivo Nude mice were injected withBxPC3 cells transfected with LINC00514 shRNA compared with NCLINC00514the oppositeshRNA orthe 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig LINC00514 was a sponge for miR285p ab Subcellular fractionation assay was used to determine the subcellular localization ofLINC00514 a BxPC3 cells b SW1990 cells C Sequence of WTLINC00514 MutLINC00514 and miR285p were conducted dg Luciferasereporter assay and RIP assay was performed to demonstrate that miR285p was a downstream target of LINC00514 h Pulldown assay wasconducted to detect the reaction between miR285p and WTLINC00514 or MutLINC00514 ij Relative miR185p expression level in BxPC3and SW1990 were determined by qRTPCR K The expression of miR285p in PC tissue and normal tissue were detected by qRTPCR LSpearman™s rank correlation test was utilized to analyze the correlation between the levels of LINC00514 and miR285p MN The miR285pexpression levels under LINC00514 silencing and LINC00514 overexpression were evaluated in vivo op CCK8 assay was performed to detectproliferation of cells transfected with LINC00514 shRNA and cells cotransfected with LINC00514 and miR285p inhibitor qr Transwell migrationand invasion assay were carried out to detect cell migration and invasion abilities p05 p01 p001 All experiments were repeated atleast for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtime polymerase chainreaction RIP RNA immunoprecipitation SD standard deviation WTlINC00514 wild type LINC00514 MutLINC00514 mutant LINC00514 CCK8cell counting kitoverexpression plasmid compared with the empty plasmid into the tail vein Images of pulmonary metastasiswere acquired at the endpoint Fig 3ij There was anobviously lower incidence of pulmonary metastasis and asmaller number of metastatic tumors per lung in the shLINC00514 group compared with the NC groupwhereas the LINC00514 overexpression group showedthe opposite results Fig 3klin the celldetected the expression level of miR285p in the tumorswe collected before and the results showed that miR285p expression was higherlines withLINC00514 knockdown while miR285p was lower inthe cells with LINC00514 overexpression Fig 4mnLINC00514 promoted cell proliferation migration andinvasion at least partially by sponging miR285p Fig4or In summary LINC00514 accelerates PC progression by sponging miR285pLINC00514 acted as a sponge for miR285pTo explore the underlying molecular mechanism of theoncogenic effects of LINC00514 on PC we determinedthe subcellular localization of LINC00514 The resultsshowed that LINC00514 was mostly distributed in thecytoplasm Fig 4ab which suggested that LINC00514might exert its biological function by sponging miRNAStarBase30 was utilized to identify a candidate microRNA miR285p and predict the potential downstreamtargets of LINC00514 Fig 4c The luciferase reporterassay results confirmed that the luciferase activity ofWTLINC00514 was clearly decreased by miR285pmimics while the luciferase activity of MutLINC00514did not change significantly Fig 4de In addition theRIP assay further revealed that LINC00514 and miR285p were enriched in beads conjugated to Ago2 comparedwith the IgG group Fig 4fg Furthermore overexpression of WTLINC00514 but not MutLINC00514 decreased miR285p expression in BxPC3 and SW1990cells Fig 4h Additionally overexpressing LINC00514dramatically decreased miR285p levels in both BxPC3and SW1990 cells while silencing LINC00514 increasedmiR285p levels with NC shRNA used as an internalreference Fig 4ij Then we further detected miR285p expression in tumor tissue The results revealed alower level of miR285p in PC tissue than in normal tissue and a negative correlation between LINC00514 expression and miR285p levels Fig 4kl To obtainmore evidence in vivo experiments were performed WeRap1b was a downstream target of miR285p in PCThe posttranscriptional function of miRNAs is usually toinhibit protein synthesis by base pairing with the ²untranslated region [] Next to ascertain the detailed regulatory mechanism of LINC00514 in PC we searchedStarBase30 and observed that Rap1b was predicted to bea downstream target of miR285p Fig 5a MutRap1bor WTRap1b and miR285p or NCmiRNA were transfected into BxPC3 and SW1990 cells A luciferase reporter assay and RIP assay were used to confirm thehypothesis that Rap1b is a direct target of miR285p Fig5be Then we found that Rap1b expression was downregulated by LINC00514 silencing while cotransfectingmiR285p and shLINC00514 inhibited the effect ofLINC00514 knockdown on Rap1b at both the transcriptional and translational levels Fig 5fgThen we detected Rap1b levels in tumor tissue Rap1bwas obviously increased in PC tissue compared with adjacent normal tissue and there was a positive relationshipbetween Rap1b expression and LINC00514 levels while anegative correlation was observed between Rap1b expression and miR285p levels Fig 5hj In addition we alsodetected the expression of Rap1b in vivo and the transfection efficiency was examined previously As expected theexpression of Rap1b was clearly decreased in BxPC3 cellsby LINC00514 knockdown while increased expressionwas observed in LINC00514overexpressing cells Fig 5kl Thus far we have proven that Rap1b is a direct target 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig Rap1b was a downstream target of miR285p in PC a The sequence of WTRap1b MutRab1b and miR285p were conducted efLuciferase reporter assay and RIP assay were performed to determine the association between miR285p and Rap1b fg QRTPCR and westernblot were used to detect Rap1b expression in cells of LINC00514 silencing and cells of cotransferring miR285p and LINC00514 shRNA attranscription and translation level h Relative Rap1b expression in tumor tissue and normal tissue were detected by qRTPCR ig Thecorrelation between Rap1b and LINC00514 as well as the correlation between Rap1b and miR285p were analyzed by Spearman™s rankcorrelation test kl The Rab1b expression levels under LINC00514 silencing and LINC00514 overexpression were evaluated in vivo mn CCK8assay was performed to detect proliferation of cells transfected with miR285p inhibitor and cells cotransfected with miR285p inhibitor andRap1b shRNA OP Transwell migration and invasion assay were carried out to detect cell migration and invasion abilities p05 p01p001 All experiments were repeated at least for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtime polymerase chain reaction RIP RNA immunoprecipitation SD standard deviationof miR285p Thereafter functional experiments werecarried out to investigate the bioeffects of Rap1b The resultsdemonstrated that Rap1b silencing remarkably suppressedthe promoting effects of the miR285p inhibitor on cell proliferation migration and invasion capacities Fig 5mppatients which indicated that LINC00514 might be involved in PC progression In addition it was determinedthat LINC00514 facilitated PC cell proliferation migration and invasion in vitro and tumor growth and metastasis in vivo The underlying molecules however havenot yet been revealedRap1b inhibition inhibited the tumorigenesis effects ofLINC00514Finally we explored the role of LINC00514 mediatedby Rap1b in promoting tumor growth We knockeddown Rap1b in BxPC3 and SW1990 cells to determine whether Rap1b inhibition can reverse the oncogenic effects of LINC00514 Based on the rescueassays Rap1b inhibition partially inhibited the effectof LINC00514 overexpression on cell proliferationmigration and invasion Fig 6adIn conclusionthesethatLINC00514 acted as a key tumor promotor of PC bycompetitively binding to miR285p and then upregulating the expression of Rap1bdemonstratedcollectivelyresultsthrough the miR1883pBRD4 axisDiscussionIn recent years PC has received increasing attention dueto its high incidence and extremely poor prognosis []Accumulated studies have shown that lncRNAs play animportant role in the initiation and development of cancers including PC [] For instance LINC00346 accelerated PC progression and gemcitabineresistancepartially[]lncRNA GLSAS mediated the feedback loop of Mycand GLS and provided a potential therapeutic strategyfor metabolic reprogramming in PC [] AFAP1AS1was shown to exert inhibitory effects on the stemness ofPC cells and ultimately PC tumorigenicity in vivo via themiR384ACVR1 axis [] LINC00514 has been previously reported in papillary thyroid cancer [] and neuroendocrine prostate cancer [] butthere are noreports in PC Our study revealed that LINC00514 expression was markedly elevated in PC tissues and PC celllines and that increased expression of LINC00514 wasassociated with the progression and prognosis of PCIn recent years increasing evidence has proven the hypothesis that lncRNAs exert their biological impact by acting as competitive endogenous RNAs ceRNAs to affectthe development of cancers [] There has been considerable progress in the study of ceRNAs in PC For examplelncRNAPVT1 promotes PC cell proliferation and migration by sponging miR448 [] cucurbitacin B inhibits PCcell proliferation both in vitro and in vivo throughlncRNAAFAP1AS1 binding with miR146b5p [] andPXNAS1 acts as a ceRNA of miR3064 which upregulates PIP4K2B expression and suppresses the progressionof pancreatic cancer [] In our research subcellular fractionation assays indicated that LINC00514 was mostly located in the cytoplasm which provided a basis forLINC00514 to act as a ceRNA in the initiation and progression of PC Then the online software StarBase30 wasutilized to predict the possible downstream target miR285p for LINC00514 Luciferase reporter assay RIPassay and pulldown assay were used to confirm the interaction between LINC00514 and miR285p Overexpression of LINC00514suppressed miR285p whileLINC00514 silencing upregulated miR285p expressionFurther investigation was carried out to demonstrate themigration and invasion promoting effect of miR285p inthe initiation and development of PC which suggested atumorpromotingeffectthat wasdependent on miR285pLINC00514ofAccording to the ceRNA hypothesis mRNA expression is upregulated due to lncRNA competitively bindingto miRNA Rap1b was first reported in the study of Chajut [] and was found to be related to various cancers such as thyroid cancer [] breast cancer []gastric cancer [] and colorectal cancer [] Howeverthere is only a limited number of reports about Rap1b inPC [] In our current study Rap1b was predicted to be 0cHan Journal of Experimental Clinical Cancer Research Page of Fig Rap1b inhibition restrained the tumorigenesis effects of LINC00514 ab CCK8 assay was performed to detect proliferation of cellstransfected with LINC00514 overexpression plasmids and cells cotransfected with LINC00514 overexpression plasmids and Rap1b shRNA cdTranswell migration and invasion assay were carried out to detect cell migration and invasion abilities p05 p01 p001 All experimentswere repeated at least for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtimepolymerase chain reaction SD standard deviation CCK8 cell counting kit8a direct target of miR285p by StarBase30 Luciferase reporter assay and RIP assay confirmed thedirect binding of Rap1b with miR285p Rap1bacting as a cancerpromoting gene had a positivecorrelation with LINC00514 while there was anegative relationship between Rap1b and miR285p Moreover silencing Rap1b partially abolishedthe tumorigenic effects of LINC00514 based on therescue assayIn conclusion ourstudy provides evidence thatLINC00514 promotes PC development by spongingmiR285p and increasing Rap1b expression Thishighlights the LINC00514miR285pRap1b axis as anovel diagnostic and therapeutic strategy for PCpatientsresults highlighted the significantConclusionsOurtheLINC00514miR285pRap1b axis in PC progressionsuggesting that LINC00514 may serve as a potential biomarker and therapeutic target in PCrole ofAbbreviationsPC Pancreatic cancer LncRNA Long noncoding RNA LINC00514 Longnoncoding RNA00514 MiR285p MicroRNA285p CeRNA Competingendogenous RNA EMT Epithelialmesenchymal transition QRTPCR Quantitative realtime PCR RIP RNA immunoprecipitation 0cHan Journal of Experimental Clinical Cancer Research Page of AcknowledgmentsNot applicableAuthors™ contributionsQH and ZWS designed the study QH JHL and JPX collated the data carriedout data analyses and produced the initial draft of the manuscript QH andZWS contributed to drafting the manuscript All authors have read andapproved the final submitted manuscriptFundingThis study was funded by Jiangxi Provincial Education Fund Project youth Science Foundation of Jiangxi Province20202BAB216027Availability of data and materialsAll the data and materials supporting the conclusions were included in themain paperEthics approval and consent to participateThe study was conducted in accordance with the Declaration of Helsinkiprinciples It was approved by the Ethics Committee of the First AffiliatedHospital of Nanchang UniversityConsent for publicationNot applicableCompeting interestsThe authors declare no competing interestsReceived May Accepted July ReferencesPei X Song F Wang Z Emerging incidence trends and application ofcurative treatments of pancreatic cancer in the USA Medicine e17175Ansari D Tingstedt B Andersson B Holmquist F Sturesson C Williamsson CSasor A B D Bauden M Andersson R Pancreatic cancer yesterdaytoday and tomorrow Future Oncol “Shin SJ Park H Sung YN Yoo C Hwang DW Park JH Kim KP Lee SS RyooBY Seo DW Prognosis of pancreatic Cancer patients with synchronousor Metachronous malignancies from other ans is better than those withpancreatic Cancer only Cancer Res Treat “Halbrook CJ Lyssiotis CA Employing metabolism to improve the diagnosisand treatment of pancreatic Cancer Cancer Cell “Beermann J Piccoli MT Viereck J Thum T Noncoding RNAs indevelopment and disease background mechanisms and therapeuticapproaches Physiol Rev “ Wei

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

coronavirus disease COVID19 pandemic access to surgical care for patients with head and neck cancer HNC is limited and unpredictable Determining which patients should be prioritized is inherently subjective and difficult to assess The authors have proposed an algorithm to fairly and consistently triage patients and mitigate the risk of adverse outcomes METHODS Two separate expert panels a consensus panel participants and a validation panel participants were constructed among international HNC surgeons Using a modified Delphi process and RAND CorporationUniversity of California at Los Angeles methodology with consensus rounds and meetings groupings of highpriority intermediatepriority and lowpriority indications for surgery were established and subdivided A pointbased scoring algorithm was developed the Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN Agreement was measured during consensus and for algorithm scoring using the Krippendorff alpha Rankings from the algorithm were compared with expert rankings of case vignettes using the Spearman rank correlation coefficient RESULTS A total of indications for surgical priority were rated Weights for each indication ranged from ˆ’ to scale range ˆ’ to The response rate for the validation exercise was The SPARTANHN demonstrated excellent agreement and correlation with expert rankings Krippendorff alpha [ CI ] and rho [ CI ] S The SPARTANHN surgical prioritization algorithm consistently stratifies patients requiring HNC surgical care in the COVID19 era Formal evaluation and implementation are required Cancer American Cancer Society LAY SUMMARY ¢ Many countries have enacted strict rules regarding the use of hospital resources during the coronavirus disease COVID19 pandemic Facing delays in surgery patients may experience worse functional outcomes stage migration and eventual inoperability¢ Treatment prioritization tools have shown benefit in helping to triage patients equitably with minimal provider cognitive burden¢ The current study sought to develop what to the authors™ knowledge is the first cancer“specific surgical prioritization tool for use in the COVID19 era the Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN This algorithm consistently stratifies patients requiring head and neck cancer surgery in the COVID19 era and provides evidence for the initial uptake of the SPARTANHN KEYWORDS coronavirus disease COVID19 delivery of health care head and neck cancer health priorities patient selection surgical procedures waiting listsCorresponding Author John R de Almeida MD MSc Division of Surgical Oncology Department of Otolaryngology“Head and Neck Surgery 8NU883 Toronto General Hospital University Health Network Elizabeth St Toronto ON M5G 2C4 Canada Johndealmeidauhnca Division of Surgical Oncology Department of Otolaryngology“Head and Neck Surgery Princess Margaret Cancer Center University Health Network University of Toronto Toronto Ontario Canada Institute of Health Policy Management and Evaluation Dalla Lana School of Public Health University of Toronto Toronto Ontario Canada Division of Otolaryngology“Head and Neck Surgery Dalhousie University Halifax Nova Scotia Canada Division of Otolaryngology“Head and Neck Surgery McMaster University Hamilton Ontario Canada Department of Otolaryngology“Head and Neck Surgery Western University London Ontario Canada Department of Otolaryngology“Head and Neck Surgery Memorial Sloan Kettering Cancer Center New York New York Head and Neck“Endocrine Oncology Moffitt Cancer Center Tampa Florida Department of Otolaryngology“Head and Neck Surgery Medical University of South Carolina Charleston South Carolina Department of Otolaryngology“Head and Neck Surgery Stanford University Palo Alto California Department of Otolaryngology“Head and Neck Surgery University of Michigan Ann Arbor Michigan Department of Otolaryngology“Head and Neck Surgery The University of Texas MD Anderson Cancer Center Houston Texas Head and Neck Unit The Royal Marsden Hospital London United Kingdom Department of Otolaryngology“Head and Neck Surgery Icahn School of Medicine at Mount Sinai New York New York Department of Otolaryngology“Head and Neck Surgery Sunnybrook Health Sciences Centre University of Toronto Toronto Ontario Canada Department of Otolaryngology“Head and Neck Surgery Sinai Health System University of Toronto Toronto Ontario CanadaThe first authors contributed equally to this Additional supporting information may be found in the online version of this 101002cncr33114 Received June Revised June Accepted June Published online Month in Wiley Online Library wileyonlinelibrarycomCancer Month 0cOriginal INTRODUCTIONOn March the World Health anization declared a global pandemic due to the novel coronavirus severe acute respiratory syndrome coronavirus SARSCoV2 and the resulting coronavirus disease COVID191 As a result in many jurisdictions operating room capacity has been limited to only emergent or urgent surgical procedures2 Several advisory bodies have issued recommendations to safeguard access to oncologic surgery while still acknowledging that treatment delays may be necessary The American College of Surgeons has recommended postponing elective surgery including for patients with lowrisk cancers while recommending that other urgent cancer surgeries proceed34 Cancer Care Ontario has issued similar guidance recommending that hospitals include cancer surgery in their care delivery plan5The time from the diagnosis of head and neck cancer HNC to surgery is a metric with prognostic importance with treatment delays portending poorer oncologic outcomes68 In a recent systematic review evaluating delays in time from diagnosis to treatment initiation of studies demonstrated a decrease in survival to be associated with treatment delays68 These data support the urgency of initiating treatment for patients with HNC but to our knowledge do not inform a stratification schema when operating room access is not available for all patientsAs a result of these new imposed constraints difficult decisions regarding prioritization for cancer surgery are obligatory and require the consideration of broader principles regarding scarce resource allocation9 Key among these is the need for consistency and transparency to achieve fairness and to avoid engendering disparities in both access and outcomes1011 Prioritization on a casebycase basis using expert clinical judgment can be logistically challenging carries a cognitive burden and is susceptible to the biases of practitionersSurgical prioritization tools or algorithms offer decisionmaking transparency and provide equitable and timesensitive access to care to the patients who need it most1213 Although tools for surgical prioritization in the era of COVID19 continue to emerge to our knowledge oncology patients have not been explicitly considered14 Herein we have presented the development and validation of a novel algorithm Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN for the prioritization of surgery for patients with HNCMATERIALS AND METHODSThe current study was granted a waiver from the research ethics board at the University Health NetworkParticipants and SettingFor instrument development a group of expert HNC surgeons JRD DPG RG JCI DBC DB AE DJE KMH EM and IJW from institutions University Health Network Sinai Health Systems and Sunnybrook Health Sciences Centre at the University of Toronto participated in the consensus process consensus panel At the time of the consensus process all institutions were operating under significant resource constraints with limited availability of operating room time For instrument validation a group of participants JRD CWN DF DPG and EM completed the scoring algorithm designed after the consensus process Fifteen external head and neck surgeons HZ ACN RJW MAC CM EMG VD AGS AJR CML EYH JM VP BM and EG from institutions across Canada institutions the United States institutions and the United Kingdom institution participated in a ranking exercise of clinical vignettes validation panelScopeThe scope of variables considered in the prioritization algorithm was established and vetted by the consensus panel see Supporting Information All indications for prioritization were presented to the consensus panel using an online survey platform Google Forms httpsdocsgooglecomforms With exceptions survey respondents were asked to consider each of the indications in isolation For wait times panel members were asked to also consider histologic grade Similarly for surgical site the panel was asked to simultaneously consider extent of surgery Related indications were presented sequentially to facilitate pairwise comparison eg stage I and II vs stage III and IV were presented in sequence AJCC 8th edition The list of indications was pilot tested by surgeons JRD DPG EM and RG for sensibility readability content validity language and comprehensibilityConsensus ProcessThe consensus panel participated in a Delphi consensus process with rounds of rating see Supporting Information The first rounds aimed to achieve consensus regarding the priority grouping high intermediate or low High priority was defined as an indication to Cancer Month 0cproceed to surgery within weeks The second rounds of rating involved ranking each indication less important neutral or more important within their respective priority grouping Two teleconference meetings were conducted between the first and second rounds and between the third and fourth rounds with anonymized results from the prior round presented for discussion and to address inconsistencies and misinterpretationsA modification of the RANDUniversity of California at Los Angeles UCLA method was used to achieve consensus15 This methodology typically is used to determine the appropriateness of an intervention but in this setting was used to determine surgical priority We used a scale ranging from to in rounds and to indicate the decision to not operate or low priority scores intermediate priority scores or high priority scores For rounds and we used a scale from to to rate each indication compared with other indications within each of the priority groupings as either less important neutral or more important Consensus was determined based on RANDUCLA criteria15 For the first rounds to determine surgical priority a hierarchical logic was adopted to determine consensus regarding whether surgery should be performed and to then determine the priority of surgery based on the given indication Agreement on the decision to not operate was defined as a minimum of of the panelists rating a given indication with a zero score If there was no agreement to avoid surgery agreement for surgical priority then was defined as ‰¤ panelists rating the indication outside the 3point range containing the median as per RANDUCLA guidelines15 For rounds and any indication that failed to achieve consensus was classified as being of intermediate priority and for rounds and any indication failing to achieve consensus was classified as neutral within the priority groupingDevelopment of the SPARTANHNThe algorithm uses a pointbased system to assign a total score based on the sum of the individual indication scores see Supporting Information with higher scores corresponding to higher priority Scoring weights were based on consensus from both sets of rounds such that highpriority indications were assigned scores ranging from to intermediatepriority indications were assigned scores ranging from ˆ’ to and lowpriority indications were assigned scores ranging from ˆ’ to ˆ’ Within each priority grouping 3point range the scores were assigned based on the consensus ratings from the third and fourth rounds For any patients with the same total score the SPARTANHNde Almeida et alpatient with the longer surgical wait time was assigned the higher priority rankClinical VignettesTwelve clinical vignettes were constructed see Supporting Information after the consensus rounds to validate the SPARTANHN The vignettes described a variety of clinical scenarios incorporating multiple prioritization indications and additional clinical information Experts were asked to consider only the patientlevel information provided to them and not their own unique clinical and community practice environments Twelve scenarios were selected for diversity of cases The number was considered appropriate while avoiding the excessive cognitive burden associated with ranking too many scenariosStatistical AnalysisAgreementAgreement between raters during the Delphi process was calculated at each round and within each priority grouping using the Krippendorff alpha Kalpha Because typical coefficients of reliability are not suitable for coded data agreement for the rank orders generated by coders JRD CWN DF EM and DPG applying the SPARTANHN algorithm to the clinical vignettes was assessed using Kalpha calculated with bootstrap samples16 The Kalpha allows for estimation of reliability for any number of raters and categories and may be used when there are missing data17Validity of the SPARTANHN AlgorithmConvergent validity of the median rankings from the coders of each of the vignettes using the SPARTANHN and the expert panel rankings were assessed using the Spearman rank correlation coefficient The strength of the correlation was considered weak if the rho was moderate if the rho was between and and strong if the rho was In addition to SPARTANHN a second algorithm using a decisionmaking flowchart was developed SPARTANHN2 The tool and associated performance characteristics are included in Supporting Information Sample Size ConsiderationsFor determination of an adequate sample size for the expert panel we assumed that for model validity there was a strong correlation between the model rank order and expert rank order ie rho ‰¥ an alpha of power of and a nonresponse rate of Therefore the calculated sample size requirement was participantsCancer Month 0cOriginal All analyses were 2sided and statistical significance was set at P\xa0‰¤\xa0 Analyses were conducted using SAS University Edition statistical software SAS Institute Inc Cary North CarolinaRESULTSEstablishing Consensus Priority Groupings First Consensus RoundsAfter the first rounds the panel failed to achieve consensus for any indications that would result in a decision to not operate More than respondents indicated that they would not operate for the following indications the availability of alternative nonsurgical treatment with a similar prognosis respondents poor performance status ie Eastern Cooperative Oncology Group [ECOG] performance status of respondents and very severe comorbidity as indicated by a non“cancerspecific survival rate of at year respondents In the first round consensus was achieved for indications for surgical prioritization of which were considered high priority of which were considered intermediate priority and of which were considered low priority After review of firstround results consensus was achieved for an additional indications indications were rated as being of intermediate priority and indications were rated as low priority Table Establishing Ranking Within Each Priority Grouping Second Consensus RoundsOf the lowpriority indications consensus for the importance of factors was achieved for scenarios both of which were deemed less important Table Of the intermediatepriority indications consensus for the importance of factors was achieved for of scenarios Of highpriority factors consensus for the importance of factors was achieved for scenarios all of which were deemed to be more importantAgreement during consensus rounds was found to be weak to moderate for all rounds ranging from to The agreement was similar when measured as per priority grouping in which the Kalpha ranged from to Table SPARTANHN Surgical Prioritization Scoring SystemPriority weights for each indication ranged from ˆ’ to spanning a 9point range and translated from the rounds of priority groupings into categories Four indications were assigned a weight of based on consensus that these factors were both high priority and more important Supporting Information Table All other highpriority indications were assigned a weighted score because there was no consensus that they were either less or more important For intermediatepriority indications a weighted score of was assigned for of the indications deemed to be more important by consensus The other indication deemed to be more important thyroid cancer with tracheal invasion was assigned a score of because of the fact that this indication can be associated with lowgrade histology which is assigned a negative weighted score Three intermediatepriority indications that were rated as more important were resource use indications which generally are colinear As such the decision was made to assign a maximum score of for the presence of any or all of these indications One intermediatepriority indication was deemed to be less important by consensus and was assigned a score of ˆ’ All other intermediatepriority indications were assigned scores of For the lowpriority indications those deemed to be less important were assigned a weight of ˆ’ and all other indications were assigned a weight of ˆ’ The total scale score ranged from ˆ’ to Fig Reliability and Validity AssessmentAgreement between the coders for the SPARTANHN was excellent Kalpha Agreement between the expert raters was moderate Kalpha Convergent validity was demonstrated by a strong correlation between the rank orders generated by the SPARTANHN and external experts rho CI [P\xa0 a0 ] Agreement between expert rankings and SPARTANHN rankings for the vignettes is shown in Figure DISCUSSIONIn the setting of the COVID19 pandemic in which the availability of operating room time as well as hospital and intensive care unit beds is limited the prioritization of surgical oncology cases is imperative to mitigate downstream adverse outcomes1920 The current methodology was adopted based on expert consensus In the current study we have proposed the SPARTANHN with the objective of providing transparency and facilitating surgical prioritization for treatment providersCreating COVID19“era allocation schemas that are ethically sound is both critical and challenging Emanuel et al have advocated ethical principles with which to Cancer Month 0cSPARTANHNde Almeida et ali ytidbrom lanoitcnufi tnacifings laitnetoP fi ytilibareponi rohtw romut fi tnemriapmi citem etaredom laitnetoPsoc ro lanoitcnuf htiw recnac doryhTiinosavni laehcarthtw romut inossergorp esaeisd citamotpmySENEtsil tiawno e lihwTR suoverPi dedeecxe emit tiaW dedeecxe emit tiaWihgh rof kw‰¥ yb liygootsh edargihgh rof kw yb liygootsh edarg igngami ro lacniilC hpmyl decnavdAegats gncnavdai ei inossergorpi cpocsorcam roN ge esaesd edoni esaesdV inoitceserI ot III egatSnoitceser enob htiw recnac ytivac larOnoitide ht CCJA yregrus fo htgneL latot ro latotraen gniriuqer recnac ytivac larO yats fo htgnel latipsoHh tinu erac evsnetni ioNtinu nwodpets rod yregrus larosnart htiw recnac laegnyrahporOymotcessogllymotoubdnam htiiw recnac laegnyrahporO ymotcegnyral latot htiw recnac laegnyrahpopyHymotcegnyral latot gniriuqer recnac laegnyraLi cpocsodne gniriuqer recnac laegnyrahposaNymotcegnyrahp laitrapdna odne gniriuqer recnac suns lasanarap roi lasaNymotolli xamgniriuqer recnac laegnyrahposaNnoitceser cpocsinoitceseri noitceser nks gniriuqer recnac nks decnavdAinoitcurtsnocer palf lanoger dnai edon hpmyl detimil htiw renac kcen dna daeH edon hpmyl on htiw recnac kcen dna daeHycnangilam enob laropmeTesaesdiII ot I egatSy egAy egAy egAesaesdi SPGOCE y ‰¥ egAycnangil amditorap edarghgHinoitcurtsnocer palfeerf gniriuqer recnac nks decnavdAi laitrapgniriuqeryregrus laegnyral recnac laegnyraL gniriuqer recnac laegnyrahpopyHnyrahpognyral latotymotcegriuqer recnac sunsi roiretna nepogn i lasanarap ro lasaN ‰¥i lacgrus lacafonarciili ygootsh edargwol rof dedeecxe emit tiaWkwnoitceser eussittfos htiw recnac ytivac larOi rof gnhcaorppa tubdedeecxe ton em it tiaW dedeecxe emit tiaWliygootsh edarghghiwoliygootsh edargl rof kw deecxe ton emit tiaWromoc ereves yreV SP GOCE ei sutats ecnamrofrep rooP ypareht evitanretlAlebaliava ni dehcaorppa tub edargwol rof kw ditorap edargwoLycnangilam edon hpmyl htiw recnac doryhTiesaesdilygootshi recnacnonge ytidb i ta i lavvrusy srotcaF ytiroirPhgHisrotcaF ytiroirPetademretniIsrotcaF ytiroirPwoLˆ’ˆ’ˆ’ˆ’i gnknaR fo sdnuoR retfA serocSdna snoitacdn iI noitazitiroirP ELBATCancer Month 0cOriginal TRj tnavuda dna esaesddecnavda hti iw tneitaPderiuqer tinu nwodpets ro tinu erac evsnetniIderiuqer ebut ymotsoehcart oNderiuqer palf eerf oNd yats fo htgnel latipsoHh yregrus fo htgneLd yats fo htgnel latipsoHderiuqer palf eerFh yregrus fo htgneLnoitpo na si SPGOCEˆ’ˆ’ˆ’ˆ’srotcaF ytiroirPhgHisrotcaF ytiroirPetademretniIsrotcaF ytiroirPwoLdeunitnoC ELBATyparehtodar iTRi nosnetxe ladonartxe ENE sutats ecnamrofreppu ygoocnO evitarepooCnretsaE l SPGOCE snoitaverbbAiTABLE Agreement Between Experts During the Delphi ProcessRoundOrdinal ScaleaLCL UCLPer Priority GroupLCL UCLAbbreviations LCL lower confidence limit UCL upper confidence limitThere were raters and agreement was measured using the Krippendorff alphaaœOrdinal scale refers to the scale of to used to rate priority of surgery and œPer Priority Group refers to the lowpriority mediumpriority and highpriority groups related to the scoring scaleguide the allocation of scarce resources maximizing the benefits produced by scarce resources treating people equally promoting and rewarding instrumental value and giving priority to those patients who are worst off9 These have been contextualized for cancer care more broadly and are manifest in the SPARTANHN algorithm21 The highpriority indications implicitly embrace an underlying premise of saving the most lives andor preserving the most lifeyears Many procedures for patients with HNC are aerosolgenerating and increase the risk to health care workers and other hospitalized patients22 Our process accounted for these by giving consideration to these factors during the consensus process although indications associated with potential exposure to health care workers did not emerge as lowpriority ones Indications associated with lower resource use did achieve consensus for higher importance This may help to avoid the opportunity cost of treating fewer patients with longer surgeriesAnecdotal and institutionspecific prioritization schemas for patients with HNC and general otolaryngology have been suggested213 These parallel similar efforts for general surgery cardiac surgery and orthopedic surgery12132328 In many of these patients are prioritized by the scoring of several criteria and summing of the scores to achieve a total patient score Many of these systems have been validated against expert rankings of surgical priority2728We used a methodology for developing a pointbased prioritization system similar to those previously described29 To the best of our knowledge pointbased surgical prioritization systems have been very well studied to date Hansen et al previously proposed a methodology for developing a pointbased prioritization system using the following steps ranking patient case vignettes Cancer Month 0cSPARTANHNde Almeida et alFIGURE The Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN scoring system ECOG indicates Eastern Cooperative Oncology Group ENE extranodal extensionusing clinical judgment drafting the criteria and categories within each criteria pretesting the criteria and categories consulting with patient groups and other clinicians determining point values for criteria and categories checking the testretest reliability and face validity and revising the points system as new evidence emerges29 Our approach to the development of the SPARTANHN was similar However given the relatively expedited nature of the process we did not directly involve patientsOne method proposed for establishing the priority of all indications in a pointbased scoring system is known as Potentially All Pairwise Rankings of all Alternatives PAPRIKA30 In the current study we chose to use the RANDUCLA process instead of pairwise comparison to minimize computational burden We established Cancer Month 0cOriginal FIGURE External validation rank results A total of experts were asked to rate the scenarios provided shown on the xaxis and the results were compared with the rank generated by models and shown on the yaxis Green shading reflects high priority ranked yellow shading indicates medium priority ranked and red shading indicates low priority ranked Asterisk denotes ties from the algorithm SPARTANHN indicates Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancerindications for surgical prioritization that would create an enormous computational burden using pairwise comparison methodology One problem inherent in the PAPRIKA methodology is the assumption that all indications are not equal and can be ranked However clinically certain indications may be equivalent in priority Furthermore pairwise comparisons assume mutual exclusivity of each of the indications which is not always the case Use of the RANDUCLA consensus process avoids the need for multiple pairwise comparison and allows for consideration of each factor in isolation The goal of the consensus rounds was not to establish a rank order for all indications but mainly to understand which indications result in high intermediate or low priorityThe SPARTANHN algorithm has demonstrated preliminary reliability and validity We demonstrated good agreement between raters and the SPARTANHN algorithm suggesting minimal interpretive error Many of the highpriority indications accounted for some component of interpretation because raters were forced to consider imminent disease progression that may result in an adverse outcome Despite the subjective decisions that must be made as part of SPARTANHN agreement remained high In fact true interrater reliability was found to be higher because the Kalpha is a conservative measure of reliability Other measures of reliability such as the Kendall coefficient of concordance tend to overestimate reliability and cannot be applied to missing data31 Perhaps most important the SPARTANHN correlated highly with expert rankings With established validity this algorithm may be ready for preliminary clinical use although further testing against realworld data to validate it with other cancer outcomes such as survival is neededThe results of the current study must be interpreted within the context of the study design Although externally validated by other surgeons across North America and the United Kingdom the criteria for which consensus was achieved for prioritization were not vetted by patients advocacy groups or other stakeholders such as medical or radiation oncologists The latter groups represent essential providers in the multidisciplinary care of patients with HNC and may have important insight into the availability and effectiveness of nonsurgical treatments1920 Nonetheless the actual prioritization of surgical waitlists remains the sole responsibility of surgeons and their practice partners In addition the SPARTANHN algorithm is intended to assist in making difficult prioritization decisions and is not intended to make recommendations for the time frame in which patients should receive treatment Instead established guidelines should be adhered to for treatment targets Patient wait times as they relate to those targets should be considered when using the SPARTANHN algorithm The validation process in the current study used expert opinion as the gold standard of prioritization which is potentially biased and reflected the opinions of surgeons practicing in academic medical centers from resourcerich nations Subsequently use of the SPARTANHN algorithm in other geographic regions Cancer Month 0cand health care systems requires additional investigation because local treatment paradigms and risk factors may vary substantiallyThe current study has presented the development and validation of a novel algorithm for the prioritization of surgery for patients with HNC Further evaluation of its implementation in various practice settings will be obligatory However the results of the current study have provided data with which to inform realworld use as the current pandemic has obviated our ability to more rigorously study the instrument prior to making necessary and difficult realtime allocation decisionsFUNDING SUPPORTNo specific funding was disclosedCONFLICT OF INTEREST DISCLOSURESEvan M Graboyes has received grants from the National Cancer Institute and the Doris Duke Charitable Foundation for work performed outside of the current study Vinidh Paleri offers his services as a proctor for a transoral robotic surgery proctoring program run by Intuitive Surgical and has been remunerated for his time Antoine Eskander has received a research grant from Merck and acted as a paid consultant for BristolMyers Squibb for work performed outside of the current study Ian J Witterick has stock in Proteocyte Diagnostics Inc and has received honoraria from Sanofi Genzyme and Medtronic Canada for work performed outside of the current study The other authors made no disclosuresAUTHOR CONTRIBUTIONSJohn R de Almeida Study idea and design writing and editing Christopher W Noel Study design writing data analysis and editing David Forner Study design writing data analysis and editing Han Zhang Data acquisition and editing Anthony C Nichols Data acquisition and editing Marc A Cohen Data acquisition and editing Richard J Wong Data acquisition and editing Caitlin McMullen Data acquisition and editing Evan M Graboyes Data acquisition and editing Vasu Divi Data acquisition and editing Andrew G Shuman Writing data acquisition and editing Andrew J Rosko Data acquisition and editing Carol M Lewis Data acquisition and editing Ehab Y Hanna Data acquisition and editing Jeffrey Myers Data acquisition and editing Vinidh Paleri Data acquisition and editing Brett Miles Data acquisition and editing Eric Genden Data acquisition and editing Antoine Eskander Data acquisition and editing Danny J Enepekides Data acquisition and editing Kevin M Higgins Data acquisition and editing Dale Brown Data acquisition and editing Douglas B Chepeha Data acquisition and editing Ian J Witterick Data acquisition

Thyroid_Cancer

immune‘related genes pairs signature predict the prognosis of cervical cancer patientsHan Nie1 Fanqin Bu2 Jiasheng Xu1 Taoshen Li1 Jun Huang2To screen the key immune genes in the development of cervical cancer construct immune related gene pairs IRGPs and evaluate their influence on the prognosis of cervical cancer Tumor Genome Atlas TCGA database and geo database were downloaded as training set and validation set respectively and immune related gene data were downloaded from immport IRGPs model is established by machine learning and the model is analyzed and evaluated Using the Uclcan to analyze the immune genes expression in cervical cancer and to further explore the association with the expression level and the clinical stage and prognosis of cervical cancer According to the analysis of training set we identified IRGPs as key gene pairs and constructed the model The AUC value of the model was greater than and the model group survival rate was conspicuous different P The reliability of the model was confirmed in the validation group Our IRGPs play an important role in the occurrence and development of cervical cancer and can be used as a prognostic marker and potential new target of cervical cancerCervical cancer is one of the four most common gynecological tumors1 Every year at least women in the world are diagnosed with cervical cancer and more than people are killed2 In recent years the incidence rate of cervical cancer has decreased significantly through universal screening and health knowledge However the incidence rate of cervical cancer is still high in developing countries3 For women with low education in less developed areas the coverage rate of cervical cancer screening is still very low4 Squamous cell carcinoma is the most common type of cervical cancer accounting for of cervical cancer cases while adenocarcinoma only accounts for about In developing countries of cervical cancer patients have local infiltration or metastasis which has led to the high mortality of cervical cancer in developing countries Early cervical cancer is usually treated by radical hysterectomy When there are risk factors such as lymph node metastasis and endometriosis that may lead to recurrence they will be treated with chemotherapy5 The standard treatment for patients with locally advanced cervical cancer is conventional radiation therapyCRT6 The fiveyear survival rate of patients with locally advanced cervical cancer can be as high as “ after surgical resection radiotherapy chemotherapy CRT and so on7 However at present all treatment methods are not effective for patients with paraaortic lymph node metastasis and their threeyear progression free survival time PFS and total survival time OS are and respectively The fiveyear survival rate of cervical cancer patients with recurrence and metastasis was as low as Limited treatment is the main reason for this situation Now palliative chemotherapy is the most commonly used for patients with metastatic and recurrent cervical cancer10 The median survival time of patients with metastatic or recurrent cervical cancer treated with platinumtaxane chemotherapy and bevacizumab can be extended to a0months11 However these treatments are far from enough for most locally advanced and metastatic cervical cancer patients with positive lymph node metastasisIn recent years immunotherapy has been developed and increasingly used in cancer patients For example PDL1 is overexpressed in a variety of tumor cells including liver cancer cells and lung cancer cells and plays an important role in regulating the immune response of tumor cells12“ Currently there are several clinical trials involving FDAapproved immunosuppressive checkpoint inhibitors which attack tumor cells expressing PDL1 by blocking the PDL1PD1 signaling pathway so as to improve the treatment and prognosis of patients From the current situation immunosuppressive therapy has achieved good results in many solid tumors16 The 1Department of Vascular Surgery The Second Affiliated Hospital of Nanchang University No Minde Road Nanchang Jiangxi Provence China 2Department of Gastrointestinal Surgery The Second Affiliated Hospital of Nanchang University No Minde Road Nanchang Jiangxi Provence China email junhuangncu163comScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cresults of PD1PDL1 inhibition in cervical cancer are also satisfactory However at present immunoassay sites with a therapeutic effect are scarce and the research on tumor immunotherapy is far from sufficient In this study we screened immune genes that are significantly related to the prognosis of cervical cancer constructed an immune gene pair IRGP model based on these genes and used it to verify the unique prognostic markers of cervical cancerMethodData acquisition Gene expression profile data of patients with cervical squamous cell carcinoma were obtained from cancer and tumor gene map TCGA wwwtcga and gene expression profile data set gse4400116 was obtained from gene expression compilation GEO wwwncbinlmnihgovgeo database including samples of cervical cancer patientsAcquisition of sample immune gene expression immune related genes were downloaded from immport wwwimmpo rthome including antigen presenting cells chemokines and their receptors cytokines and their receptors interferon interleukin etc Using limma package in R we compared the gene expression data of cervical cancer samples downloaded from TCGA database and geo database as training set and verification set with immune related genes and extracted the expression amount of immune related genes in cervical cancer samplesConstruction of immune related gene pairs IRGPs In the two groups of data processed in the previous step the IRGP of the sample is calculated and the relatively high change is selected according to the standard of media absolute deviation IRGP values are calculated by comparing gene expression levels in specific samples or profiles in pairs The immune related genes are matched to compare the IRGPs If the first IRG is larger than the second IRG the output of the IRGP is otherwise the output is If the ratio of IRGP score of or in training set and verification set is higher than then remove the IRGP and retain the remaining IRGP as candidate IRGP for prognosis prediction The logistic rank test was used to screen the prognosis IRGP FDR Cox risk regression analysis and glment in R were used to perform tenfold cross validation to analyze the candidate IRGP and obtain the IRGP index We constructed the best gene pairs as immune gene pair model We use ROC to calculate the optimal cutoff value of IRGP index and use it as the basis to distinguish high and low risk groupsIRGPs model validation The single factor and multi factor Cox proportional risk analysis and survival analysis of TCGA and gse44001 cervical cancer samples were carried out with IRGPs modelInfiltration of immune cells in cervical cancer samples In order to study the infiltration of immune cells in the high and low risk groups of cervical cancer we used CIBERSORT17 to evaluate and predict the enrichment of immune cells in the samples CIBERSORT is a tool for deconvolution of the expression matrix of immune cell subtypes based on the principle of linear support vector regression RNA SEQ data can be used to estimate the infiltration of immune cells CIBERSORT can analyze the relative abundance of immune infiltrating cells in each sample including NK cells T cells B cells and macrophagesFunctional enrichment analysis of GSEA go and KEGG Gene set enrichment analysisGSEA enrichment analysis was carried out for each gene related to immune prognosis using the fgsea package in R Cluster profiler19 was used to enrich Gene ontologyGOfunction and KEGG pathway Significant enrichment criteria the absolute value of NES is greater than the nomp value is less than and the fdrq value is less than Expression of immune gene in cervical cancer Ualcan were used to analyze the expression of immune genes in cervical cancerStatistical analysis Measured data were expressed as mean ± standard deviation x ± s and data were compared using t test Kaplan Meier method was used for survival analysis The receiver operating characteristic curve ROC curve and ROC analysis were completed by survivalROC103 Single factor and multi factor analysis using Cox proportional risk regression model P was statistically significant P a0 as the difference has very significant statistical significanceEthical approval and consent to participate This article does not contain any studies with patients or animals performed by any of the authorsResultsExpression of immune related genes and construction of IRGPs in cervical cancer samples We obtained gene expression data of cervical cancer samples from TCGA database as training set cervical cancer samples from gse44001 as verification set immune related genes from immport and immune related genes from cervical cancer samples by comparing the two Through these immune related genes we constructed IRGPs We remove more than of the IRGP with a score of or from the training set and validation set leaving IRGP as candidates Combine TCGA clinical data with training set data Scientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cTCGA clincial dataAge ‰¥ ‰¥ GradeG1G2G3G4TT1T2T3T4MM0M1NN0N1Table TCGA clinical dataIRG1APOBEC3HARG2BTCCCL2CCL20CCL20CCL20CCL28CXCL1CXCL2DESDESDLL4FLT3LGHCKHCKHLADQA2IL1BIL1BJAK1NOD1NRP1PLXNB3PSMD7RBP7RBP7STC1TLR3VAV3Immune processesAntimicrobialsAntimicrobialsCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesAntimicrobialsCytokinesAntimicrobialsAntimicrobialsAntigen_Processing_and_PresentationAntimicrobialsAntimicrobialsAntimicrobialsAntimicrobialsCytokine_ReceptorsCytokine_ReceptorsAntigen_Processing_and_PresentationAntimicrobialsAntimicrobialsCytokinesAntimicrobialsBCRSignalingPathwayTable Model information about IRGPIImmune processesCytokinesCytokinesCytokinesCytokine_ReceptorsIRG2BTCCLCF1IL16FGFR3APOBEC3C AntimicrobialsARAFPLXNA1MAP3K14TNFSF10PTAFREPORVEGFCDESINHBASAA2STC2LTB4R2DUOX1EDN1APOBEC3C AntimicrobialsCSF2RBCD3DFGFR2SHC1CXCR3DESTNFRSF18CXCR6NRP1NaturalKiller_Cell_CytotoxicityChemokine_ReceptorsTCRsignalingPathwayTNF_Family_MembersChemokine_ReceptorsCytokine_ReceptorsCytokinesCytokinesTGFb_Family_MemberChemokinesCytokinesCytokine_ReceptorsAntimicrobialsChemokinesCytokine_ReceptorsTCRsignalingPathwayCytokine_ReceptorsNaturalKiller_Cell_CytotoxicityChemokine_ReceptorsCytokinesCytokine_ReceptorsAntimicrobialsCytokine_ReceptorsCoefficient““““““““““Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure A Timedependent ROC curve for IRGPI in the training cohort B Timedependent ROC curve for IRGPI in a0year C Timedependent ROC curve for IRGPI in a0year D Timedependent ROC curve for IRGPI in a0yearTable a0 prognosis related IRGPs were screened by lasso Cox proportional risk regression analysis After iterations we selected optimal IRGPs to build the immune prognosis model Table a0IRGPs model validation The immune prognosis model was applied to the training set and the patients in each training set were scored According to ROC curve analysis the optimal cutoff value for patients to be divided into high and low risk groups is Fig a01A After evaluating the model we found that AUC value of model and a0years is Fig a01B Fig a01C and Fig a01D The results show that our immune prognosis gene has a high reliability for the model The training set was divided into highrisk group Fig a02A and highrisk group Fig a02B The results showed that the overall survival rate OS of highrisk group was significantly lower than that of lowrisk group For TCGA training set data single factor and multi factor Cox risk regression analysis showed that only IRGPs model showed significant prognostic effect in single factor Cox Fig a02C while age and IRGPs could be significant independent prognostic factors in multi factor Cox Fig a02D Applying this model to the validation set of gse44001 Fig a03A Table a0 survival analysis showed that the OS of patients in the highrisk group was significantly lower than that in the lowrisk group Fig a03B In the single factor and multi factor Cox analysis IRGPs model and tumor size were significantly correlated with prognosis Fig a03CDInfiltration of immune cells in cervical cancer samples Most studies believe that the occurrence and development of tumor are closely related to immune cells so it is an ideal method to study the infiltration of immune cells in tumor We used CIBERSORT to analyze the infiltration of kinds of immune cells in patients with high and low risk groups Figure a04A shows the expression of immune cells in different risk groups Macrophage M0 Fig a04B activated mast cells Fig a04C were significantly overexpressed in the highrisk group while stationary dendritic cells Fig a04D stationary mast cells Fig a04E activated CD4T cells Fig a04F and cd8t cells Fig a04G were overexpressed in the lowrisk groupScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A The model divides the training set patients into lowrisk or highrisk groups B Kaplan Meier curve between high and low risk groups C Training set single factor Cox regression analysis forest map D Training set multivariate Cox regression analysis forest mapFunctional enrichment analysis of GSEA go and KEGG We analyzed the function enrichment of IRGP in the model The results of go analysis showed that IRGP in the model was mainly enriched in the binding of cytokines and their receptors the binding of chemokines and their receptors the binding of growth factors and their receptors the binding of epidermal growth factor receptors the binding of fibroblast growth factors and the activity of tyrosine kinase Fig a05AB KEGG results showed that these IRGP were involved in cytokine cytokine receptor interaction chemokine signaling tumor necrosis factor signaling MAPK signaling NF kappa B signaling natural killer cellmediated cytotoxicity viral proteins and cytokines and Th1 and Th2 cell differentiation Fig a05CD The results of GSEA Fig a06A showed that these IRGP were significantly enriched in trace ribonucleoprotein complex Fig a06B neurotransmitter transporter activity Fig a06C endopeptidase activity Fig a06D fibroblast growth factor receptor binding Fig a06E hormone activity Fig a06F fibroblast cell proliferation Fig a06G and growth factor receptor binding Fig a06HExpression of immune gene in cervical cancer We explored the expression of IRGP in cervical cancer using the ualcan model Table a0 There were lowlevel expression of IRGP in cervical cancer Fig a0 and highlevel expression of IRGP Fig a0 There were differences in the expression of low expression IRGP and high expression IRGP in different age groups Fig a0 and there were differences in the expression of IRGP in different stages of cervical cancer Fig a0DiscussionCervical cancer is one of the most common gynecological malignancies HPV infection is considered to be the main cause of cervical cancer2122 although the incidence rate of cervical cancer has been significantly decreased due to the development and promotion of HPV vaccine23 But incidence rate of cervical cancer is still high in developing countries and China™s low income countries24 At present for cervical cancer patients without invasion and lymphatic metastasis the effect of surgery combined with radiotherapy and chemotherapy is better If metastasis and infiltration occur the treatment effect of cervical cancer patients will become very unsatisfactory In recent years immunotherapy has performed well in a variety of cancers including cervical cancer25“ Blocking PDL1 PD1 signaling pathway to attack tumor cells expressing PDL1 is the current mainstream method28 Although the anticancer activity of PD1 and PDL1 inhibitors is exciting such immunotherapy is not effective for all patients and a metaanalysis shows that patients who receive PD1 PDL1 inhibitors have a Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure A The model divides the validation set patients into lowrisk or highrisk groups B Kaplan Meier curve between high and low risk groups C Validation set single factor Cox regression analysis forest map D Validation set multivariate Cox regression analysis forest mapGSE44001 clincial dataStageLargest diameter cm ‰¥ ‰¥ ‰¥ Table GSE44001 clincial datahigher risk of rash thyroid dysfunction pruritus pneumonia and colitis29“ Therefore it is of great significance for the detection and treatment of cervical cancer to predict and find more biomarkers that may be related to immune prognosisAt present most of the prognostic genes need to be standardized to reduce the errors caused by sequencing platform and samples In this study the scores of IRGPs constructed by us are calculated from the gene expression data of the same sample which can not only ignore the impact of different platforms but also do not need to standardize and scale the data This method has been used in many studies including cancer molecular classification with high reliability3233In this study we screened pairs of IRGP to construct the immune prognosis model related to the overall survival rate of cervical cancer patients The AUC values of the model in and a0years were all greater than According to these pairs of IRGP they were divided into highrisk group and lowrisk group In TCGA training group and GSE44001 verification group the OS of highrisk group was significantly lower than that of lowrisk group P These pairs of IRGP have a good effect on sample discrimination We found that macrophage Mo and activated mast cells were significantly over expressed in highrisk group by immunocyte infiltration analysis of samples The existing research shows that mast cells and macrophages play an important Scientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Immune infiltration status within IRGPI risk groups B Expression of Macrophage M0 C Expression of Mast cells activated D Expression of Dendritic cells resting E Expression of Mast cells resting F Expression of T cells CD4 memory activated G T cells CD8role in cervical cancer which can promote the development of cervical cancer by promoting lymphangiogenesis and angiogenesis34“ However in the lowrisk group the expression of static dendritic cells static mast cells activated CD4T cells and cd8t cells is high Although the effect of CD4T cells on cervical cancer has not been agreed the cd8t cells are closely related to the better prognosis of cervical cancer patients37“ there is evidence that dendritic cells will decrease in patients with high HPV infection which indicates that high expression of dendritic cells is beneficial to resist cervical cancer40 which is consistent with our results The enrichment analysis of go and GSEA showed that these immune genes were mainly involved in the binding of cytokines and their receptors the binding of chemokines and their receptors the binding of growth factors and their receptors the binding of epidermal growth factor receptors the activity of metalloendopeptidase the binding of fibroblast growth factors and their receptors hormone activity fibroblast proliferation and the binding process of growth Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Histogram graph of Immunerelated genes GO analysis results B Point graph of Immunerelated genes GO analysis results C Histogram graph of Immunerelated genes KEGG pathway analysis results D Point graph of Immunerelated genes KEGG pathway analysis resultsfactor receptorsAs we all know cytokines and chemokines are the key factors in the immune response for example In cervical cancer IL10 can interfere with the differentiation of dendritic cells and thus play a strong immunosuppressive effectTGF”β can inhibit T cell proliferation and attenuate immune response41 Research shows that growth factors and epidermal growth factors are closely related to the growth of cervical cancer and the survival rate of cervical cancer patients High expression of growth factors and epidermal growth factors often predict poor prognosis42“ Growth of fibroblasts can stimulate angiogenesis at the early stage of tumor The proliferation and invasion of cancer cells and the remodeling of extracellular matrix promote the growth of cervical cancer4546 KEGG results showed that these immune genes were mainly enriched in chemokine signaling pathway tumor necrosis factor signaling pathway MAPK signaling pathway NF kappa B signaling pathway natural killer cellmediated cytotoxicity viral protein and cytokine and Th1 and Th2 cell differentiation Th1 and Th2 may be involved in the pathogenesis and growth of cervical cancer Th1 may be the target of predicting chemotherapy response of advanced cervical cancer47“ while other pathways are classical signal pathways related to cancer Immune cytokines play an important role in cervical lesions Torres et a0al Found that IL10 is highly expressed in the cervix of women with persistent HPV which may be related to the persistence of HPV and the promotion of disease progression Further research by their team showed that copy individuals of IL4 IL6 IL10 and TGFB1 were significantly associated with cervical cancer and could be used as biomarkers for susceptibility to the disease5152These pairs of IRGP have different immune genes most of which are cytokines antimicrobial agents and natural killer cells which are involved in various stimulation reactions and play a key role In cervical cancer HPV can inhibit the apoptosis of cervical cancer cells by down regulating NOD153 In our sample we also found that the expression of NOD1 in tumor tissue is low and there are differences in different ages and stages Figs a07D 9C 10I Sang Yeon Cho et a0al Found that duox1 is highly expressed in cervical squamous cell carcinoma and can play a good prognostic role by increasing the amount of innate immune cells54 The analysis also showed that DUOX1 is highly expressed in tumor tissues and related to age and grade Figs a08B 9G 10D Stc2 can promote the proliferation of cervical cancer cells and increase the resistance to cisplatin55 while high expression of DDL4 is usually associated with low pelvic lymph node metastasis and survival rate of cervical cancer56 Therefore we believe that the IRGP constructed in this study plays an important role in the development and prognosis of cervical cancerThere are also some deficiencies in our research Although we select data samples from two databases for analysis and use more advanced methods to reduce the errors caused by platforms samples etc this is still a retrospective analysis If we can carry out a prospective study or obtain clinical samples and evaluate them with Western blot or immunohistochemistry it will be more convincingScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A GSEA analysis of immune signature genes B“H In the high immune risk group of cervical cancer cancer marker genes were abundant P FDR Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cCLCF1DLL4INHBANOD1NRP1RBP7CXCR3DUOX1FGFR3AgeNormalvsAge4160YrsAge2140YrsvsAge4160YrsAge2140YrsvsAge6180YrsAge2140YrsvsAge81100YrsAge4160YrsvsAge6180YrsAge4160YrsvsAge81100YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsAge2140YrsvsAge81100YrsAge4160YrsvsAge6180YrsAge4160YrsvsAge81100YrsAge6180YrsvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsNormalvsAge2140YrsHLADQA2NormalvsAge4160YrsLTB4R2STC2TNFSF10VAV3NormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge4160YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsAge4160YrsvsAge6180YrsPval777E05123E02483E04115E07209E02433E07270E06354E05281E02982E03260E03384E04322E02163E05600E04868E03106E02109E02122E04396E03281E04459E02421E04876E03441E02105E02384E04566E05218E05803E09139E06225E08111E16204E13725E10305E02422E07693E10174E05433E15162E12550E10341E02803E04159E06458E04192E02524E10415E11559E10162E12162E12934E14108E02208E03StageNormalvsStage2NormalvsStage3Stage1vsStage2Stage1vsStage3Stage1vsStage4NormalvsStage1NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage4Stage3vsStageNormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Pval185E06220E04542E03304E02460E04696E07546E03252E03210E02439E02390E03186E02395E02317E02122E03164E03396E04984E03222E02315E06226E02387E07734E03433E01118E04173E05355E04222E02160E05259E09131E09258E04162E12199E12176E05202E04663E12390E05241E04162E12860E10479E09175E04148E02240E04435E05119E04463E02230E13297E09263E07840E04170E02162E12493E12284E12125E04Table P value of IRGPs expression in different ages and stagesScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues B Expression of DLL4 in cervical cancer and normal tissues C Expression of INHBA in cervical cancer and normal tissues D Expression of NOD1 in cervical cancer and normal tissues E Expression of NRP1 in cervical cancer and normal tissues F Expression of RBP7 in cervical cancer and normal tissuesScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Expression of CXCR3 in cervical cancer and normal tissues B Expression of DUOX1 in cervical cancer and normal tissues C Expression of FGFR3 in cervical cancer and normal tissues D Expression of HLADQA2 in cervical cancer and normal tissues E Expression of LTB4R2 in cervical cancer and normal tissues F Expression of STC2 in cervical cancer and normal tissues G Expression of TNFSF10 in cervical cancer and normal tissues H Expression of CESC in cervical cancer and normal tissuesScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues at different ages B Expression of DLL4 in cervical cancer and normal tissues at different ages C Expression of NOD1 in cervical cancer and normal tissues at different ages D Expression of NRP1 in cervical cancer and normal tissues at different ages E Expression of RBP7 in cervical cancer and normal tissues at different ages F Expression of CXCR3 in cervical cancer and normal tissues at different ages G Expression of DUOX1 in cervical cancer and normal tissues at different ages H Expression of FGFR3 in cervical cancer and normal tissues at different ages I Expression of HLADQA2 in cervical cancer and normal tissues at different ages J Expression of LTB4R2 in cervical cancer and normal tissues at different ages K Expression of STC2 in cervical cancer and normal tissues at different ages L Expression of TNFSF10 in cervical cancer and normal tissues at different ages M Expression of VAV3 in cervical cancer and normal tissues at different agesConclusionWe constructed an immune gene pair model which is closely related to the prognosis of cervical cancer patients The model contains IRGP and immunerelated genes The biological functions of these immunerelated genes are closely related to the occurrence and development of cervical cancer Therefore we think that these IRGPs may be the target of predicting or diagnosing cervical cancer and suggest that immunotherapy can improve the prognosis of cervical cancer patients by regulating these IRGPsScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues at different stages B Expression of CXCR3 in cervical cancer and normal tissues at different stages C Expression of DLL4 in cervical cancer and normal tissues at different stages D Expression of DUOX1 in cervical cancer and normal tissues at different stages E Expression of FGFR3 in cervical cancer and normal tissues at different stages F Expression of HLADQA2 in cervical cancer and normal tissues at different stages G Expression of INHBA in cervical cancer and normal tissues at different stages H Expression of LTB4R2 in cervical cancer and normal tissues at different stages I Expression of NOD1 in cervical cancer and normal tissues at different stages J Expression of NRP1 in cervical cancer and normal tissues at different stages K Expression of RBP7 in cervical cancer and normal tissues at different stages L Expression of STC2 in cervical cancer and normal tissues at different stages M Expression of TNFSF10 in cervical cancer and normal tissues at different stages N Expression of VAV3 in cervical cancer and normal tissues at different stagesData availabilityAll data are available Please contact us to access if it is neededReceived May Accepted July References Stewart Bernard W et al Cancer prevention as part of precision medicine œplenty to be done Carcinogenesis “ 101093carci nbgv16 Bray F et al Global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “ 103322caac21492 Passos Camila M Sales Jacqueline B Maia Emanuella G Caldeira Thaís C M Rodrigues Roberta D Figueiredo N Claro Rafael M Trends in access to female cancer screening in Brazil “ J Public Health Oxf 101093pubme dfdaa0 Asrabuddhe V V Parham G P Mwanahamuntu M H Vermund S H Cervical cancer prevention in low and middleincome countries feasible affordable essential Cancer Prevent Res “ 10115819406207CAPR110540 Koh WuiJin AbuRustum Nadeem R Bean Sarah Bradley Kristin Campos Susana M Cho Kathleen R Chon Hye Sook Chu Christina Clark Rachel Cohn David Crispens Marta Ann Damast Shari Dorigo Oliver Eifel Patricia J Fisher Christine M Frederick Peter Gaffney David K Han Ernest Huh Warner K Lurain John R Mariani Andrea Mutch David Nagel Christa Nekhlyudov Larissa Fader Amanda Nickles Remmenga Steven W Reynolds R Kevin Tillmanns Todd Ueda Stefanie Wyse Emily Yashar Catheryn M McMillian Nicole R Scavone Jillian L2019 Cervical Cancer Version NCCN Clinical Practice Guidelines in Oncology J Natl Compr Canc Netw “ 106004jnccn Chen J et al Nanotechnology in the management of cervical cancer Rev Med Virol 25Suppl “ 101002 Varia M A et al Cervical carcinoma metastatic to paraaortic nodes extended field radiation therapy with concomitant 5fluorouracil and cisplatin chemotherapy a Gynecologic Oncology Group study Int J Radiat Oncol Biol Phys “ 101016s0360 Randall Leslie M Monk Bradley J Darcy Kathleen M Tian C Burger Robert A Liao SY Peters William A Stock Richard J Fruehauf John P Markers of angiogenesis in highrisk earlystage cervical cancer a Gynecologic Oncology Group study Gynecol Oncol 101016jygyno Boussios S et al Management of patients with recurrentadvanced cervical can

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

"unrestricted use distribution and reproduction in any medium provided the original work is properly citedPurpose The present study was aimed at determining the serum levels of actinin4 ACTN4 in cervical cancer CC andinvestigating the diagnostic and prognostic value of serum ACTN4 in CC Materials and Methods We included CC patients cervical intraepithelial neoplasia CIN patients and healthy women Serum ACTN4 levels were assessed using an ELISAmethod A receiver operating characteristic ROC curve was performed to evaluate the diagnostic value of serum ACTN4 Thesurvival curves were used to display the overall survival distributions Results Serum ACTN4 levels in CC patients were ± pgmL which is significantly higher than those in CIN patients ± pgmL P and those in healthycontrols ± pgmL P The ROC analysis demonstrated that the area under the curve AUC of ACTN4 was 95CI “ with sensitivity of and specificity of Serum ACTN4 levels were associated with theFIGO stage lymph node metastasis and lymphovascular space invasion of CC all P The survival curve suggested thathigh serum ACTN4 levels were related to poor prognosis Conclusion Our findings suggest that serum ACTN4 levels may bevaluable diagnostic and prognostic biomarkers for CC IntroductionCervical cancer CC is the second most common femalemalignancy globally and it is the most common femalemalignancy in developing countries which has high morbidity and mortality rates [] In recent years the incidence ofCC has increased greatly in young women under the age of [] Despite great advances in surgical and adjuvant therapy the overall survival of CC patients especially that ofadvanced patients is still very poor [] At present a Papsmear combined with an HPV test has been used for the earlyscreening of cervicalthe screeningmethods are invasive and costly leading to lower screeningcoverage in China [] Previous studies have reported thatthe human papillomavirus HPV screening results have arelatively high falsepositive rate and a relatively low specificity [ ] In addition the results of TCT interpretation byfilmreading doctors are uneven which might cause somelesions Howevermisleadingness in the choices of prevention measures andtreatment for CC [] Noteworthily when applying the sametreatment plan to patients with similar pathological types theefficacy and prognosis are quite diï¬erent Therefore it is necessary to identify new biomarkers directly related to the progression and prognosis of CCAlphaactinins ACTNs are actinbinding proteins inthe spectrin gene superfamily [] which are known to becrosslinked with filamentous actin Factin to maintainthe integrity of cytoskeleton and to control cell motility []The ACTN family has four members numbered ACTN1“which are present in humans and other mammals [“]ACTN4 is encoded by the ACTN4 gene and is widelyexpressed in many tissues especially in glomerular podocytes[] ACTN4 has an actinbinding domain at the Nterminus and ACTN4 monomers can form a homodimer throughreverse binding forming a dumbbellshaped structure []As an actinbinding protein ACTN4 is closely related to 0cDisease Markersenhancing cell viability and tumor invasion and metastasis[] Recent researches have reported that the expression ofACTN4 is significantly elevated in multiple cancers including breast cancer [] pancreatic cancer [] ovarian cancer[] and lung cancer [] In addition the ACTN4 levels aremarkedly associated with the poor prognosis of lung cancer[ ] thyroid cancer [] and salivary gland carcinoma[] An [] have found that the expression level ofACTN4 in human cervical tumors is dramatically higherthan that in normal cervical tissues Their finding demonstratedepithelialtomesenchymal transition and tumorigenesis by regulatingSnail expression and the Akt pathway in CC [] Thereforethe expression of ACTN4 in cervical tissues may be used inthe clinical diagnosis and prognosis prediction of CCthat ACTN4promotestheHowever up to now the significance of the serumACTN4 levels in CC has not been evaluated Hence in thecurrent study the serum levels of ACTN4 in patients withCC were measured In addition we estimated the potentialdiagnostic and prognostic value of serum ACTN4 expressionin CC Materials and Methods Study Population A retrospective study was designed toevaluate serum actinin4 as a biomarker for CC Between July and June newly diagnosed female CC patientsand newly diagnosed female cervical intraepithelial neoplasia CIN patients who received treatment at Huai™anMaternal and Child Health Care Hospital Huai™an JiangsuChina were recruited The diagnoses of all patients were verified by the histopathological examination The patients withother types of tumor or autoimmune atherosclerotic andhematologic diseases were excluded The mean age of CCpatients was years with a range of years Meanwhile healthy women with no evidence of neoplasmsand other serious diseases were enrolled from the physicalexamination center in the same hospital There was no significant diï¬erence in age among the CC CIN and healthy control groups This study was consistent with the Helsinkideclaration and was authorized by the Ethics Committee ofHuai™an Maternal and Child Health Care Hospital approvalnumber H20130504 All participantssigned writteninformed consent Clinicopathologic Feature Collection and FollowUp Byreviewing the medical records we collected the clinicopathologic characteristics of the patients including age at diagnosis pathological type FIGO stage tumor diï¬erentiationpelvic lymph node metastasis tumor size and lymphovascular space invasion The CC patients were classified based onthe revised FIGO staging system for CC in The tumorsize was the maximum tumor diameter determined by agynecologic oncologist during pelvic examination Thepatients in stage 1A1 received hysterectomy the patients instages IB1 and IIB received radical hysterectomy and pelviclymph node dissection the patients with ‰¥stage IIB receivedradiotherapy or radiotherapy combined with chemotherapyA regular telephone followup was conducted after treatmentto obtain the overall survival OS time of CC patients andthe OS was defined as the time from diagnosis to death orthe last followup The followup was in accordance withthe FIGO guidelines Blood Sample Collection and Detection of Serum Actinin and SCCA A mL peripheral blood sample from eachpatient was collected before receiving any treatment Afterstanding at room temperature for minutes the blood samples were centrifugated at gmin for min and then°the supernatant was stored at ˆ’C until further usageThe serum actinin4 concentration was measured by a quantitativeELISAmethod Uscn Life Science Inc Wuhan China The levelsof SCCA in serum were determined using an ELISA kitRD Systems Minneapolis MN The detection of all samples was strictly in accordance with the instructions providedby the manufacturer and was performed in duplicatesenzymelinked immunosorbentassay Statistical Analysis All statistical analyses were conducted by using SPSS and GraphPad Prism The continuous data following normal distribution were expressed asthe mean ± standard deviation°SDž A ttest was used tocompare serum ACTN4 levels between the two subgroupsof each clinicopathological parameters and the serumACTN4 levels of CC patients CIN patients and healthy controls were compared by the SNKq test Receiver operatingcharacteristic ROC curves were performed to assess thediagnostic value of serum ACTN4 levels for diï¬erentiatingCC patients from CIN patients and healthy controls TheKaplanMeier method and logrank test were used to plotsurvival curves The Cox proportional hazards models in univariate and multivariate analyses were used for evaluating theprognostic value of serum ACTN4 expression A twotailed Pvalue was considered to be statistically significant Results Serum ACTN4 Levels Are Higher in Patients with CCSerum concentrations of ACTN4 were detected to rangefrom to pgmL with a mean ±SD of ± pgmL for CC patientsto range from to ngmL with a mean ±SD of ± pgmL forCIN patients and to range from to ngmL witha mean ±SD of ± pgmL for healthy controlsSerum ACTN4 levels in CC patients were significantly higherthan those in CIN patients and healthy controls P However no significant diï¬erence in serum ACTN4 wasfound between CIN patientscontrolsP as shown in Figure and healthy The Diagnostic Value of Serum ACTN4 Levels for CC Wenext used ROC curve analysis to estimate the diagnostic valueof serum ACTN4 expression for CC The ROC curve showedthat the serum levels of ACTN4 were robust for discriminating CC patients from benign and healthy control subjectswith an area under the curve AUC value of 95CI “ as demonstrated in Figure index we usedAccordingto maximum Youden™s 0cDisease MarkersŽŽlymph node metastasis were the independent prognostic factors for CC all P Table Lmgp NTCAnsCCCINCON DiscussionCervical cancer is a heterogeneous disease with complicatedetiology Genetic and environmental factors play a crucialrole in the pathogenesis of CC [] Although the diagnosisand prognosis of CC have improved greatly over the pastfew decades it is necessary to improve early detection andscreening methods to determine additional promising circulating biomarkers for better patient selection and more personalized treatments [] As far as we know this studyrepresented the first eï¬ort to evaluate the serum expressionof ACTN4 as a new biomarker for CCAs an actinbinding protein ACTN4 can participate inregulating cell migration invasion and metastasis via regulating the actin filament flexibility at the leading edge ofinvading cancer cells [ ] ACTN4overexpressing cancercells have the potential to metastasize because the overexpression of ACTN4 protein in cancer cells can stimulate thedynamic reconstruction of the actin cytoskeleton [] Upto now numerous studies have reported the associationbetween ACTN4 and multiple cancers Okamoto []observed that ACTN4 is expressed in smallcell lung cancerNSCLC and it had a significant correlation with invasionand distant metastasis Additionally ACTN4 was reportedto be a potential predictive biomarker for the efficacy of adjuvant chemotherapy in patients with NSCLC [] Watabe [] revealed that the copy number increase of ACTN4is a novel indicator for poor overall survival of patients withsalivary gland carcinoma and the copy number variationwould aï¬ect the expression of protein A recent study demonstrated that serum ACTN4 levels were dramatically elevated in patients with breast cancer when compared tohealthy controls and serum ACTN4 may be an eï¬ective clinical indicator for diagnosing or predicting the clinical outcomes of breast cancer patients [] In addition ACTN4was proven to be associated with the pathogenesis of CCAn [] proposed a novel mechanism for epithelialtomesenchymal transition and tumorigenesis in CC whichcould be induced by ACTN4 through regulating Snail expression and βcatenin stabilization Hence it is significant toinvestigate the role of serum ACTN4 in CCIn the current study we observed that serum levels ofACTN4 in CC patients were statistically higher than thosein CIN patients and those in healthy controls Howeverserum ACTN4 levels were not significantly diï¬erent betweenthe CIN group and the control group It was shown thatserum ACTN4 expression could strongly diï¬erentiate CCpatients from CIN patients and healthy controls The ROCanalysis demonstrated that the AUC of ACTN4 was and at the optimal cutoï¬ of pgmL the sensitivity andspecificity were respectively and suggestingthat serum ACTN4 might be a potential diagnostic biomarker for CC In a recent study which included Chinesewomen Hu [] reported that the sensitivity and specificity of HPV screening in the diagnosis of CC were and The sensitivity of the HPV test was a litter higherFigure The serum ACTN4 levels in CC patients CIN patientsand healthy controls ˆ—P pgmL as the cutoï¬ value and the sensitivity and specificity were and respectively Association between Serum ACTN4 Levels andClinicopathological Parameters of CC Patients We furtherinvestigated the correlations between serum levels of ACTN4and clinical pathological data of CC patients and theresults are demonstrated in Table We observed that serumACTN4 levels were related to the FIGO stage lymph nodemetastasis and lymphovascular space invasion all P Nevertheless no significant association was found betweenserum ACTN4 levels and age pathological type diï¬erentiation degree and tumor size in CC patients all P Survival Analysis of Serum ACTN4 Levels in CC Duringthe followup period nine CC patients were lost and thefollowed up rate is Finally the prognostic value ofserum ACTN4 was assessed in patients The patients werefollowed up to December The range of followup timewas to months with the median time of months andmean time of months According to the median serumlevels of ACTN4 in CC patients pgmL the CCpatients were divided into the high ACTN4 level group pgmL N and low ACTN4 level group‰¥ pgmL N The estimated 5year OS of patientswith high serum ACTN4 levels and low serum ACTN4 levelswere and respectively The KaplanMeier survival curve and logrank test indicated that CC patients withhigh serum ACTN4 levels had a worse prognosis than thosewith low serum ACTN4 levels P Figure Univariate Cox regression analyses showed that theserum ACTN4 levels P FIGO stage P diï¬erentiation degree P lymph node metastasisP and lymphovascular space invasion P had significant prognostic value for OS Multivariate analysiswas further performed to evaluate the prognostic value ofserum ACTN4 as an independent factor for CC All the statistically significant factors from univariate analyses wereincluded and the results indicated that the FIGO stage and 0cDisease MarkersytivitisneS ˆ’ specificityFigure ROC curve analysis assessed the diagnostic performance of serum ACTN4 in CC The AUC was P Table Serum ACTN4 levels in CC patients according toclinicopathological parametersParametersAge years‰¤Pathological typeSquamous cell carcinomaAdenocarcinomaFIGO stageIA1IB1‰¥IB2Diï¬erentiationWell and moderatelydiï¬erentiatedPoorly diï¬erentiatedLymph node involvementNegativePositiveTumor size‰¤Lymphovascular space invasionNegativePositiveN ACTN4pgmLP ± ± ± ± ± ± ± ± ± ± ± ± ± ± than that of serum ACTN4 detection though the specificityof serum ACTN4 detection was well above that of the HPVtest Hence comparing with the HPV test in diagnosingCC detecting serum ACTN4 has some advantages Furthermore serum ACTN4 levels have been indicated to be a greatbiomarker for diagnosing multiple cancers Fang [] intheir study reported that serum ACTN4 was a promisingindicator for diagnosing breast cancer with the AUC of Wang [] used ACTN4 expression in peripheralblood to diï¬erentiate NSCLC patients from healthy individuals in two groups of participants and they obtained bothsatisfactory eï¬ects Furthermore we investigated the correlation between serum ACTN4 and clinical characteristics ofCC patients The serum ACTN4 levels were significantlyassociated with the FIGO stage lymph node metastasis andlymphovascular space invasion of CC which suggests thatACTN4 could contribute to the development invasion andmetastasis of CC In addition our results indicated that highACTN4 levels were associated with the poor survival of CCpatients In the multivariate analysis although ACTN4 levelsdid not reach the statistical significance it still seems to beable to influence the OSHowever several limitations in the present study should betaken into consideration First the sample size was relativelysmall which was likely to reduce the statistical power of ourresults Second we only explored the relationship betweenserum ACTN4 and OS and other prognostic indicators werenot examined due to the incomplete data which needs to beimproved in the future Third this study was a primary studyto determine the clinical significance of serum ACTN4 levelsfor the diagnosis and prognosis of CC but the specific molecular mechanisms remain unclear Hence further experimentsshould be conducted to elucidate the mechanismsIn conclusion our study showed that serum ACTN4levels were increased in CC patients and were related to the 0cDisease Markers lavivrus muCLog rank P Overall survival monthsLow ACTN4 groupHigh ACTN4 groupFigure KaplanMeier curve compared OS of CC patients with high serum ACTN4 levels versus those with low serum ACTN4 levelsTable Univariate and multivariate Cox regression analysis of OS in CC patientsUnivariate CIVariablesAge vs ‰¤ yearsPathological type squamous cell carcinoma vs adenocarcinomaFIGO stage ‰¥IB2 vs IA1IB1Diï¬erentiation poorly diï¬erentiated vs well and moderately diï¬erentiated “Lymph node involvement positive vs negativeTumor size vs ‰¤ cmLymphovascular space invasion positive vs negativeSerum ACTN4 levels high vs low levelsHR “ “ “ “ “ “ “ “ “ “ “ “P”””Multivariate CIPHRFIGO stage lymph node metastasis and lymphovascularspace invasion of CC patients In addition serum levels ofACTN4 have great diagnostic and prognostic value in CCNevertheless further studies with a larger sample size shouldbe carried out to confirm our resultsAcknowledgmentsWe thank all the patients and blood donors who participatedin our study This study was funded by grants from the Science and Technology Project of Traditional Chinese Medicine Bureau of Jiangsu province China YB2015128Data AvailabilityReferencesThe datasets used andor analyzed during the present studyare available from the corresponding author on reasonablerequestConflicts of InterestAll authors declare that they have no conflicts of interestAuthors™ ContributionsXigui Ma and Huiying Xue contributed equally to this workand should be considered as cofirst authors[] M H Forouzanfar K J Foreman A M Delossantos et alœBreast and cervical cancer in countries between and a systematic analysis The Lancet vol no pp “ [] E Pelkofski J Stine N A Wages P A Gehrig K H Kimand L A Cantrell œCervical cancer in women aged yearsand younger Clinical Therapeutics vol no pp “ [] Y Zhou W Wang R Wei œSerum bradykinin levels as adiagnostic marker in cervical cancer with a potential mechanism to promote VEGF expression via BDKRB2 International Journal of Oncology vol pp “ [] Y J Hu H P Zhang B Zhu H Y Chen L H Ma andY Wang œThe role of FH detection combined with HPV 0cDisease Markers[] N Miura M Kamita T Kakuya œEfficacy of adjuvantchemotherapy for nonsmall cell lung cancer assessed by metastatic potential associated with ACTN4 Oncotarget vol no pp “ [] N Tanaka T Yamashita S Yamamoto œHistologicalgrowth pattern of and alphaactinin4 expression in thyroidcancer Anticancer Research vol no pp “[] Y Watabe T Mori S Yoshimoto œCopy numberincrease of ACTN4 is a prognostic indicator in salivary glandcarcinoma Cancer Medicine vol no pp “ [] HT An S Yoo and J Ko œÎ±Actinin4 induces theepithelialtomesenchymal transition and tumorigenesis viaregulation of Snail expression and βcatenin stabilization incervical cancer Oncogene vol no pp “[] F Niu T Wang J Li œThe impact of genetic variants inIL1R2 on cervical cancer risk among Uygur females fromChina a casecontrol study Molecular Genetics GenomicMedicine vol no article e00516 [] W Li Y Zhao L Ren and X Wu œSerum human kallikrein represents a new marker for cervical cancer Medical Oncology vol no p [] H Shao J HC Wang M R Pollak and A Wells œÎ±Actinin4 is essential for maintaining the spreading motility andcontractility of fibroblasts PLoS One vol no articlee13921 [] K Honda T Yamada Y Hayashida œActinin4 increasescell motility and promotes lymph node metastasis of colorectalcancer Gastroenterology vol no pp “ [] D G Thomas and D N Robinson œThe fifth sense mechanosensory regulation of alphaactinin4 and its relevance forcancer metastasis Seminars in Cell Developmental Biologyvol pp “ screening on the diagnostic significance of cervical cancer andprecancerous lesions European Review for Medical and Pharmacological Sciences vol no pp “ [] KH Wang C J Lin C J Liu œGlobal methylationsilencing of clustered protocadherin genes in cervical cancerserving as diagnostic markers comparable to HPV CancerMedicine vol no pp “ [] T Li Y Li G X Yang œDiagnostic value of combination of HPV testing and cytology as compared to isolatedcytology in screening cervical cancer a metaanalysis Journal of Cancer Research and Therapeutics vol no pp “ [] K Honda T Yamada R Endo œActinin4 a novel actinbundling protein associated with cell motility and cancer invasion The Journal of Cell Biology vol no pp “ [] E de Almeida Ribeiro N Pinotsis A Ghisleni œThestructure and regulation of human muscle αactinin Cellvol no pp “ [] D Wang X W Li X Wang œAlphaactinin4 is a possible target protein for aristolochic acid I in human kidneycellsin vitro The American Journal of Chinese Medicinevol no pp “ [] I V Ogneva N S Biryukov T A Leinsoo and I M Larina œPossible role of nonmuscle alphaactinins in musclecell mechanosensitivity PLoS One vol no articlee96395 [] K Honda œThe biological role of actinin4 ACTN4 in malignant phenotypes of cancer Cell Bioscience vol no p [] X Zhao K S Hsu and J H Lim œÎ±Actinin potentiatesnuclear factor κlightchainenhancer of activated BcellNFκB activity in podocytes independent of its cytoplasmic actin binding function The Journal of BiologicalChemistry vol no pp “ [] H Shams J Golji K Garakani and M R Mofrad œDynamicRegulation of α Actinin's Calponin Homology Domains on FActin Biophysical Journal vol no pp “[] C Fang J J Li T Deng B H Li P L Geng and X TZeng œActinin4 as a diagnostic biomarker in serum ofbreast cancer patients Medical Science Monitor vol pp “ [] T Watanabe H Ueno Y Watabe œACTN4 copynumber increase as a predictive biomarker for chemoradiotherapy of locally advanced pancreatic cancer British Journal of Cancer vol no pp “ [] S Yamamoto H Tsuda K Honda œACTN4 gene amplification and actinin4 protein overexpression drive tumourdevelopment and histological progression in a highgrade subset of ovarian clearcell adenocarcinomas Histopathologyvol no pp “ [] M C Wang Y H Chang C C Wu œAlphaactinin is associated with cancer cell motility and is a potential biomarker in nonsmall cell lung cancer Journal of ThoracicOncology vol no pp “ [] N Okamoto H Suzuki K Kawahara œThe alternativelyspliced actinin4 variant as a prognostic marker for metastasisin smallcell lung cancer Anticancer Research vol no pp “ 0c"

Thyroid_Cancer

catalytic activity of human Telomerase Reverse Transcriptase TERT compensates for the loss of telomere length eroded during each cell cycle to ensure a correct division of stem and germinal cells In human tumors ectopic TERT reactivation most frequently due to hotspot mutations in the promoter region TERTp ie c1124 C T c1146 C T confers a proliferative advantage to neoplastic cells In gliomas TERTp mutations TERTpmut mainly occur in oligodendroglioma and glioblastoma We screened for TERTp hotspot mutations adult patients with gliomas and identified heterozygous mutations in cases of oligodendroglioma of glioblastoma and of diffuseanaplastic astrocytoma Besides the recurrent c1124 C T and c1146 C T two cases of glioblastoma harbored novel somatic TERTp variants which consisted of a tandem duplications of nucleotides ie a TERTp c1100_179dup and TERTp c1110_189 both located downstream c1124 C T and c1146 C T In silico analysis predicted the formation of and new transcription factor™s recognition sites for TERTp c1100_179dup and TERTp c1110_189 respectively TERTp duplications TERTpdup mainly affected the binding capacity of two transcription factors™ families ie the members of the Etwentysix and the Specificity ProteinKr¼ppelLike Factor groups In fact these new TERTpdup significantly enhanced the Etwentysix transcription factors™ binding capacity which is also typically increased by the two c1124 C Tc1146 C T hotspot TERTpmut On the other hand they were distinguished by enhanced affinity for the Kr¼ppel proteins The luciferase assay confirmed that TERTpdup behaved as gainoffunction mutations causing a fold increase of TERT transcription The present study provides new insights into TERTp mutational spectrum occurring in central nervous system tumors with the identification of new recurrent somatic gainoffunction mutations occurring in of glioblastoma IDHwildtypeKeywords TERT Gliomas Gainoffunction mutation ETS and Kr¼ppel transcription factorsIntroductionThe abnormal reactivation of human Telomerase Reverse Transcriptase TERT is a common hallmark of human solid tumors Although it may be caused by Correspondence cristinamecucciunipgit robertalastarzaunipgit Cristina Mecucci Roberta La Starza have equally contributed to this work Molecular Medicine Laboratory Centro di Ricerche EmatoOncologiche CREO S Maria della Misericordia Hospital University of Perugia Ple Menghini Perugia ItalyFull list of author information is available at the end of the several mechanisms ie methylation mutations rearrangementsfusions and DNA copy number amplifications TERT promoter TERTp methylation and gainoffunction mutations are the most frequent [ ] In particular two recurrent hotspot mutations are respectively located at TERTp124 and TERTp146 base pairs bp from the TERT ATG start site [ “ ] Both mutations generated from a cytidine to thymidine dipyrimide transition C T are usually heterozygous mutually exclusive and produce The Authors 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 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 0cPierini a0et a0al acta neuropathol commun Page of an identical a0bp ˜CCC CTT CCGGG™ sequence resulting in the creation of de novo consensus binding motifs for Etwentysix ETS transcription family members These new binding sites recruit a larger number of ETS factors enhancing the transcription of TERT []TERT promoter mutations TERTpmut typically occur in tumors that arise from low selfrenewal tissue such as melanomas thyroid hepatobiliary carcinoma and central nervous system CNS tumors with a variable frequency that range from to of cases in diverse histological subtypes [ ] In CNS tumors TERTpmut are typically associated with glioblastoma GBM “ and oligodendroglioma ODG “ whereas their frequency decreases in other glioma subtypes such as diffuseanaplastic astrocytoma DAAA “ medulloblastoma and meningioma about [ ] Although the clinical value of TERTpmut in refining the diagnostic classification of gliomas is widely accepted [] its role as prognosticpredictive biomarker is still largely debated TERTpmut have been associated with a poor disease outcome in GBM IDHwildtype GBM IDHwt but there is no full agreement on its impact on DAAA [ ] It is worth noting however that DAAA IDHwildtype DAAA IDHwt harboring genomic abnormalities typically associated with GBM ie TERTp mutations or EGFR amplification or gain of whole chromosome in combination with monosomy of chromosome have a clinical outcome similar to or only slightly longer than GBM [] Thus the cIMPACT NOW Update recommended to use one of these molecular criteria to classify this subgroup of astrocytomas as œdiffuse astrocytic glioma IDHwildtype with molecular features of glioblastoma WHO grade IV and to revise the classification of DAAA IDHwt accordingly []Herein we report two new TERTp mutations that were identified in two patients with GBM IDHwt Both these new variants originated from the duplication of a stretch of nucleotides at TERTp TERTpdup and although slightly different shared an overlapping sequence of nucleotides We demonstrated the somatic nature of one of these TERTpdup and that enhancing the binding affinity for ETS transcription factors TFs they both elicit the TERT transcription thus widening the spectrum of recurrent gainoffunction mutations of TERTp in GBMCase presentationCohortThe study was carried out on a cohort of patients affected by primary CNS tumours and referred to our laboratory during the last a0years Table a0 There were males and females ratio with a median age of range age According to the WHO the diagnosis was grade II DA IDHwt cases and DA IDHmutant DA IDHmut cases grade III AA IDHwt cases and AA IDHmut grade IV GBM IDHwt and GBM IDHmut grade IIIII ODG Three patients had a diagnosis of uncommon glioma Table a0 The study was approved by Institutional Bioethics Committee University of Perugia and Santa Maria della Misericordia Hospital of PerugiaItaly Protocol no284316 all patients gave informed consent for sample collection and molecular analyses in agreement with the Declaration of HelsinkiIndex casesA 71yearold male UPN131 had a left frontal lesion of a0mm diameter partially infiltrating the corpus callosum the second case UPN171 a male of a0years presented with a right frontal lesion Histopathology and immunohistochemistry were consistent with a diagnosis of GBM IDHwt in both patients In case UPN131 neoplastic cells showed marked cytoplasmic and nuclear pleomorphism there was a discrete number of atypical mitotic figures widespread necrosis a diffuse GFAP positivity and few neoplastic elements with strong nuclear TP53 stain Case UPN171 was characterized by striking atypia of neoplastic cells diffuse necrosis vascular proliferation strong and diffuse positivity for GFAP and nuclear TP53 Fig a0 No IDH1IDH2 hotspot mutations were detected while both cases showed MGMT promoter methylation Monosomy of chromosome cooccurred with EGFR amplification UPN131 or with gain of the whole chromosome UPN171Materials and a0methodsTERT promoter mutational analysisGenomic DNA was extracted from FormalinFixed ParaffinEmbedded FFPE tumor tissue and from peripheral blood PB by QIAamp DNA FFPE and AllPrep DNARNA kits respectively following the manufacturer™s instructions QIAGEN Milan Italy Hotspot TERTpmut were investigated by Sanger sequencing using ABI Genetic analyzer instrument Applied Biosystems Monza Italy Primers were reported in Table a0S1 Additional file a0 Table a0S1 and referred to GRCh37 genomic coordinate system NM_0000059 for regulatory core promoter a0 bp wwwncbinlmnihgovgene [] wwwensem blHomo_sapie ns [] Sequences™ alignments and their analyses were supported by Clustal wwwebiacukTools msaclust alo Ensembl Omega httpwwwensem blHomo_sapie ns and [] 0cPierini a0et a0al acta neuropathol commun Page of Table Epidemiological and a0clinical features of a0our cohort of a0patientsEpidemiologicalclinical dataTotal cohortGenderAge yearsDiagnosis WHO Common GliomasUncommon GliomasAnatomic locationMaleFemaleMFRangeMedian years‰¥ yearsDiffuse astrocytoma IDHwt grade IIDiffuse astrocytoma IDHmut grade IIAnaplastic astrocytoma IDHwt grade IIIAnaplastic astrocytoma IDHmut grade IIIGlioblastoma IDHwt grade IVGlioblastoma IDHmut grade IVOligodendroglioma IDHmut and 1p19qcodeleted grade IIAnaplastic oligodendroglioma IDHmut and 1p19qcodeleted grade IIIPilocytic astrocytoma grade IPleomorphic xanthoastrocytoma grade IIAnaplastic pleomorphic xanthoastrocytoma grade IIIFrontalFrontalparietalFrontaltemporalParietalParietaloccipitalTemporalTemporalparietalTemporaloccipitalOccipitalCerebellar hemisphereCorpus callosumThalamusPituitary glandInsularMulticentric pts pts pts pts pts patients wt wildtype mut mutantCOSMIC https cance rsange racukcosmi c websites []In silico TERTpmut functional analysis JASPAR toolThis bioinformatic tool estimates the binding affinity and the number of TFs binding sites for the input sequence provided in FASTA format A relative threshold score of and Δ relative score ‰¥ mutant™s relative score”wildtype™s relative score were chosen to define the statistically significant changes induced by TERTpmut as previously reported [] The JASPAR CORE predicted the effects of the four different TERTpmut that we detected in our patients ie the two new TERTpdup the TERTp and the TERTp146 on TFs binding capacity JASPAR CORE Collection httpjaspa rgener egnet 8th version [ ] JASPAR was also used to analyze two TERTpdup which have been previously reported in a case of MDS c1110_1101dup and in a case of thyroid cancer c1104_183dup [ ] According to JASPAR data we used the Venn diagram to plot TFs for which a significant enhanced probability of binding capacity or an 0cPierini a0et a0al acta neuropathol commun Page of Fig Histological and immunohistochemical analysis in patient UPN171 a HematoxylinEosin staining original magnification 200X enlarged neoplastic cells with multiple often bizarre hyperchromatic nuclei and high number of mitoses Vascular proliferation as seen in these œglomeruloids lower half of the image is a specific pattern of microvascular growth b HematoxylinEosin staining original magnification 400X multiple mitotic figures are evident in the middle field œGeographic pattern of necrosis detail in insert panel b c Positive GFAP staining highlights high neoplastic cells with astrocytic differentiation d Intense and diffuse nuclear TP53 stainingincrease of the number of binding sites was predicted httpbioin forma ticspsbugent bewebto olsVennIn vitro TERTpmut functional study luciferase assayTo study the effect of TERTpmut on the expression of TERT a luciferase assay was done for the TERTpdup detected in case UPN171 the TERTp146 UPN205 and the TERTp124 UPN216 The TERTdup of case UPN131 could not be studied due to lack of material A TERTp wildtype TERTpwt construct already available in the laboratory was also used as reference Additional file a0 Table a0S2 [] TERT core promoter a0bp was amplified with specific primers reported in Table a0 S3 Additional file a0 Table a0 S3 introducing cleavage sites for BglII forward and HindIII reverse restriction enzymes Then TERTpmut constructs were inserted in pGEMT easy plasmid Promega Madison WI USA and cloned in Electromax DH10BT1 cells Invitrogen Milan Italy to increase the amount of mutant DNA Finally the inserts were subcloned in pGL410[luc2] vectors Promega Madison WI USA upstream of LUC2 gene encoding for luciferase enzyme of Photinus Pyralis and resequenced An empty pGL410[luc2] vector was also used as negative control Luciferase assay was performed using the GBM U87MG cell line maintained in Dulbecco™s Modified Eagle Medium Thermo Fisher Scientific Monza Italy with fetal bovine serum and streptomycinpenicillin at a0°C5 CO2 U87MG cells were seeded in a 6multiwell plate — cellsml cotrasfected with a0 µg of modified pGL410[luc2] plasmids and with of pGL474[hRlucTK] a vector containing the luciferase gene of Renilla Reniformis by Viafect Transfection Reagent Promega Madison WI USA After 24h incubation cells were lysed and fluorescence emission was assessed using DualGlo Luciferase assay kit Promega following manufacturer™s instructions All experiments were performed in triplicate in three independent experiments 0cPierini a0et a0al acta neuropathol commun Page of ResultsNew somatic TERT promoter variantsTERTpmut were detected in cases including ODG DAAA and GBM Additional file a0 Table a0 S4 In GBM and DAAA TERTpmut were prevalent in IDHwt cases GBM IDHwt vs GBM IDHmut DAAA IDHwt vs DAAA IDHmut Chi square P Additional file a0 Table a0S5 Thus in agreement with the diagnostic criteria recommended by the cIMPACTNOW Update the DAAA IDHwt with TERTpmut were referred to as œdiffuse astrocytic glioma IDHwildtype with molecular features of glioblastoma WHO grade IV []In GBM TERTpmut there was a significant enrichment of cases harbouring EGFR amplification vs Chi square P andor monosomy 10PTEN deletions vs Chi square P Likewise EGFR amplification or gain of whole chromosome in combination with monosomy occurred in of TERTpmut DAAA IDHwtThe most common variant TERTp124 was detected in cases while the TERTp146 was found in cases TERTpmut were mutually exclusive heterozygous and equally distributed among the different histological subtypes Additional file a0 Table a0S5 Besides the TERTp124 and TERTp146 we uncovered two new TERTp variants in two cases of GBM IDHwt UPN131 and UPN171 These novel TERTpmut consisted of a nucleotide tandem duplication occurring in a genomic region starting at and a0bp from the ATG starting site ie c1100_179dup TERTp10079 in case UPN131 and c1110_189dup TERTp11089 in case UPN171 Fig a02a b wwwncbinlmnihgovgene wwwensem blHomo_sapie ns cancersangeracukcosmic [ ] They shared a region of duplication of nucleotides from “ to “ nucleotides from the ATG start site The absence of TERTp10079 in the PB DNA demonstrated the somatic origin of this variant in case UPN131In silico analysis predicts TERTpmut effectsIn silico analysis predicted that both TERTpdup created new binding sites ie for TERTp10079 and for TERTp11089 which were respectively recognized by and TFs Instead TERTp124 and TERTp146 were predicted to increase the binding affinity for and sites and to enhance the probability of binding for and TFs respectively Additional file a0 Table a0S6 Although all TERTpmut affected the binding sites for diverse families of TFs the ETS group emerged as one of the most frequently involved in TERTp10079 for TERTp11089 in TERTp124 and in TERTp146 Fig a0 2c Additional file a0 Table a0 S7 Other recurrently involved TFs in TERTpdup variants were the Specificity ProteinKr¼ppelLike Factor SpKLF family ie in TERTp10079 and in TERTp11089 and the More than adjacent zinc finger factors family in TERTp10079 and TERTp11089 Additional file a0 Table a0S7The Venn diagram showed a close interrelationship between all TERTp mutations Namely all TERTp mutations shared an increase of the binding affinity or the number of binding motifs for common TFs Fig a03a including ETS members ETS1 ETS2 ERG ELK1 ETV6 FLI1 ELK4 SPIB ELF1 ELF3 ETV4 ETV1 FEV EHF ETV5 ELF5 SPI1 and GABPA and TEAD1 Fig a03a Additional file a0 Table a0S8 The Venn diagram also showed that the new TERTpdup were characterized by the exclusive involvement of common TFs Specifically there were SpKLF members ie KLF2 KLF3 KLF4 KLF5 KLF10 KLF11 KLF14 KLF15 KLF16 SP1 SP2 SP3 SP4 SP8 SP9 and EGR1 Fig a03a Additional file a0 Table a0 S8 and TFs that belong to different families Fig a03a Additional files and Tables S7 and S8 Matching our TERTpdup with the two cases of TERTpdup previously reported Additional files and Tables S9 and S10 [ ] JASPAR predicted that all variants determined an increase of binding sites for common TFs and confirmed that the SpKLF family was the most frequently involved Fig a0 3b Additional file a0 Table a0S11In vitro analysis confirms the a0increasing of a0TERT transcriptional activity induced by a0its promoter mutationsIn vitro luciferase assay was carried out to evaluate whether the new TERTp11089 variant induced an increase of TERT transcriptional activity enhancing its expression similarly to TERTp124 and TERTp146 [ ] In Table a0S12 Additional file a0 Table a0S12 we reported raw data referred to the fluorescence emission values expressed in Relative Luciferase Activity RLA of both Photinus Pyralis and Renilla Reniformis luciferase enzymes for all samples Our experiments demonstrated that all three variants caused a significant increase of TERT transcription by fold than wildtype TERTp11089 vs TERTpwt P TERTp124 vs TERTpwt P TERTp146 vs TERTpwt P Mann“Whitney U test Fig a0 On the other hand no differences on the levels of TERT expression were present between the diverse TERTp variants indicating they may all behave as gainoffunction mutations likely exerting the same consequences on TERT transcription 0cPierini a0et a0al acta neuropathol commun Page of Fig Schematic representation of TERTp mutations a TERT promoter electropherogram in case UPN131 The arrow indicates the start point of the c1100_179dup b TERT promoter electropherogram in case UPN171 The arrow indicates the start point of the c1110_189dup c Overview of all TERTp variants detected in our cases Upper arrow wildtype TERT core promoter with the normal location of ETS binding sites The vertical black lines indicate the genomic positions of TERTp variants Lower arrow positions and types of TERTp variants and their predicted effects on transcription factors binding sitesDiscussionAbnormal genomic events that alter telomere elongation are common in gliomas Particularly mutually exclusive mutations affect the TERT or the ATRX chromatin remodeler ATRX genes a critical regulator of telomere homeostasis by chromatin remodeling []Our studies on a cohort of patients confirmed previous data on the incidence and distribution of TERTpmut in diverse subtypes of CNS tumors As expected we found that TERTpmut were highly recurrent in ODG and GBM and less frequent in DAAA Additional file a0 Table a0 S4 TERTpmut were significantly enriched in GBM IDHwt cases Chi square P Additional file a0 Table a0S5 where they mainly occurred together with EGFR amplification Chi square P andor monosomy 10PTEN deletions Chi square P Similarly in DAAA TERTpmut were highly recurrent in IDHwt cases thus allowing the reclassification of of these subgroup of astrocytomas as œdiffuse astrocytic glioma 0cPierini a0et a0al acta neuropathol commun Page of ODG [] Afterwards TERTpdup were found in a case of myelodysplastic syndrome MDS c1110_1101dup and in a case of papillary thyroid carcinoma c1104_183dup [ ] Published TERTpdup as well as our cases are located in the same core promoter region that span a0 bp from the ATG start site Furthermore they are all located downstream TERTp124 and TERTp ie at “ nucleotides from TERTp124 and nucleotides from TERTp146 in a region that contains the binding sites for the TFs modulating TERT transcription Interestingly in silico analysis predicted these new TERTdup affect the transcriptional regulation of the gene through the creation of new binding sites for TFs that mainly belong to the ETS family Fig a02c Additional file a0 Table a0S7 Likewise an increased number of binding sites or an enhanced affinity for the ETS TFs has been previously reported in a thyroid cancer harbouring a TERTp c1104_183dup variant and in cases bearing TERTp124 or TERTp146 mutations [ ] Bioinformatic analyses were consistent with the luciferase data showing a significant increase of TERT expression in cells transfected with the new TERTp11089 variant as well as with the two recurrent TERTpmutThen we sought to assess the possible interrelationship between the four diverse TERTp mutations using the Venn diagram Fig a03a All four TERTp variants were predicted to share an increase binding capacity for ETS members Fig a03a Additional file a0 Table a0S8 which included GABPA a putative oncogene in GBM Namely in a0vitro studies on GBM cell lines have demonstrated that this transcription factor is needful in mediating the transcriptional reactivation of TERT dependent from TERTp or TERTp146 [ ] Besides ETS TFs all TERTp variants affected the binding capacity for TEAD1 a protein that belongs to TEF1related factors family and that has been demonstrated to act as a putative oncogene in GBM favoring cell infiltration in a0 vitroin vivo models []Although TERTp124 and TERTp146 and the new TERTp10079 and TERTp11089 variants shared the same effects on the binding capacity for ETS members the latters were characterized by the exclusive involvement of TFs mainly belonging to SpKLF family Fig a0 3a Additional files and Tables S7 and S8 SpKLF TFs are involved in a plethora of cellular processes ranging from proliferation and differentiation pluripotency and apoptosis in normal and tumoral tissues []Fig The Venn diagrams show all possible relations among a four TERTp variants reported in our cases refer to Additional file Table S8 and b TERTpdup described in this study c1100_179dup and c1110_189dup and those reported in literature c1104_183dup and c1110_1101dup refer to Additional file Table S11IDHwildtype with molecular features of glioblastoma WHO grade IV []Besides the two known TERTp124 and TERTp146 variants we uncovered two new TERTp variants in two cases of GBM IDHwt UPN131 and UPN171 These novel TERTpmut consisted of a nucleotide tandem duplication sharing a duplicated region of nucleotides from “ to “ from the ATG start site Hitherto somatic TERTpdup has been reported in three human tumors The first one a duplication of nucleotides in the TERT core promoter was detected in a case of Altogether these data support the hypothesis that the recruitment of ETS family TFs plays a pivotal role in mediating the reactivation of TERT transcription in human tumors bearing different types of TERTpmut However they also indicate that slight differences mark TERTpdup variants whose activities appear to be 0cPierini a0et a0al acta neuropathol commun Page of Fig Luciferase assay The histogram reports the relative luciferase activities RLA of TERTp wildtype and for the variants c1110_189dup c1124 C T and c1146 C T p value refers to probability obtained using Mann“Whitney U testalso dependent from Kr¼ppelrelated factors Indeed among the TFs shared by all TERTpdup Fig a03b belonged to SpKLF family as reported in Tables S10 and S11 Additional files and Hence the precise definition of mutationspecific profiles would strengthen the definition of TERTdependent oncogenesis mechanismsOur study contributes to enrich the spectrum of recurrent somatic TERTpdup variants reporting for the first time two new gainoffunction mutations ie TERTp10079 and TERTp11089 in of GBM IDHwt cases These new mutations can be reliably detected by diagnostic assays used to investigate hotspot TERTp and TERTp146 Although the assessment of TERTp mutational status is not an essential diagnostic criterion it can be a relevant information to assist histological diagnosis [] As a matter of fact the status of TERTp together with IDH mutations and 1p19q codeletion classify gliomas in distinct subcategories ie triple negative triple positive cases with IDHTERT mutations and cases with a unique mutation either IDH or TERT that are typified by unique demographic clinical and biological characteristics [] Moreover TERTpmut has been proposed as one of the most relevant molecular marker to stratify DAAA IDHwt [] Thus we consider that molecular testing of TERTp mutations should be included in the clinical workup of GBM and DAAA in order to provide a precise diagnosis prospective multicentric studies on large cohort of patients will clarify the value of TERTp mutations as prognostic markerSupplementary informationSupplementary information accompanies this paper at https doi101186s4047 Additional file a0 Table a0S1 Primer set used for Sanger sequencing Additional file a0 Table a0S2Samples used for in vitro luciferase assay Additional file a0 Table a0S3 Primer set used to create constructs for luciferase assay Additional file a0 Table a0S4 Incidence and distribution of TERTp variants in the main glioma subgroups Additional file a0 Table a0S5 Incidence and distribution of TERTp variants in glioma subtypes according to WHO guidelines Additional file a0 Table a0S6 JASPAR analysis for the TERTp c1124 CT c1 CT and the new TERTpdupc1100_179dup c1110_189dup Additional file a0 Table a0S7 Transcription Factors predicted to be involved in TERTp variants Additional file a0 Table a0S8 Transcription Factors predicted to be involved in different TERTp variants Additional file a0 Table a0S9 JASPAR analysis for the two published TERTp duplications c1110_1101dup and c1104_183dup [ref ] Additional file a0 Table a0S10 Transcription factors predicted to be involved in the TERTpdup c1110_1101dup and c1104_183dup [ref ] Additional file a0 Table a0S11 Transcription factors predicted to be involved in all TERTp duplications Additional file a0 Table a0S12 Luciferase assay raw dataAbbreviationsTERT Telomerase Reverse Transcriptase TERTp TERT promoter TERTpmut TERT promoter mutation TERTpdup TERT promoter duplication TERTp124 c1124 TERT promoter mutation TERTp146 c1146 TERT promoter mutation bp base pair ETS Etwentysix transcription factor CNS central nervous system GBM glioblastoma ODG oligodendroglioma DA diffuse astrocytoma AA anaplastic astrocytoma GBM IDHwt glioblastoma IDHwildtype DA IDHwt diffuse 0cPierini a0et a0al acta neuropathol commun Page of astrocytoma IDHwildtype AA IDHwt anaplastic astrocytoma IDHwildtype TFs transcription factors DA IDHmut diffuse astrocytoma IDHmutant AA IDHmut anaplastic astrocytoma IDHmutant GBM IDHmut glioblastoma IDHmutant FFPE formalinfixed paraffinembedded PB peripheral blood MDS myelodysplastic syndrome TERTpwt TERTp wildtype TERTp10079 c1100_179dup TERTp11089 c1110_189dup SpKLF Specificity ProteinKr¼ppelLike Factor RLA relative luciferase activity ATRX ATRX chromatin remodelerAcknowledgementsNot applicable Authors™ contributionsTP RLS conceived the study planned the experiments and wrote the paper TP carried out and evaluated mutational analysis and in vitro functional studies CN made in silico analysis AGLF contributed in the analysis of in vitro luciferase assay MM and SA performed DNA extraction and FISH experiments FP VN and PG performed sequencing analysis PG SA and MEL provided the diagnosis and the tissue sections for molecularcytogenetic studies CC and RC ML GM and CM provided all clinical data VP GR and CM were involved in drafting the manuscript All the authors read and approved the final manuscript FundingThe project was supported by Comitato per la vita œDaniele Chianelli Perugia Italy Sergio Luciani Association Fabriano Italy and Fondazione Cassa di Risparmio Perugia Italy Grant numbers to RLS Availability of data and materialsAll data generated or analyzed during this study are included in this published [and in its supplementary information files]Ethics approval and consent to participateThis study was approved by the local ethic committee CEAS code number August 8th Consent for publicationAll participants signed an institutional informed consentCompeting interestsThe authors declare that they have no competing interestsAuthor details Molecular Medicine Laboratory Centro di Ricerche EmatoOncologiche CREO S Maria della Misericordia Hospital University of Perugia Ple Menghini Perugia Italy Hematology and Center of Bone Marrow Transplants Medicine and Surgery Department University and Hospital of Parma Via Gramsci Parma Italy Diagnostic Cytology and Histology Unit S Maria della Misericordia Hospital Ple Giio Menghini Perugia Italy Division of Radiotherapy S Maria della Misericordia Hospital Ple Giio Menghini Perugia Italy Medical Oncology S Maria della Misericordia Hospital Ple Giio Menghini Perugia Italy Division of Neurosurgery S Maria della Misericordia Hospital Ple Giio Menghini Perugia Italy Pathology Unit S Maria Hospital V Tristano di Joannuccio Terni Italy Received June Accepted August References Allory Y Beukers W Sagrera A Fl¡ndez M Marqu©s M M¡rquez M et al Telomerase Reverse Transcriptase promoter mutations in bladder cancer high frequency across stages detection in urine and lack of association with outcome Eur Urol “ Barthel FP Wei W Tang M MartinezLedesma E Hu X Amin SB et al Systematic analysis of telomere length and somatic alterations in cancer types Nat Genet “ Bell RJ Rube HT Kreig A Mancini A Fouse SD Nagarajan RP et al The transcription factor GABP selectively binds and activates the mutant TERT promoter in cancer Science “ Brat DJ Aldape K Colman H Holland EC Louis DN Jenkins RB et al cIMPACTNOW update recommended diagnostic criteria for œDiffuse astrocytic glioma IDHwildtype with molecular features of glioblastoma WHO grade IV Acta Neuropathol “ COSMIC Catalogue of Somatic Mutations in Cancer Database Wellcome Sanger Institute Cambridge UK https cance rsange racukcosmi c Accessed May EckelPassow JE Lachance DH Molinaro AM Walsh KM Decker PA Sicotte H et al Glioma groups based on 1p19q IDH and TERT promoter mutations in tumors N Engl J Med “Ensembl DatabaseHomo Sapiens European Molecular Biology Laboratory™s European Bioinformatics Institute Cambridge UK httpwwwensem blHomo_sapie ns Accessed May Fornes O CastroMondragon JA Khan A van der Lee R Zhang X Richmond PA et al JASPAR update of the access database of transcription factor binding profiles Nucleic Acids Res 48D87“D92 https doi101093

Thyroid_Cancer

"Tumor microenvironment TME plays an important role in malignant tumors Our study aimed toinvestigate the effect of the TME and related genes in osteosarcoma patientsMethods Gene expression profiles and clinical data of osteosarcoma patients were downloaded from the TARGETdataset ESTIMATE algorithm was used to quantify the immune score Then the association between immune scoreand prognosis was studied Afterward a differential analysis was performed based on the high and lowimmunescores to determine TMErelated genes Additionally Cox analyses were performed to construct two prognosticsignatures for overall survival OS and diseasefree survival DFS respectively Two datasets obtained from the GEOdatabase were used to validate signaturesResults Eightyfive patients were included in our research The survival analysis indicated that patients with higherimmune score have a favorable OS and DFS Moreover genes were determined as TMErelated genes Theunsupervised clustering analysis revealed two clusters were significantly related to immune score and T cells CD4memory fraction In addition two signatures were generated based on three and two TMErelated genesrespectively Both two signatures can significantly divide patients into low and highrisk groups and were validatedin two GEO datasets Afterward the risk score and metastatic status were identified as independent prognosticfactors for both OS and DFS and two nomograms were generated The Cindexes of OS nomogram and DFSnomogram were and respectivelyConclusion TME was associated with the prognosis of osteosarcoma patients Prognostic models based on TMErelated genes can effectively predict OS and DFS of osteosarcoma patientsKeywords Tumor microenvironment Osteosarcoma Prognosis Immune features NomogramBackgroundOsteosarcoma is the most common bone tumor especiallyin children and adolescents [] It was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group [] Currently surgery and Correspondence 407404159qqcom4Wenzhou Medical University Wenzhou ChinaFull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years However due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable[] Previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively [] Thereforeit is necessary to 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 0cHu BMC Cancer Page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsRecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified For example Chen [] reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients Moreover Huang [] discovered that dysregulated circRNAs serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microRNA In addition lncRNA [] microRNA [] and many clinical data [] were also identified asprognostic biomarkers for osteosarcoma patients However osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humansTherefore it is necessary to find accurate biomarkers forosteosarcomaIn recent years researchers have paid more and moreattention to the role of the tumor microenvironmentTME in malignant tumors The function of TME inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] More importantly Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data ESTIMATE an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in [] Based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [“] Generally the above research indicatedthat TME can serve as the prognostic biomarker in tumorsand many TMErelated genes were determined as the prognostic genes However the role of TME and TMErelatedgenes in osteosarcoma patients remains unclearIn the present study gene expression data and corresponding clinicopathologic data were obtained from TheTherapeutically Applicable Research to Generate EffectiveTreatments TARGET dataset Then the ESTIMATEalgorithm was performed to quantify the immune score ofosteosarcoma and the TMErelated genes were identifiedby the differential expression analysis Subsequently theprognostic value of TME and TMErelated genes weredetermined by a series of bioinformatics methodsMethodsGene expression datasetsLevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom TARGET dataset ocgcancergovprogramstarget accessed on Oct The correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus After data were extracted from the public domainthe ESTIMATE an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in R software version [] Meanwhile the messenger RNAmRNA expressionprofiles and clinical data ofincludingGSE21257 [] and GSE39055 [] were obtained fromthe Gene Expression Omnibus as external validationcohortstwo cohortsSurvival analysis and correlation analysisAfter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score The KaplanMeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival OS and diseasefreesurvival DFS between high and lowscore cohorts Inaddition the association between clinicopathologic dataand TME score was also studied MannWhitney signedrank test was performed to compare the differences ofimmune score between each clinical group All statisticalanalyses in the present study were performed using Rsoftware Except for the special instructions p value twoside was identified as statistically significantin the present studyDEGexpressed geneDifferentially expressed gene analysisDifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group The limma package in Rsoftware was used to perform the differential analysisand genes with log FC and adjusted pvalue qvalue were identified as DEGs []To further understand the function of DEGs identifiedin the present study Gene Ontology GOincludingbiological processes BP molecular functions MF andcellular componentsCC and Kyoto Encyclopedia ofGenes and Genomes KEGG analysis were performedby clusterProfiler package in R software []Evaluation of association with immune cellsTo further investigate the association between DEGs andimmune cells the CIBERSORT package was used toestimate the relative proportions of types of immunecells [] Meanwhile the œConsensusClusterPlus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping DEGs [] Cumulative distribution function CDF and relative change inarea under the CDF curve were used to determine theoptimal number of clusters k Then MannWhitney 0cHu BMC Cancer Page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters []Survival analysis of DEGsBased on the DEGs the univariate COX analysis was performed to determine the prognostic value of immunerelated genes Then the OSrelated genes were validatedin the GSE21257 dataset while the DFSrelated geneswere validated in the GSE39055 dataset Only genes successfully validated were selected for further analysis Afterward based on the validated genes the multivariate COXanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsThe risk score for each patient was calculated based onthe coefficient from the multivariate COX analysis and thecorresponding gene expression Meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score The survival receiver operating characteristic ROC curve was used to show the discrimination of signatures and the KaplanMeier survivalcurve with the logrank test was generated to show thedifferences of OS and DFS between high and lowriskgroups In addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature The KaplanMeier survivalcurve and survival ROC curve were generated to show thepredictive ability of the signature in the validation cohortDevelopment of a nomogram for osteosarcoma patientsNomogram is a tool to visualize the predictive model andconvenient for clinical practice Therefore we attemptedto develop a nomogram based on the TMErelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients Firstlythe univariateCOX analysis was performed to filter prognostic variableswhich will be further included in the multivariate COXanalysis Secondly based on independent prognostic variables two nomograms were established for predicting theOS and DFS respectively The Cindex was used to assessthe discriminatory performance of the nomogram whichrange from to [] A Cindex of means agreement by chance and a Cindex of represents perfectdiscriminatory performance The higher value of the Cindex the better performance of the nomogram is Furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsResultsImmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females The immunescore of the cohort range from ˆ’ to Tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group The survival analysis indicated thatpatients with higher immune score had a favorable OSand DFS Fig 1a and b After adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both OS and DFSFig 1a and b Inaddition the relationship between immune score and clinical features was also investigated However there was nosignificant relationship between immune score and clinicalvariables Supplementary Figure 1A1CDifferential expression analysisAccording to the median of the immune score patients were divided into highscore n and lowFig Association between immune score and prognosis in osteosarcoma patients a KaplanMeier survival analysis of overall survival for patientswith high vs low immune score b KaplanMeier survival analysis of diseasefree survival for patients with high vs low immune score 0cHu BMC Cancer Page of score group n There were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesFig 2a b and Supplementary Table To furtherunderstand the function of DEGs GO analysisand KEGG analysis were performed The top significant results of GO analysis among three types wereillustrated in Fig 2c Interestingly we can find that theresults of GO analysis are mostly associated with immunity which further verify that the immunerelated DEGsare associated with immune features In addition the results of KEGG also confirmed it Such as œPhagosomeœAutoimmune thyroid disease œAntigen processing andpresentation œB cell receptor signaling pathway œIntestinal immune network for IgA production œInflammatorybowel disease œPrimary immunodeficiency œTh1 andTh2 cell differentiation œTh17 cell differentiation œNatural killer cell mediated cytotoxicity and œNFˆ’kappa Bsignaling pathway Fig 2dconsensusunsupervisedEvaluation of DEGs and immune cellsTo further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns Based on the consensus matrix heat map patientswere clearly divided into two clustersFig 3a In additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined Fig 3bc The immune score betweentwo clusters was significantly different Fig 3d In additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot Fig 3e Interestinglywe can see that the T cells CD4 memory activated ofcluster is significantly higher than cluster Fig 5fPrognostic value of TMErelated genesPrevious studies indicated that TMErelated genes canserve as the prognostic biomarker for tumor patientsFig Differentially expressed genes with the immune score in osteosarcoma patients a Heatmap of significantly differentially expressed genesbased on immune score b The volcano figure to show the upregulated and downregulated genes c GO analysis of differentially expressedgenes d KEGG of differentially expressed genes GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes 0cHu BMC Cancer Page of Fig The immune landscape of the tumor microenvironment ac Unsupervised clustering of all samples based on the overlapping DEGs dComparison of immune score between two clusters e The distribution of types of immune cells in osteosarcoma patients f The comparisonof types of immune cells between clusters DEG Differentially expressed geneHence we performed the univariate COX analysis toidentify prognostic DEGs The results showed that and genes were identified as OS and DFSrelatedDEGs respectively Supplementary Table and Afterward five OSrelated genes were successfully validated inthe GSE21257 data set and five DFSrelated genes were successfully validated in the GSE39055 cohort Furthermoremultivariate COX analysis was performed and two prognostic signatures were generated for predicting the OS andDFS respectively The risk score for predicting the OS wasasrisk score FCGR2B0766 GFAP0702 MPP70387 In addition the risk score for predicting theDFS was as follows risk score CYP2S10574 ICAM3 The AUC values of OSrelated signature were follows 0cHu BMC Cancer Page of and in and 3year respectively Fig 4aand the AUC values of DFSrelated signature were and in and 3year respectively Fig 5aMoreover survival curves showed that patients in the highrisk group had worse OS and DFS compared with the lowrisk patients Figs 4b and 5b Heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients Figs 4ceand 5ce Then both signatures were validated in independent cohorts For OS signature the AUC values ofvalidation cohort were and at and3year Fig 4f For DFS signature the AUC values ofvalidation cohort were and at and3year Fig 5f Additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients Figs 4g and 5gHeat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsFigs 4hj and f 5hjDevelopment of a nomogram for osteosarcoma patientsTo generate a nomogram for clinical use the COX analysiswas performed to select the clinical prognostic variables InFig Establishment and validation of the prognostic model for overall survival based on significant DEGs a Receiver operating characteristiccurves of prognostic signature in the training cohort b The survival curve showed the different overall survival status between high and lowriskpatients c The heat map showed the expression of prognostic genes in the training cohort d The risk curve of each sample reordered by riskscore e The scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f Receiver operating characteristiccurves of prognostic signature in validation cohort g The survival curve showed the different overall survival status between high and lowriskpatients h The heat map showed the expression of prognostic genes in the validation cohort i The risk curve of each sample reordered by riskscore j The scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0cHu BMC Cancer Page of Fig Establishment and validation of the prognostic model for diseasefree survival based on significant DEGs a Receiver operatingcharacteristic curves of prognostic signature in the training cohort b The survival curve showed the different diseasefree status between highand lowrisk patients c The heat map showed the expression of prognostic genes in the training cohort d The risk curve of each samplereordered by risk score e The scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f Receiver operatingcharacteristic curves of prognostic signature in validation cohort g The survival curve showed the different diseasefree status between high andlowrisk patients h The heat map showed the expression of prognostic genes in the validation cohort i The risk curve of each sample reorderedby risk score j The scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate COX analysis risk score and metastatic status were identified as both OS and DFSrelated variablesFig 6a and e Afterward risk score and metastatic statuswere determined as both independent OS and DFSrelated variables in the multivariate COX analysis Fig 6band f Based on independent variables two nomogramswere established for predicting the OS and DFS in osteosarcoma patients respectively Fig 6c and g The Cindexvalues were and in OS nomogram and DFSnomogram respectively The results of Cindex mean thatboth two nomograms have good discrimination Meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve Fig 6dand h which indicated a good calibrationDiscussionThe relationship between TME and tumor have beenwidely studied in recent years In the present study ESTIMATE algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from TARGET database We confirmed that theTME is significantly associated with the prognosis ofosteosarcoma patientsInadditionfunctional enrichment analyses of TMErelated genes indicated that immunerelated processesincluding OS and DFS 0cHu BMC Cancer Page of Fig Nomograms based on the tumor microenvironment related genes for osteosarcoma patients a Univariate COX analysis of overall survivalrelated variables b Multivariate COX analysis of overall survivalrelated variables c Nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curveS of overall survival nomogram e Univariate COX analysis of diseasefree survivalrelated variables fMultivariate COX analysis of diseasefree survivalrelated variables g Nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curveS of diseasefree survival nomogramknown to contribute to tumor progression More importantly DEGs based on the TME were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the OS and DFS of osteosarcoma patientsrespectivelyIn recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the TME and the ESTIMATE algorithm is oneof the most important quantitative tools for this researchfield Based on the ESTIMATE algorithm the association between the prognosis and TME has been initially 0cHu BMC Cancer Page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ “] However previousstudies indicated that TME scores serve as a differentrole in different tumors For example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ “] However for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] Therefore we can find great heterogeneity among different tumors from the perspectiveof TME For osteosarcoma patients the present studyindicated that patients with higher immune score had abetter OS and DFS Hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionIn our research TMErelated genes were identified by comparing the highscore and lowscore osteosarcoma patients The functional enrichment includingGO and KEGG analyses showed that TMErelated geneswere mainly involved in the immune features such asregulation of leukocyte activation MHC protein complex MHC protein and complex binding More importantly the unsupervised cluster analysis based on DEGswas performed and all patients were divided into twoclusters Immune score and T cell CD4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between DEGs and immune featuresDue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantThe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients Emilie etal []performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma Recently the prognostic signature based onTMErelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients [] Compared with the study performedby Zhang [] we think that our research have someadvantages Firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts Secondlythe outcome of DFS was not reported in the previousstudy As reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]Hence the DFS nomogram can improve the managementof osteosarcoma patients Finally two nomograms incorporated TMErelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsIn our research five genes were incorporated into thefinal prognostic signatures FCGR2B GFAP and MPP7were identified and validated as OSrelated biomarkerswhile CYP2S1 and ICAM3 were DFSrelated biomarkersThe role of these genes in tumor prognosis had beenwidely reported in previous studies [“] FCGR2Bhas been confirmed as an immunerelated gene previously [] Although the relationship between FCGR2Band prognosis in sarcoma patients had not been reported the prognostic value of FCGR2B had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] In addition NewM etal [] demonstrated that MPP7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomaCYP2S1 described as Cytochrome P450 Family Subfamily S Member was reported significantly associatedwith colorectal cancer In primary colorectal cancerCYP2S1 was present at a significantly higher level ofintensity compared with normal colon [] More importantly the presence of strong CYP2S1 immunoreactivity was associated with poor prognosis [] The roleof ICAM3 in cancer was also widely reported in published studies and the Akt pathway plays an importantrole in the impact of ICAM3 on tumors YG Kim etal[] reported that ICAM3 can induce the proliferationof cancer cells through the PI3KAkt pathway Additionally JK Park etal showed that the ICAM3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing MMP2 andMMP9 via Akt pathway [] showed that the ICAM3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingMMP2 and MMP9 via Akt pathwayAlthough the role of TME and TMErelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated Firstly the treatmentinformation cannot be obtained from the TARGET database which may influence the prognosis of osteosarcomapatients Secondly two nomograms were generated andshowed good performance in our study However externalvalidation by a large cohort is needed Thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear Finally inthe training cohort and DEGs were identified asOS and DFSrelated DEGs respectively However onlyfive OS and five DFSrelated genes were identified in thevalidation cohort The different age structures smaller 0cHu BMC Cancer Page of sample sizes and the platform covering only part of thegenes may contribute to this resultReceived February Accepted July ConclusionIn conclusion TME plays an important role in osteosarcoma patients and related with the progression of thetumor Moreover TMErelated genes can serve as prognostic biomarkers in osteosarcoma patients Howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studySupplementary informationSupplementary information accompanies this paper at doi101186s12885020072162Additional file Additional file Additional file Additional file AbbreviationsTME Tumor microenvironment DEG Differentially expressed genesOS Overall survival DFS Diseasesfree survival ROC Receiver characteristiccurve ESTIMATE Estimation of STromal and Immune cells in MAlignantTumor tissues using Expression data TARGET Therapeutically ApplicableResearch to Generate Effective Treatments GO Gene Ontology BP Biologicalprocesses MF Molecular functions CC Cellular components KEGG KyotoEncyclopedia of Genes and Genomes CDF Cumulative distribution functionAcknowledgementsNoneAuthors™ contributionsC H L Y Sq T C L and Yh W conceived of and designed the study C H R Sand C L performed literature search R S L Y and B C generated the figuresand tables L Y Hl R X Y and Jy L analyzed the data C H wrote themanuscript and Sq T and L Y critically reviewed the manuscript L Ysupervised the research All authors have read and approved the manuscriptFundingWe received no external funding for this studyAvailability of data and materialsThe data of this study are from TARGET and GEO databaseEthics approval and consent to participateThe research didn™t involve animal experiments and human specimens noethics related issuesConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Joint Surgery the Affiliated Hospital of Qingdao UniversityQingdao China 2Department of Medical Oncology the First Hospital ofChina Medical University Shenyang China 3Department of Nursing Sir RunRun Shaw Hospital Affiliated to Zhejiang University Hangzhou China4Wenzhou Medical University Wenzhou ChinaReferencesJaffe N Bruland OS Bielack S Pediatric and adolescent osteosarcoma vol New York Springer Science Business Media Vander RG Osteosarcoma and its variants Orthopedic Clin North Am “Biermann JS Adkins D Benjamin R Brigman B Chow W Conrad EU 3rdFrassica D Frassica FJ Gee S Healey JH Bone cancer J Natl ComprCancer Netw “Simpson S Dunning MD de Brot S GrauRoma L Mongan NP Rutland CSComparative review of human and canine osteosarcoma morphologyepidemiology prognosis treatment and genetics Acta Vet Scand Chen X Cates JM Du YC Jain A Jung SY Li XN Hicks JM Man TKMislocalized cytoplasmic p27 activates PAK1mediated metastasis and is aprognostic factor in osteosarcoma Mol Oncol “Huang X Yang W Zhang Z Shao Z Dysregulated circRNAs serve as prognosticand diagnostic markers in osteosarcoma by sponging microRNA to regulatethe downstream signaling pathway J Cell Biochem “Liu M Yang P Mao G Deng J Peng G Ning X Yang H Sun H Long noncoding RNA MALAT1 as a valuable biomarker for prognosis in osteosarcoma asystematic review and metaanalysis Int J Surg “Xu K Xiong W Zhao S Wang B MicroRNA106b serves as a prognosticbiomarker and is associated with cell proliferation migration and invasionin osteosarcoma Oncol Lett “Zheng W Huang Y Chen H Wang N Xiao W Liang Y Jiang X Su W WenS Nomogram application to predict overall and cancerspecific survival inosteosarcoma Cancer Manag Res Kahlert C Kalluri R Exosomes in tumor microenvironment influence cancerprogression and metastasis J Mol Med “ Binnewies M Roberts EW Kersten K Chan V Fearon DF Merad M CoussensLM Gabrilovich DI OstrandRosenberg S Hedrick CC Understanding thetumor immune microenvironment TIME for effective therapy Nat Med“ Yoshihara K Shahmoradgoli M Martínez E Vegesna R Kim H TorresGarcia WTreviño V Shen H Laird PW Levine DA Inferring tumour purity and stromaland immune cell admixture from expression data Nat Commun Yang S Liu T Nan H Wang Y Chen H Zhang X Zhang Y Shen B Qian PXu S Comprehensive analysis of prognostic immunerelated genes inthe tumor microenvironment of cutaneous melanoma J Cell Physiol “ Deng Z Wang J Xu B Jin Z Wu G Zeng J Peng M Guo Y Wen Z MiningTCGA database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma Biomed Res Int Zhao K Yang H Kang H Wu A Identification of key genes in thyroid Cancermicroenvironment Med Sci Monit Xu WH Xu Y Wang J Wan FN Wang HK Cao DL Shi GH Qu YYZhang HL Ye DW Prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment AgingAlbany NY Chen B Chen W Jin J Wang X Cao Y He Y Data Mining of PrognosticMicroenvironmentRelated Genes in clear cell renal cell carcinoma a studywith TCGA database Dis Markers Li X Gao Y Xu Z Zhang Z Zheng Y Qi F Identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods Cancer Med “ Pan XB Lu Y Huang JL Long Y Yao DS Prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma Aging Albany NY Wang H Wu X Chen Y Stromalimmune scorebased gene signature aprognosis stratification tool in gastric Cancer F

Thyroid_Cancer

"Immune checkpoint inhibitors ICIs can induce immunerelated adverse events irAEs includingthyroid dysfunction There are only a few reports on Graves™ disease induced by ICIs We report a case of newonsetGraves™ disease after the initiation of nivolumab therapy in a patient receiving gastric cancer treatmentCase presentation The patient was a 66yearold Japanese man who was administered nivolumab mg every weeks as a thirdline therapy for stage IVb gastric cancer His thyroid function was normal before the initiation ofnivolumab therapy However he developed thyrotoxicosis before the third administration of nivolumab Elevatedbilateral and diffuse uptake of radioactive tracer was observed in the 99mTcpertechnetate scintigraphyFurthermore the thyroidstimulating hormone receptor antibody TRAb and thyroidstimulating antibody TSAbtest results which were negative before the first administration of nivolumab were positive after starting thetherapy The patient was diagnosed with Graves™ disease and the treatment with methimazole and potassiumiodide restored thyroid functionConclusions This is the first complete report of a case of newonset Graves™ disease after starting nivolumabtherapy confirmed by diffusely increased thyroid uptake in scintigraphy and the positive conversion of antibodiesagainst thyroidstimulating hormone receptor It is important to perform thyroid scintigraphy and ultrasonographyto accurately diagnose and treat ICIinduced thyrotoxicosis because there are various cases in which Graves™ diseaseis developed with negative and positive TRAb titresKeywords Graves™ disease Nivolumab Thyrotoxicosis Immune checkpoint inhibitor 99mTcpertechnetatescintigraphy Thyroidstimulating hormone receptor antibodyBackgroundImmune checkpoint inhibitors ICIs such as cytotoxicTlymphocyteassociated protein CTLA4 programmed cell death protein1 PD1 and programmeddeath ligand PDL1 inhibitors have been widely usedas a standard cancer treatment during recent yearsimmunerelatedHowever occasionallycauseICIs Correspondence yhiroshinmsacjp1Department of Endocrinology Diabetes and Metabolism Graduate Schoolof Medicine Nippon Medical School Sendagi Bunkyoku Tokyo JapanFull list of author information is available at the end of the adverse events irAEs which affect different anssuch as the lung gastrointestinal tract liver nervous system skin and endocrine glands The endocrine irAEsinclude hypophysitis thyroid dysfunction adrenal insufficiency and type diabetes While endocrine irAEs dueto CTLA4 inhibitors such as ipilimumab and tremelimumab mainly include pituitary dysfunction those dueto PD1 inhibitors such as nivolumab and pembrolizumab are mainly related to thyroid dysfunction [“]The PD1 inhibitorinduced thyroid dysfunction oftenincludes hypothyroidism rather than hyperthyroidism [ ] Thyrotoxicosis following ICI therapy is caused 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 0cYamada BMC Endocrine Disorders Page of spontaneouslyrecover withmostly by thyroiditis syndrome which has been reportedsubsequenttohypothyroidism in many cases[ ] HoweverGraves™ disease induced by ICI treatment has not beenextensively explored Here we present a case of Graves™disease shortly after the initiation of nivolumab therapyfor gastric cancerthecancerCase presentationA 66yearold man was diagnosed with stage IVbT4bN0M1 human epidermal growth factor receptor HER2positive gastricat Nippon MedicalSchool Chiba Hokusoh Hospital one and a half yearsbefore the onset of thyrotoxicosis After diagnosis hewas not referred for surgery because of liver metastasiswith a portal tumour thrombus rather the patient received cycles of first line chemotherapy with a combination of tegafurgimeraciloteracil S1 cisplatin andtrastuzumab However the patient presented with progressive disease assessed based on the computed tomography CT and oesophagogastroduodenoscopy OGDevaluations following the first line therapy Hence he received a second line chemotherapy with paclitaxel andramucirumab After cycles of the second line chemotherapy although there was a reduction in tumour sizeafter cycles the patient presented with progressivedisease as assessed by CT At this stage nivolumab mg every weeks was started The patient had anormal thyroid function before the first administrationHowever TSH suppression was observed before the second administration and thyrotoxicosis occurred beforethe third administration of the drug hence nivolumabtherapy was discontinued and the patient was referred toour departmentThe patient had complained of fatigue and shortnessof breath during exertion His height was cm bodyweight was kg heart rate was beats per minute and blood pressure was mmHg There wasno evidence of Graves™ orbitopathy or pretibial myxedema He and his family members had no history ofandTgAbantibody ngmL Thyroidthyroid diseases Thethyroidstimulating hormoneTSH free triiodothyronine FT3 and free thyroxineFT4 levels were μIUmL pgmL and ngdL respectively Table The titres of thyroidstimulating hormone receptor antibody TRAb andthyroidstimulating antibody TSAb were positive IUL and respectively whereas those of antithyroglobulinantithyroidperoxidase antibody TPOAb were negative IULrespectively The thyroglobulin Tgand IULlevel wasultrasonographyshowed slight goitre Fig 1a and rich blood flow in theparenchyma Fig 1b 99mTcpertechnetate scintigraphywhich was performed on the first consultation day of thepatient at our department showed elevated bilateraland diffuse uptake of the radioactive tracer Fig Wemeasured antithyroid autoantibodiesin preservedserum samples The titres of TRAb and TSAb werenegative before the first administration of nivolumabwhereas they were positive IUL and respectively before the second administration Thus we diagnosed his thyrotoxicosis as newonset Graves™ diseaseafter the initiation of nivolumab therapy The humanleukocyte antigen HLA typing of the patient showedthe following allelic variants A24022601 B510154 C01021502 DRB104051501 DQA1010203 DQB104010602 DPA10202 and DPB10501We treated the patient with methimazole MMI at adose of mgday and potassium iodide KI at a doseof mgday One month after the initiation of the therapy when the FT3 and FT4 levels of the patient werenormal we discontinued KI Gradually we reduced thedosage of MMI and the continued administration tillthe death of the patient of MMI at a dose of mg everyalternate day stabilised his thyroid function Fig Furthermore as nivolumab was found to be ineffectivebased on the CT and OGD evaluations the patient received irinotecan therapy However after cycles ofchemotherapy the patient was diagnosed with brain metastasis by magnetic resonance imaging MRIforTable TSH FT3 FT4 and Tg levels and TRAb and TSAb titres in our patientTSH μIUmLFT3 pgmLFT4 ngdLTRAb IULTSAb Tg ngmLDay of nivolumab administration NA NANA NANANANANADay first administration of nivolumab Day second administration of nivolumabThe normal range of the thyroid parameters is as follows TSH “ μIUmL FT3 “ pgmL FT4 “ ngdL TRAb IUL TSAb ‰ and Tg ‰ ngmL 0cYamada BMC Endocrine Disorders Page of Fig Thyroid ultrasonography of the patient a Slight swelling in isthmus b Rich blood flow in parenchymawhich he received gamma knife and steroid therapyThe patient died months after his first visit to ourdepartmentDiscussion and conclusionsWe present a case of newonset Graves™ disease after theinitiation of nivolumab therapy in a patient receivinggastric cancer treatment Thyrotoxicosisinduced byICIs is mainly a form of destructive thyroiditis Threecases of newonset Graves™ disease during nivolumabtherapy other than the present case have been reported[“] Table Iadarola [] reported a case ofGraves™ diseaselike hyperthyroidism after the second administration of nivolumab in a patient with left lungIn this case 99mTcpertechnetate scintigcarcinomaraphy in the patient with T3toxicosis showed diffusethyroid uptake of the radionuclide suggesting Graves™diseaselike hyperthyroidism whereas the TRAb testswere consistently negative Thyroid ultrasonographyshowed a multinodular goitre with a normoechoic pattern and normal vascularity ofthe parenchyma []Brancatella [] reported a case similar to that ofIadarola [] with diffuse thyroid uptake and negative TRAb titre In this case ultrasonography showed anenlargement of the thyroid with a hypoechoic patternand mild hypervascularity Kurihara [] reported acase of simultaneous development of Graves™ disease andtype diabetes mellitus during nivolumab therapy InFig 99mTcpertechnetate scintigraphy showing elevated bilateral and diffuse uptake of the radioactive tracer 0cYamada BMC Endocrine Disorders Page of Fig Clinical course of the patient MMI methimazole KI potassium iodide Day first administration of nivolumab Day secondadministration of nivolumabTable Comparison of case reports on newonset Graves™ disease during nivolumab therapyStudyTSHμIUmL FT3pgmLFT4ngdLTRAb IULBeforeNAIadarola []AfterNegativeTSAb BeforeNANAAfterNANANAUSNormalHypervascularNormalRAIU99mTcuptakeHigh99mTcHighRAIUNAHLANANADRB104Brancatella []NANegativeKurihara []NAPositive NAYamada presentcaseUS ultrasonography RAIU radioactive iodine uptake Before before the initiation of nivolumab therapy After after the initiation of nivolumab therapy at theonset of the thyrotoxicosisNegative NegativeHypervascular PositivePositiveHigh99mTcDPB105 0cYamada BMC Endocrine Disorders Page of this case thyrotoxicosis was detected after the sixth administration of nivolumab with positive TRAb titreHowever ultrasonography showed no enlargement ofthe thyroid and a normal vascularisation pattern Thispatient was clinically diagnosed as mild Graves™ diseaseand treated with MMI [] Unlike these cases our caseis important in terms of confirmation of both positiveTRAb titre and diffuse thyroid uptake in scintigraphyMoreover titres of TRAb and TSAb were convertedfrom negative to positive after starting nivolumab therapy It seems reasonable to presume that Graves™ diseasewas induced by nivolumab although there is a possibilityof coincidence Furthermore our patient had HLADPB10501 which has been reported to be associatedwith Japanese Graves™ disease [ ] Although the involvement of HLA cannot be argued based only on asingle case accumulating similar cases might help clarifythe mechanism of development of rare ICIinducedGraves™ diseaseGraves™ disease induced by ICIs other than nivolumabhas been rarely reported Azmat [] reported aipilimumabinduced thyrotoxicosis caused bycase ofGraves™ disease Gan et al[] reported a case oftremelimumabinduced Graves™ hyperthyroidism Yajima [] reported a case of Graves™ disease induced bypembrolizumab a PD1 inhibitor In this case TRAbwas positive after the fifth administration of pembrolizumab and thyroid ultrasonography showed a mild increase in the intrathyroidal blood flow A thyroidscintigraphy was not performed because of the iodinetreatment [] The cases of nivolumabinduced Graves™disease with negative TRAb titre suggest that performingthyroid scintigraphy and ultrasonography can help to accurately diagnose and treat ICIinduced thyrotoxicosisA relationship between thyroid antibodies and PD1inhibitorinduced thyroid dysfunction has not been explained Kimbara [] suggested that patients withpreexisting TgAb and an elevated TSH level at baselineare at a higher risk of thyroid dysfunction induced bynivolumab Osorio [] reported an association between positive thyroid antibodies antithyroglobulin orantimicrosomal antibodies and thyroid dysfunction induced by ICIs In the studies on newonset Graves™ disease during nivolumab therapy it is interesting to notein two caucasian patients with Graves™ diseasethatTRAb was negative [ ] Furthermore TRAb waspositive in two Japanese patients including our patient[] Table However additional evidence is requiredto reveal the role of TRAb in the pathogenesis of ICIinduced hyperthyroidismA limitation of our case was radioactive iodine uptakeRAIU was not performed Because imaging with99mTcpertechnetate reflects both blood flow and uptakevia the symporter and does not assess anificationmalignant nodules may appear hyperfunctioning in pertechnetate imaging but hypofunctioning in 123IimagingIn our study tumours were not detected although a partof 99mTc uptake was strongerIn conclusion we reported a case of Graves™ diseaseshortly after the initiation of nivolumab therapy for gastric cancer Our case presented a typical Graves™ diseasewith both positive TRAb titre and diffuse thyroid uptakein scintigraphy Moreover our case is valuable in termsof confirming the conversion of TRAb and TSAb fromnegative to positive titres after starting the therapy It isimportant to perform thyroid scintigraphy and ultrasonography because there are cases of nivolumabinducedGraves™ disease with negative TRAb titre as previouslyreported To revealthe pathogenesis of ICIinducedGraves™ disease it is necessary to study additional casesof similar natureAbbreviationsCT Computed tomography CTLA4 Cytotoxic Tlymphocyteassociated protein FT3 Free triiodothyronine FT4 Free thyroxine HER2 Humanepidermal growth factor receptor HLA Human leukocyte antigenICI Immune checkpoint inhibitor IrAE Immunerelated adverse eventKI Potassium iodide MMI Methimazole MRI Magnetic resonance imagingOGD Oesophagogastroduodenoscopy PD1 Programmed cell deathprotein1 PDL1 Programmed death ligand RAIU Radioactive iodineuptake Tg Thyroglobulin TgAb Antithyroglobulin antibody TPOAb Antithyroidperoxidase antibody TRAb TSH receptor antibody TSAb Thyroidstimulating antibody TSH Thyroidstimulating hormoneAcknowledgmentsNot applicableAuthors™ contributionsHY FO and NE interpreted the data drafted the manuscript andparticipated in the endocrinological treatment of the patient HS revised themanuscript TO and SF participated in the gastroenterological treatment ofthe patient All authors have read and approved the final version of themanuscript for publicationFundingNot applicableAvailability of data and materialsThe data that support the findings of this study are stored in NipponMedical School Chiba Hokusoh Hospital Inzai Chiba and available from thecorresponding author on reasonable requestEthics approval and consent to participateThis case report was approved by the ethics committee of Nippon MedicalSchool Chiba Hokusoh HospitalConsent for publicationWritten informed consent was obtained from the patient™s next of kin forpublication of this case report and any accompanying images A copy of thewritten consent is available for review by the editor of this journalCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Endocrinology Diabetes and Metabolism Graduate Schoolof Medicine Nippon Medical School Sendagi Bunkyoku Tokyo Japan 2Department of Gastroenterology Nippon Medical SchoolChiba Hokusoh Hospital Kamagari Inzai Chiba Japan 0cYamada BMC Endocrine Disorders Page of Received April Accepted August ReferencesGonzalezRodriguez E RodriguezAbreu D Spanish Group for CancerImmunoBiotherapy GETICA Immune checkpoint inhibitors review andmanagement of endocrine adverse events Oncologist “ Michot JM Bigenwald C Champiat S Collins M Carbonnel F PostelVinay S Immunerelated adverse events with immune checkpoint blockade acomprehensive review Eur J Cancer “Bertrand A Kostine M Barnetche T Truchetet ME Schaeverbeke T Immunerelated adverse events associated with antiCTLA4 antibodies systematicreview and metaanalysis BMC Med Faje A Immunotherapy and hypophysitis clinical presentation treatmentand biologic insights Pituitary “Topalian SL Hodi FS Brahmer JR Gettinger SN Smith DC McDermott DF Safety activity and immune correlates of antiPD1 antibody in cancerN Engl J Med “Robert C Schachter J Long GV Arance A Grob JJ Mortier L et alPembrolizumab versus ipilimumab in advanced melanoma N Engl J Med“Orlov S Salari F Kashat L Walfish PG Induction of painless thyroiditis inpatients receiving programmed death receptor immunotherapy formetastatic malignancies J Clin Endocrinol Metab “Kimbara S Fujiwara Y Iwama S Ohashi K Kuchiba A Arima H et alAssociation of antithyroglobulin antibodies with the development ofthyroid dysfunction induced by nivolumab Cancer Sci “Iadarola C Croce L Quaquarini E Teragni C Pinto S Bernardo A et alNivolumab induced thyroid dysfunction Unusual clinical presentation andchallenging diagnosis Front Endocrinol Lausanne Brancatella A Viola N Brogioni S Montanelli L Sardella C Vitti P et alGraves' disease induced by immune checkpoint inhibitors a case reportand review of the literature Eur Thyroid J “Kurihara S Oikawa Y Nakajima R Satomura A Tanaka R Kagamu H et alSimultaneous development of Graves' disease and type diabetes duringantiprogrammed cell death1 therapy a case report J Diabetes Investig“ Dong RP Kimura A Okubo R Shinagawa H Tamai H Nishimura Y et alHLAA and DPB1 loci confer susceptibility to graves™ disease Hum Immunol“ Ueda S Oryoji D Yamamoto K Noh JY Okamura K Noda M et alIdentification of independent susceptible and protective HLA alleles inJapanese autoimmune thyroid disease and their epistasis J Clin EndocrinolMetab “ Azmat U Liebner D JoehlinPrice A Agrawal A Nabhan F Treatment ofipilimumab induced graves™ disease in a patient with metastatic melanomaCase Rep Endocrinol Gan EH Mitchell AL Plummer R Pearce S Perros P Tremelimumab inducedgraves hyperthyroidism Eur Thyroid J “ Yajima K Akise Y A case report of Graves' disease induced by the antihuman programmed cell death1 monoclonal antibody pembrolizumab ina bladder cancer patient Case Rep Endocrinol Osorio JC Ni A Chaft JE Pollina R Kasler MK Stephens D Antibodymediated thyroid dysfunction during Tcell checkpoint blockade in patientswith nonsmallcell lung cancer Ann Oncol “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

Thyroid_Cancer

Primary squamous cell carcinoma ofis anextremely rare aggressive malignancy with a poor prognosis However almost noreportthus far has investigated the microvasculature of ThyPSCC imaged usingcontrastenhanced ultrasoundthe thyroid ThyPSCCCase Report A 59yearold male patient presented to our hospital with progressivelyworsening hoarse voice symptoms for days and was diagnosed with left unilateralvocal fold palsy Ultrasonography revealed a solitary marked hypoechoic thyroid nodulewith an unclear boundary in the inferior part of the left lobe Color Doppler flow imagingshowed a poor blood flow signalinside this nodule Contrastenhanced ultrasoundimages showed a persistent low peak enhancement of the nodule from its periphery to itscenter The timeintensity curve displayed a washin time of s a time to peak of s apeak signal intensity of and a washout time of s for the thyroid tumor Finallyleft hemithyroidectomy of the thyroid tumor was performed and histopathologic andimmunohistochemical evaluations confirmed the diagnosis of ThyPSCC Postoperativelythe patient received a combination therapy of chemotherapy radiotherapy and targetedtherapy but the patient died months after surgeryPrimary squamous cell carcinoma ofConclusionthe thyroid is a rare butaggressive malignancy of the thyroid Herein we reported a case of ThyPSCC and itsultrasonography and pathologic findingsKeywords thyroid cancer thyroid nodules TNs thyroid ultrasound US primary squamous cell carcinomacontrast enhanced ultrasound CEUSINTRODUCTIONPrimary squamous cell carcinoma of the thyroid ThyPSCC is a rare thyroid malignancy withhigh aggressiveness and poor prognosis comprising ˆ¼“ of all primary thyroid carcinomas“ Owing to the rapidly progressing and highly invasive nature of the malignancy patients withThyPSCC often present at an advanced stage and are difficult to diagnose in the early stage becauseof its rare incidence and lack of typical imaging findings Thyroid ultrasonography and fineneedle aspiration biopsy FNAB are the diagnostic tools ofchoice for evaluating patients with suspected thyroid nodules Contrastenhanced ultrasoundCEUS as a relatively novel US technique is used to investigate the microvasculature of thyroidnodules and improve the diagnostic accuracy of thyroid nodules accompanied by the use of ThyroidEdited byChristoph ReinersUniversity HospitalW¼rzburg GermanyReviewed byPasqualino MalandrinoUniversity of Catania ItalyDaniela PasqualiUniversity of Campania LuigiVanvitelli ItalyCorrespondenceChengcheng NiuniuchengchengcsueducnSpecialty sectionThis was submitted toCancer Endocrinologya section of the journalFrontiers in EndocrinologyReceived February Accepted June Published August CitationChen S Peng Q Zhang Q and Niu C ContrastEnhanced Ultrasoundof Primary Squamous Cell Carcinomaof the Thyroid A Case ReportFront Endocrinol 103389fendo202000512Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaImaging Reporting and Data Systems for ultrasonographicfeatures “ However very few published studies havereported the use of ultrasonography for ThyPSCC To ourknowledge this is the first case describing the CEUS findingsof ThyPSCCreached its peak [time to peak TTP] at s with a peakintensity of Then the nodule slowly declined until allthe microbubbles washed out at s Figures 1CD Based onits malignant conventional ultrasound features and the poormicrovasculature revealed by CEUS we inferred that the nodulewas a malignant tumorCASE REPORTA 59yearold male patient presented to our hospital withprogressively worsening hoarse voice symptoms for daysand was diagnosed with left unilateral vocal fold palsy Ahighresolution ultrasound instrument Siemens Acuson S3000Mountain View CA USA equipped with a to 9MHz linearprobe was used Thyroid ultrasonography revealed a solitary — — 26cm3 marked hypoechoic thyroid nodule with anunclear boundary in the inferior part of the left lobe AThis nodule exhibited many malignant ultrasound featuressuch as solid components hypoechogenicity and microlobulatedmargins Color Doppler flow imaging CDFI showed poorblood flow signals in the nodule B Contrastenhancedultrasound was performed with a bolus intravenous injectionof mL of SonoVue Bracco Milan Italy followed by mLof saline Contrast pulse sequencing technology was used andthe timeintensity curves TICs of the nodule were calculatedThe nodule began to be slowly enhanced from the peripheryto the center at s washin time and the enhancementAfterneckthepositronultrasonographyemissiontomography“computed tomography was carried for evaluatingthesituation of distant metastases Positron emissiontomography“computed tomography showed a mass withincreased glucose metabolism in the inferior part of the leftthyroid lobe A which indicated it as a malignantmass whereas there was no evidence of lymph nodes metastasisand distant metastases Then ultrasonographyguided FNABwas performed for the left thyroid mass immediately Cytologicexamination by fineneedle aspiration FNA revealed sheets oftumor cells with giant deepstained nuclei Bethesda categoryV B Finally a left hemithyroidectomy of the thyroidtumor was undertaken The lower edge of the tumor reachedthe upper mediastinum and the depth of the tumor invadedthe esophagus and trachea which could not be completelyremoved According to the eighth edition of the AmericanJoint Committee on CancerTumor Lymph Node MetastasisTNM staging system the patient was in TNM stage IIIT4a N0 M0 Histopathological examination of hematoxylinand eosin staining showed that a carcinoma in the inferiorFIGURE Ultrasonography images of primary squamous cell carcinoma of the thyroid A Longitudinal grayscale sonography revealed a solid marked hypoechoicthyroid nodule in the inferior part of the left lobe B Color Doppler flow imaging showed a poor blood flow signal inside this nodule C Contrastenhanced ultrasoundimage showed a persistent low peak enhancement of the nodule at s D Timeintensity curves displayed the washin time of s TTP of s peak signalintensity of and washout time of s for the thyroid tumorFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaFIGURE A A positron emission tomography“computed tomography scan showed increased 18Ffluorodeoxyglucose metabolism in the left neck mass BPreoperative fineneedle aspiration cytology of the mass demonstrated a few sheets of malignantlooking tumor cells with giant deep stained nuclei hematoxylin andeosin magnification — FIGURE Hematoxylin and eosin staining of primary squamous cell carcinoma of the thyroid A magnification — B magnification — C magnification — D magnification — part of the thyroid lobe A had no obvious palisadearrangementintercellular bridges or keratinization with acancer pearl Figures 3B“D Immunohistochemically tumorcells were positive for cytokeratin CK19 Acytokeratin and CK56 B epithelial membraneantigen EMA C p40 D p63 Aand Ki67 B and negative for thyroglobulinTG C and thyroid transcription factor TTF1D In view of these findings the tumor was diagnosedas poorly diï¬erentiated ThyPSCC Postoperatively the patientreceived two cycles of chemotherapy with docetaxelcisplatinintensitymodulated radiotherapy and nimotuzumabtargetedtherapy However the patient died months after surgeryDISCUSSIONPrimary squamous cell carcinoma of the thyroid is a thyroidmalignancy with extremely rare incidence and the clinicaldiagnosis and treatment guidelines for this disease have noconsensus The biological behavior of ThyPSCC is aggressiveFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaFIGURE Immunohistochemical staining of primary squamous cell carcinoma of the thyroid magnification — Immunohistochemical staining for A CK19 BCK56 C EMA D p40 all of which were deeply stained positiveFIGURE Immunohistochemical staining of primary squamous cell carcinoma of the thyroid magnification — Immunohistochemical staining for A p63 B Ki C TG D TTF1 and p63 was deeply stain positive Ki67 proliferation index was TG and TTF1 did not stain negativeFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell Carcinomaand the prognosis is poor with a median overall survival of “ months which depends on the diï¬erent tumor grades Yang et al using the Surveillance Epidemiology and End ResultsProgram database reported that poorly diï¬erentiated tumrade occupied the highest percentages of all graded tumors andthe median survival was months which is similar to the survivaltime in our case Highfrequency ultrasound as the basic imaging modality inthe diagnosis of thyroid nodules has found gradually increasingdiï¬erentiated thyroid cancers over recent years Theultrasonography imaging findings of ThyPSCC have seldombeen published Regarding the ultrasonography findings Chenet al reported that ThyPSCC presented as a thyroid masswith eggshell calcification peripheral soft tissue with a blurredmargin and minimal vascular signals on CDFI sonographyIn the case of Jang et al ThyPSCC presented as a largewelldefined lobulated heterogeneously hypoechoic mass withdiï¬use microcalcifications on ultrasonography Kondo et al reported that a welldiï¬erentiated ThyPSCC showed acystic hypoechoic mass with a smooth margin and rapidlygrew with margin change blurring in year In our case thispoorly diï¬erentiated ThyPSCC presented as a solitary markedhypoechoic thyroid mass with an irregular margin and unclearboundary with a normal thyroid The irregular margin andunclear boundary with normal thyroid corresponded to tumorinvasion with adjacent tissue infiltration which is consistentwith the findings during the operation that tumor invasion withthe esophagus cannot be completely removed Poor blood flowsignals on CDFI sonography and persistent hypoenhancement onCEUS of the mass are consistent with squamous cell carcinomawhich has no obvious vascularity on pathologic examinationMany studies have investigated the application of CEUS toimprove the diagnostic accuracy of thyroid nodules despiteits usage in ThyPSCC being scarce Zhang et al foundthat highcircularequal enhancement indicated benign thyroidnodules and low enhancement indicated malignant thyroidnodules Ma et al investigated whether incomplete noring or heterogeneous enhancement later washin time andlow peak intensity on CEUS were independent risk factorsin predicting malignantthyroid nodules Deng et al detected that papillary thyroid carcinomas PTCs exhibited lowenhancement a lower peak signal intensity and a lower areaunder the curve AUC than peripheral thyroid parenchyma onCEUS In our study the TICs of CEUS for ThyPSCCshowed a washin time of s a TTP of s a peak signalintensity as low as and a washout time of s Thisis similar to the results of PTCs with a slow washin time alower peak signal intensity and a lower AUC as in previousreports To our knowledge no reports on CEUS imagingfindings of ThyPSCC have appeared in the Englishlanguageliterature According to Jang et al ThyPSCC showed a largeheterogeneously enhancing thyroid mass with a large centralnonenhancing portion on enhanced CT which correspondedwell with the squamous cell carcinoma portion with a necroticportion in pathologic staining Because of the rapid growth ofsquamous tumor cells relatively few interstitial blood vessels intumors were related to the low peak signal intensity and low AUCon CEUSisusefulstainingWith increasing malignancy in squamous cell carcinoma thetypical squamous cell carcinoma findings of intercellular bridgesand keratinized cancer pearl can decrease or disappearImmunohistochemicalin diagnosingprimary thyroid cancer In this case positivity for CK56and EMA and negativity for TTF1 and TG expressionpredicted squamous cell carcinoma derivation and excludedthe possibility ofthese common tumors Furtherpositivity for p63 and Ki67 expression as poor prognosticmarkers was associated with its poorly diï¬erentiated tumrade CONCLUSIONPrimary squamous cell carcinoma of the thyroid is an extremelyrare tumor and very few studies describe its ultrasonographicimaging findings It is difficult to establish a clinical guidelinefor diagnosis Our case presents the CEUS features of ThyPSCCindicating that the TICs of ThyPSCC are similar to the enhancingparameters of PTCs with a slow washin time a lower peak signalintensity and a lower AUCDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studies involving human participants were reviewed andapproved by the Ethics Committee of Second Xiangya HospitalCentral South University China The patientsparticipantsprovided their written informed consentto participate inthis study Written informed consent was obtained from theindividuals for the publication of any potentially identifiableimages or data included in this AUTHOR CONTRIBUTIONSAll authors listed have made a substantial direct and intellectualcontribution to the work and approved it for publicationFUNDINGof ChinaThis project was funded by the National Natural ScienceFoundationProvincialNatural Science Foundation of China 2018JJ2575 andHunan Provincial Health Commission Research FoundationProject B2019166 HunanFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alREFERENCES Yang S Li C Shi X Ma B Xu W Jiang H et al Primary squamous cellcarcinoma in the thyroid gland a populationbased analysis using the SEERdatabase World J Surg “ 101007s00268019049062 Limberg J Ullmann TM Stefanova D Finnerty BM Beninato T Fahey TJet al Prognostic characteristics of primary squamous cell carcinoma of thethyroid a national cancer database analysis World J Surg “ 101007s00268019050985Thyroid Primary Squamous Cell Carcinomacontrastenhanced ultrasound Ultrasound Med Biol “ 1016jultrasmedbio201810020 Casella C Ministrini S Galani A Mastriale F Cappelli C Portolani NThe new TNM staging system for thyroid cancer and the risk of diseasedownstaging Front Endocrinol 103389fendo201800541 Zhang Y Zhou P Tian SM Zhao YF Li JL Li L Usefulness of combineduse of contrastenhanced ultrasound and TIRADS classification for thediï¬erentiation of benign from malignant lesions of thyroid nodules EurRadiol “ 101007s003300164508y Koyama S Fujiwara K Nosaka K Fukuhara T Morisaki T MiyakeN et al Immunohistochemical features of primary pure squamous cellcarcinoma in the thyroid an autopsy case Case Rep Oncol “ Zhang YZ Xu T Gong HY Li CY Ye XH Lin HJ et al Application ofhighresolution ultrasound realtime elastography and contrastenhancedultrasound in diï¬erentiating solid thyroid nodules Medicine95e5329 101097MD00000000000053290000579220161108000016 Wang SS Ye DX Wang B Xie C The expressions of keratins andP63 in primary squamous cell carcinoma ofthe thyroid gland anapplication of raman spectroscopy Onco Targets Ther “ 102147OTTS229436 Chen CY Tseng HS Lee CH Chan PW Primary squamous cellcarcinoma of the thyroid gland with eggshell calcification sonographicand computed tomographic findings J Ultrasound Med “ 107863jum201029111667 Yasumatsu R Sato M Uchi R Nakano T Hashimoto K Kogo R et al Thetreatment and outcome analysis of primary squamous cell carcinoma of thethyroid Auris Nasus Larynx “ 101016janl201707009 Kao NH Tan CS H Koh AJ The utility of immunohistochemistry indiï¬erentiating metastatic primary squamous cell carcinoma of the thyroidfrom a primary lung squamous cell carcinoma Case Rep Endocrinol “ Jang JY Kwon KW Kim SW Youn I Primary squamouscarcinoma ofandtomographic“ 1014366usg13022cellrecurrence ultrasonographicthyroid gland with localUltrasonographycomputedfindings Raggio B Barrcarcinomacell 1031486toj180002J Ghandour Z Friedlander P Primary squamousof“thyroid OchsnertheJ Kondo T Matsuyoshi A Matsuyoshi H Goto R Ono K Honda Y et alA case of primary thyroid squamous cell cancer transformation frombenign tumour associated with chronic thyroiditis BMJ Case Rep 2009bcr1020081137 101136bcr1020081137 Ma JJ Ding H Xu BH Xu C Song LJ Huang BJ et al Diagnosticand contrastmalignant101089thy201performances ofenhancedultrasonographythyroid nodules Thyroid“ color dopplergrayscalepredictingfindingsvariousin Deng J Zhou P Tian SM Zhang L Liofefficacydiagnosticofradiationdiï¬erentiating9e90674 101371journalpone0090674PONED1330329imagingthyroidandnodulescontrastenhancedimpulseforcesolidfocalJL Qian Y ComparisonacousticinuseultrasoundcombinedPLoS ONEtheir Haugen BR Alexander EK Bible KC Doherty GM Mandel SJ Nikiforov YEet al American thyroid association management guidelines for adultpatients with thyroid nodules and diï¬erentiated thyroid cancer the Americanthyroid association guidelines task force on thyroid nodules and diï¬erentiatedthyroid cancer Thyroid “ 101089thy20150020 Tessler FN Middleton WD Grant EG Hoang JK Berland LL Teefey SAet al ACR thyroid imaging reporting and data system TIRADS whitepaper of the ACR TIRADS committee J Am Coll Radiol “ 101016jjacr201701046 Kwak JY Han KH Yoon JH Moon HJ Son EJ Park SH et al Thyroidimaging reporting and data system for US features of nodules a step inestablishing better stratification of cancer risk Radiology “ 101148radiol11110206radiol11110206 Peng Q Niu C Zhang Q Zhang M Chen S Mummified thyroid nodulesconventional and contrastenhanced ultrasound features J Ultrasound Med “ 101002jum14712 Peng Q Niu C Zhang M Chen S Sonographic characteristics ofpapillary thyroid carcinoma with coexistent hashimoto™sthyroiditisconventional ultrasound acoustic radiation force impulse imaging and Struller F Senne M Falch C Kirschniak A Konigsrainer A Mullerthe thyroid case report andS Primary squamous cell carcinoma ofsystematic review of the literature Int J Surg Case Rep “ 101016jijscr201706011 Wang W Ouyang Q Meng C Jing L Li X Treatment optimization andprognostic considerations for primary squamous cell carcinoma of thethyroid Gland Surg “ 1021037gs20191107Conflict 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 Chen Peng Zhang and Niu This is an openaccess distributed under the terms of the Creative Commons Attribution License CC BYThe use distribution or reproduction in other forums is permitted provided theoriginal authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'

Thyroid_Cancer

The effects of tissue fixation on sequencingand transcript abundance of nucleic acidsfrom microdissected liver samples ofsmallmouth bass Micropterus dolomieuHeather L WalshID Adam J Sperry Vicki S BlazerUS Geological Survey National Fish Health Research Laboratory Leetown Science Center KearneysvilleWest Virginia United States of Americaa1111111111a1111111111a1111111111a1111111111a1111111111 hwalshusgsgovAbstractThere is an increasing emphasis on effectsbased monitoring to document responses associated with exposure to complex mixtures of chemicals climate change pathogens parasitesand other environmental stressors in fish populations For decades aquatic monitoring programs have included the collection of tissues preserved for microscopic pathology Consequently formalinfixed paraffinembedded FFPE tissue can be an important reservoir ofnucleic acids as technologies emerge that utilize molecular endpoints Despite the crosslinking effects of formalin its impact on nucleic acid quality and concentration amplification andsequencing are not well described While freshfrozen tissue is optimal for working withnucleic acids FFPE samples have been shown to be conducive for molecular studies Lasercapture microdissection LCM is one technology which allows for collection of specificregions or cell populations from fresh or preserved specimens with pathological alterationspathogens or parasites In this study smallmouth bass Micropterus dolomieu liver was preserved in three different fixatives including neutral buffered formalin NBF ZFix®ZF and PAXgene® PG for four time periods hr hr seven days and days Controls consisted of pieces of liver preserved in RNALater® or ethanol Smallmouth basswere chosen as they are an economically important sportfish and have been utilized as indicators of exposure to endocrine disruptors and other environmental stressors Small liversections were cut out with laser microdissection and DNA and RNA were purified and analyzed for nucleic acid concentration and quality Sanger sequencing and the NanoStringnCounter® technology were used to assess the suitability of these samples in downstreammolecular techniques The results revealed that of the formalin fixatives NBF samples fixedfor and hr were superior to ZF samples for both Sanger sequencing and the NanostringnCounter® The nonformalin PAXgene® samples were equally successful and they showedgreater stability in nucleic acid quality and concentration over longer fixation times This studydemonstrated that small quantities of preserved tissue from smallmouth bass can be utilizedin downstream molecular techniques however future studies will need to optimize the methods presented here for different tissue types fish species and pathological conditions ACCESSCitation Walsh HL Sperry AJ Blazer VS The effects of tissue fixation on sequencing andtranscript abundance of nucleic acids frommicrodissected liver samples of smallmouth bassMicropterus dolomieu e0236104 101371journalpone0236104Editor Rajakumar Anbazhagan National Instituteof Child Health and Human Development NICHDNIH UNITED STATESReceived April Accepted June Published August Copyright This is an access free of allcopyright and may be freely reproduceddistributed transmitted modified built upon orotherwise used by anyone for any lawful purposeThe work is made available under the CreativeCommons CC0 public domain dedicationData Availability Statement All relevant data arewithin the paper and its Supporting InformationfilesFunding This was fully funded internally by theUSGS Environmental Health Contaminant BiologyProgram and the USGS EcosystemsEnvironments and Fisheries Program MissionAreas There are no actual grant numbersPLOS ONE 101371journalpone0236104 August PLOS ONE 0cCompeting interests The authors have declaredthat no competing interests existEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostIntroductionGlobally environmental monitoring programs are increasingly used to identify adverse effectsof human activities on aquatic resources [“] The recognition that there are numerouschemical contaminants environmental stressors as well as new and emerging pathogensparasites cooccurring has led to an emphasis on biological environmental effectsbased assessments utilizing resident indicator fish species or caged model species [“] Histopathologyhas been used for many years to assess the health of wild fishes both for specific studies as wellas part of effectsbased monitoring programs [“] More recently genomic endpoints arealso being incorporated into environmental monitoring and risk assessment [“] Whenboth histopathology and molecular analyses are part of an assessment pieces of a tissue arecommonly preserved in buffered formalin or a similar preservative and adjacent separatepieces are preserved in RNAlater1 ethanol or frozen for molecular analyses [“]However for alterations not visible the tissue piece chosen for gene expression may not contain the same cellular components or alterations as those within the histology sectionThe use of formalin fixed paraffinembedded FFPE tissue has been regarded as a valuablereservoir of preserved nucleic acids in mammalian studies [“] Although FFPE tissuesprovide a vast source of pathologically diverse types of genetic material there are drawbackscompared to other tissue preservation methods Formalin fixation causes nucleic acids to fragment degrade and crosslink [] Frozen tissues or tissues specifically preserved for downstream nucleic acid applications do not experience the type of degradation observed fromformalin fixation Despite these setbacks nucleic acids extracted from FFPE tissue have provento be suitable for use in endpoint PCR [] realtime qPCR [ ] and Nextgenerationsequencing [ ] Optimization of FFPE tissues for downstream nucleic acid applicationshas been attempted in multiple studies by evaluation of different fixation methods [ ] tissue handling and processing times [ ] and extraction methods [ ]Laser capture microdissection LCM utilizes a microscope equipped with a laser to targetand isolate specific cells from a heterogeneous population of cells [] Single cells foci of cellpopulations within a tissue or pathogens and parasites can be microdissected Hence nucleicacids from specific cell populations of interest can be analyzed for gene expression studiestranscriptome development or molecular identification of pathogens and parasites Thisallows for a more direct connection between the histopathology and molecular analyses LCMhas been previously utilized in fishrelated studies [ “] with frozen sections Snapfrozen tissue is optimal for use with LCM for the downstream recovery of nucleic acids However the use of snapfrozen tissue is not always feasible particularly in wild fish studies whereremoval and fixation of the ans occurs in the field and it can be days before tissues arereturned to the laboratory and processed LCM of FFPE tissue can bridge the gap betweenmicroscopy and molecular analyses [] As with other species there is a vast amount of archival FFPE or similarly preserved fish tissue that could be useful for molecular analysesThe aim of this study was to determine how fixative type and fixation time affects nucleicacids in FFPE smallmouth bass liver tissue dissected with LCM Smallmouth bass Micropterusdolomieu are utilized in ongoing monitoring and assessment studies as an indicator species ofexposure to endocrinedisrupting and other contaminants Additionally they are a nonmodel but economically important species To address the utility of paraffinembedded fishtissue for molecular studies smallmouth bass liver was sampled and preserved for four timeperiods hr hr seven days and days in neutral buffered formalin NBFZFix1 ZF and the nonformalin fixative PAXgene1 PG The PAXgene1 Tissue Systemwas designed to improve tissue quality for parallel molecular and morphological analyses []Similarly ZF a zincbased formalin solution was chosen as it has been shown to producePLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleosthigher yields of DNA and RNA when compared to samples fixed in NBF [] In addition toDNA and RNA quantification downstream molecular techniques including Sanger sequencing and the Nanostring nCounter1 digital multiplexed gene expression assay [] were usedto determine if nucleic acids extracted from LCM tissue sections would have utility in futurestudies To the best of our knowledge this study provides novel research on the optimizationof fixative type and fixation time for the use of fish tissue extracts with Nanostring nCounter1technologyMaterials and methodsEthics statement and smallmouth bass sample collectionAll procedures including the handling and euthanasia of fish were approved by the US Geological Survey™s Leetown Science Center™s Institutional Animal Care and Use CommitteeIACUC protocol Five smallmouth bass approximately years old were sampledfrom a flowthrough tank at the US Geological Survey Fish Health Laboratory in Kearneysville West Virginia Fish were placed in a lethal dosage mgL of tricaine methanesulfonate TricaineS Syndel Ferndale WA for euthanasia An incision from the anus tooperculum was made the liver was excised dissected into five equal pieces and placed into fixatives consisting of NBF ZF Product Anatech Ltd Battle Creek MI and PG Product QIAGEN Valencia CA Pieces of liver from each fish were also placed into RNALater1 Product AM7021 Thermo Fisher Scientific Waltham MA and ethyl alcoholETOH to serve as controls Samples in RNALater1 were stored at ˚C for hr prior tostorage at ˚C and samples in ETOH were stored at room temperature RT until extractionswere completedHistological preparation and laser capture microdissectionSamples were fixed for hrs hrs seven days and days at RT for NBF and ZF Tissues preserved in PG were removed from the PAXgene1 Tissue FIX Product QIAGEN after hrs at RT placed in the PAXgene1 Tissue STABILIZER solution Product QIAGEN and stored at ˚C for hrs hrs seven and days Tissue processing was performed on a Shandon CitadelTM Tissue Processor Thermo FisherScientific as follows hrs in alcohol hr in alcohol hr in alcohol 2x hr in alcohol 3x hr in a solution of alcohol and histoclear 2x Product HS200 National Diagnostics Atlanta GA hr in histoclear 2x and hr in paraffin 2x at ˚C Upon completion tissues were embedded into paraffin wax and cooled tohardenTissues were cut at a thickness of μm using a new sterile razor for each sample and sections placed onto Leica Microsystems UVsterilized polyethylene napthalate PEN membraneslides Product NC0496333 Thermo Fisher Scientific Sterilized diethyl pyrocarbonateDEPC Product Millipore Sigma Burlington MA water was used in the water bathand slides were allowed to air dry after sections were placed on the PEN membrane slideUnstained tissue sections were deparaffinized with Anatech Ltd ProPar Clearant Product NC9537734 Thermo Fisher Scientific for min 2x and allowed to air dry prior to lasermicrodissection Liver sections were cut at 5x magnification with a Leica LMD6500 microscope Leica Microsystems at a pulse rate of “ nm Sections x mm2were cut and dropped into the cap of a sterile microcentrifuge tube by gravity Fig and subsequently extracted for RNA or DNAPLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostFig Laser capture microdissection of a smallmouth liver section A Liver section prior to microdissection BLiver section after microdissection C Liver section floating in buffer in the cap of a microcentrifuge tub prior tonucleic acid extraction101371journalpone0236104g001Nucleic acid extractions and downstream analysesFor RNA purification the EZNA1 FFPE RNA Kit Product R695401 Omega BioTekNorcross GA was used according to manufacturer™s protocols for the xylene extractionmethod Extraction began with the addition of GPL Buffer skipping the beginning of the protocol since the tissues were already deparaffinized Samples were digested with proteinase Kfor min and eluted in μl DEPC water As part of the assay protocol DNA contaminationwas removed with a step involving DNA Clearance Columns that binds genomic DNA andallows RNA to pass through the spin column For the controls preserved in RNALater1approximately “ mg liver was extracted with an EZNA1 Total RNA Kit I Product R683402 Omega BioTek according to manufacturer™s protocols and eluted in μl DEPCwater DNA contamination was also removed from these samples with the use of HiBind1RNA Mini Columns and RNasefree DNase Product E109102 Omega BioTek All samples were quantified with a Qubit1 Fluorometer Invitrogen Carlsbad CA using theQubit1 RNA HS Assay Kit Product Q32852 Thermo Fisher Scientific To analyze degradation RIN values were obtained with the Agilent RNA Pico Kit Product “Agilent Technologies Santa Clara CA on an Agilent Bioanalyzer Agilent Technologies Following quantification samples were stored at ˚C prior to use on the NanostringnCounter1DNA was extracted with a proteinase K digestion buffer 50mM TrisHCl pH mMEDTA Tween mgml proteinase K as described in Lehmann and Kreipe []Each sample was extracted in μl of proteinase K digestion buffer and incubated overnight at˚C The tubes were vortexed centrifuged and incubated at ˚C for min to deactivateproteinase K and stored at ˚C Approximately “ mg of control liver samples preservedin ETOH were extracted with a Qiagen DNeasy Blood Tissue Kit Product QIAGEN according to manufacturer™s protocols It is worth mentioning that in initial trialsfor this study the Qiagen DNeasy Blood Tissue Kit was also used to extract DNA from LCMsamples however no quantifiable DNA could be obtained which was why a single tube extraction method was subsequently utilized DNA was quantified with the Qubit1 dsDNA HSAssay Kit Product Q32851 Thermo Fisher Scientific Samples were analyzed for mean fragment size and distribution on an Agilent Bioanalyzer with the Agilent High SensitivityDNA Kit Product “ Agilent TechnologiesFor all LCM samples the final concentration of purified RNA and DNA was standardizedby dividing the total concentration by the total amount of tissue collected μgmm3 Since agreater amount of tissue was extracted from control samples the concentration of purifiedRNA and DNA was standardized to the amount of tissue collected for LCMTo assess the suitability of LCM samples for downstream molecular analyses Sangersequencing and the NanoString nCounter1 Technology were used For endpoint PCRPLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostprimers EF1α5F ™GAG CCC CCT TAC AGC CAG AAG3™ and EF1α5R ™TTCACC TCA GTG GTC AGG CA3™ were designed with NCBI Primer BLAST [] to amplifya bp amplicon of the smallmouth bass elongation factor alpha EF1α accession HQ4248721 gene This housekeeping transcript was chosen since it has been used in othersmallmouth bass studies [ ] and sequence data was available for both smallmouth bassand the closely related largemouth bass accession KT8277941 PCR amplification was conducted under the following conditions denaturation at ˚C for min followed by cyclesof ˚C for s ˚C for s and ˚C for min s with a final extension at ˚C for min Each reaction consisted of μl clear Go Taq Master Mix Product M7132 PromegaMadison WI μl of each primer at 10μM and μl template for LCM samples approximately “ ng and μl template for ETOH samples approximately “ ng Uponcompletion samples were analyzed on an agarose gel with a bp ladder Amplicons werecleaned with a QIAquick1 PCR purification kit Product QIAGEN and eluted in μl of Buffer EB Purified amplicons were used as template in cycle sequencing reactionswith the Applied Biosystems BigDye Terminator v31 Cycle Sequencing Kit Product Thermo Fisher Scientific for cycles of ˚C for min ˚C for s ˚C for sand ˚C for min Cycle sequencing reactions were purified with an Agencourt CleanSEQKit Product A29151 Beckman Coulter Brea CA and sequenced on an ABI 3130xl GeneticAnalyzer Applied Biosystems Foster City CA Sequences were analyzed with Geneious wwwgeneiouscom and quality was assessed by the percentage of bases with aquality score of or higher Q40 NCBI BLASTn was used to determine sequence similarityto the Micropterus spp EF1α gene HQ4248721 or KT8277941NanoString nCounter1 Technology was used with a Custom CodeSet that targeted transcripts expressed in the liver of smallmouth bass as described in Hahn et al [] The previous establishment and availability of this CodeSet was the reason liver was chosen as the tissue of focus in this study The liver is also the principal an for many chemical detoxificationpathways metabolic pathways and the production of vitellogenin In brief the nCounter1platform provides the capability to quantify up to RNA DNA or protein targets called aCodeSet in a multiplex fashion providing results similar to quantitative PCR qPCR [] Itis a costeffective method to analyze specific mRNA targets unlike RNAsequencing whichproduces a vast amount of data and captures all mRNA in a sample Sample setup for hybridizations was carried out according to manufacturer™s protocols with ng of total RNA forevery sample The limit of detection LOD was calculated as the mean of the negative controls � the standard deviation of the negative controls and was transcriptsStatisticsSignificant differences in nucleic acid concentrations and transcript abundance between fixatives for each fixation time were determined with a nonparametric KruskalWallis onewayANOVA followed by a Dunn™s multiple comparison posthoc analysis with a Bonferroni correction in the statistical program R [] Normalized transcript abundance data was used forthe analysis Transcript abundance data was normalized in nSolver Analysis Software Nanostring Technologies Seattle WA where the geometric mean of the negative controlswas subtracted to estimate background and the normalization factor was computed from thegeometric mean of the positive controls and the housekeeping transcripts Housekeeping transcripts were log2 transformed and analyzed for stability with NormFinder [] in R A KruskalWallis test was also used to identify differences amongst each fixative for each fixationtime and the template concentration used for PCR the Q40 score and sequence lengthobtained with Sanger sequencing Finally Spearman™s rank correlation analyses werePLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostconducted in R to determine if the concentration of DNA samples used for PCR sequencelength and the Q40 score were associated the number of sequences with similarity to theMicropterus spp EF1α gene with Sanger sequencing Pvalues � were considered statistically significantResultsNucleic acid concentrationsBoth RNA and DNA were recovered from samples of all fixatives and fixation times Fig Liver samples preserved in RNALater1 had more than times greater RNA concentrationsthan samples preserved in NBF ZF or PG with a mean concentration of ± ngmm3 mean ± standard error The highest concentration of RNA from LCM samples wasobtained from PG samples at hr ± ngmm3 Fig 2A The lowest concentrationsFig Nucleic acid auantification A Mean standard error of RNA and B DNA concentrations μgmm3 ofmicrodissected smallmouth bass liver samples fixed in neutral buffered formalin NBF ZFix1 ZF andPAXgene1 PG for hours hours and days Samples preserved in alcohol ETOH and RNALater1were included as controls101371journalpone0236104g002PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostwere from NBF preserved tissues at two weeks ± ngmm3 The concentration ofRNA was significantly greater in RNALater1 samples than in NBF Pvalue and PGPvalue at hr NBF Pvalue and ZF Pvalue at hr NBF Pvalue and ZF Pvalue at seven days and NBF Pvalue � ZF Pvalue and PG Pvalue at days Mean concentrations of RNA in samplesfixed in NBF and ZF decreased at seven and days while those fixed in PG remained stablethroughout the time course Fig 2AThe amount of DNA recovered was less than RNA with all mean concentrations of LCMsamples below μgmm3 Samples fixed in ETOH had more than times greater concentrations of DNA than samples fixed for LCM with a mean concentration of ± ngmm3 The concentration of DNA was significantly greater in ETOH samples than in PG at hr Pvalue NBF Pvalue and PG Pvalue at hr NBF Pvalue and PG Pvalue at seven days and NBF Pvalue and PG Pvalue at days There was little variation in DNA concentrations over time for anyof the fixatives although for all fixatives the lowest concentration was at days Fig 2BThe quality of RNA varied among fixatives Mean RIN values of samples fixed in RNALater1 were at least twice as great as samples fixed in NBF ZF and PG The highest RIN valuesfor LCM samples were observed in NBF fixed tissue at hrs seven and days Fig 3A RINvalues were significantly greater in RNALater1 samples than in PG Pvalue and ZFPvalue samples at hr PG Pvalue and ZF Pvalue samples atFig Nucleic acid quality A Mean RIN values of RNA and B fragment size bp of DNA from samples fixed in neutral buffered formalin NBF ZFix ZF and PAXgene PG for hours hours and days Samplespreserved in alcohol ETOH and RNAlater were included as controls101371journalpone0236104g003PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleost hr PG Pvalue and ZF Pvalue samples at seven days and PG Pvalue � and ZF Pvalue samples at days There were no significant differences in RIN values between LCM samplesMean fragment size of DNA varied over time within each fixative group but was not significantly different than samples fixed in ETOH Fig 3B Fragment size was only significantlygreater in NBF samples than in PG samples P value at hrDownstream analysesEndpoint PCR and Sanger sequencing were successful for the amplification and sequencingof the smallmouth bass EF1α gene although differences in sequencing success were apparentWithout trimming the ™ or ™ ends samples preserved in ETOH produced sequences with amean percentage of bases with a Q40 score or greater of while samples fixed for LCMproduced lower quality sequences Fig Of the samples fixed for LCM PG preserved samplesproduced the highest quality sequences At days there were no samples fixed in NBF or ZFthat were successful for sequencing the EF1α gene For NBF and ZF the best quality sequenceswere generated by samples fixed for hr conversely PG had the lowest quality sequencesfrom samples fixed for hr Fig It should be noted that of the five PG samples sequencedat hr two samples had much lower quality sequences than the other three samples whichmay have contributed to the decrease in the mean percentage of high quality sequences at hr Additionally multiple samples failed to sequence These included one of the ETOH samples forward and reverse sequences three NBF day samples forward sequences oneNBF seven day sample forward and reverse sequences two PG seven day samples forwardsequences one PG day sample forward sequence one PG hr sample reversesequence one ZF seven day sample reverse sequence four ZF day samples three forwardand one reverse sequence and one ZF hr sample forward sequence In order to calculatethe percentage of sequences with similarity to the Micropterus spp EF1α gene failed sequenceswere not included ie of sequences with similarity to Micropterus spp EF1α ofsequences with similarity to Micropterus spp EF1α total of sequences that were successfullysequenced � For LCM fixed samples NBF samples fixed for and hr produced theFig Sanger sequencing quality Mean SEM percentage of bases with a Q40 score or above indicative of highquality sequencing Samples preserved in ethanol ETOH were included as controls routinely used for DNApreservation101371journalpone0236104g004PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostgreatest percentage of sequences with similarity to the EF1α gene In ZF samplesthe greatest percentage of samples with similarity to the EF1α gene was at hr and in PG samples it was at seven days Although PG samples produced the leastamount of sequences with similarity to the EF1α gene at and hr it produced the greatestnumber of sequences at seven and daysA Spearman™s rank correlation analysis with all samples revealed that sequence length pvalue � rho and template concentration pvalue rho were significantly associated with the number of sequences with similarity to the EF1α gene Fig Although PCR primers were estimated to produce an amplicon size around bp manysequences were longer than bp This could be due to the high degree of fragmentation inthe samples which may have resulted in the annealing of small fragments to the original template molecules in overlapping regions [] Fragment length and the percentage of bases witha Q40 score or greater were not significantly correlated with the number of sequences withsimilarity to the EF1α gene The correlations were also examined excluding the ETOH controlssince the DNA concentration of the controls was significantly greater than those of many fixedsamples and to examine the differences amongst the fixatives only Sequence length remainedsignificant pvalue � rho however template concentration was not significantpvalue rho Fragment length and the percentage of bases with a Q40 scoreFig Sanger sequencing and NCBI blastn results A Spearman™s rank correlation analysis of sequence length bpand the number of NCBI blastn matches to the Micropterus spp elongation factor alpha EF1α gene B Meansequence length bp of the EF1α gene obtained with Sanger sequencing and the percentage of sequences withsimilarity to the Micropterus spp EF1α gene101371journalpone0236104g005PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable KruskalWallis nonparametric ANOVA results Significant differences in template concentration usedin PCR and Sanger sequencing results for each fixative between fixation timesFixativeNBFZFPGFixativeNBFNBFZFPGFixativeNBFNBFZFZFPGTemplate ConcentrationpvalueZ scoreFixation TimeNo significant differencesNo significant differences hr vs days of Bases with � Q40 ScorepvaluepvalueZ scoreFixation Time hr vs days hr vs hrNo significant differencesNo significant differencesSequence LengthZ scoreFixation Time days vs hr days vs hr days vs hr days vs hrNo significant differencesSignificant differences pvalue � in template concentration of bases with a Q40 score or greater andsequence length between samples fixed in neutral buffered formalin NBF ZFix1 ZF and PAXgene PG101371journalpone0236104t001or greater remained not significantly correlated A KruskalWallis test was used to identify significant differences between fixation times and template concentration percentage of baseswith a Q40 score or greater and sequence length for each fixative Table The Nanostring nCounter1 results revealed multiple occurrences of samples fixed forLCM with similar or greater transcript abundance compared to RNALater1 samples S1Table In samples fixed for LCM there was significantly greater transcript abundance in PGsamples than in NBF andor ZF samples for one transcript at seven days and eight transcriptsat days Interestingly there were multiple significant differences in housekeeping transcriptabundances between samples preserved in RNALater1 and samples fixed for LCM MeanEF1α transcript abundance was significantly greater in RNALater1 samples than in samplesfixed for LCM at all fixation times Conversely at seven and days 40S ribosomal protein S12transcripts were higher in NBF and ZF samples when compared to RNALater1 and ribosomalprotein L8 was higher in the PG samples Table Significant differences were also identified between fixation times for each fixative typeused to preserve LCM samples Table All significant differences were between fixationtimes hr and seven or days and hr and seven or days In some instances sampleswith longer fixation times had transcripts with significantly greater transcript abundance thansamples fixed for or hr Once again significant differences were identified amongsthousekeeping transcripts There were no significant differences in PG samples over timeNormFinder results ranked the housekeeping transcripts according to stability For all fixatives and all fixation times including RNALater1 samples the most stable housekeepingtranscript was Ribosomal Protein L8 stability followed by EF1α stability Eukaryotic Translation Initiation Factor 3D stability and 40S ribosomal protein S12stability PLOS ONE 101371journalpone0236104 August PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable KruskalWallis nonparametric ANOVA results Significant differences in transcript abundance betweenfixatives for each fixation timeTranscript NameElongation Factor Alpha�Heat Shock Protein Elongation Factor Alpha�Thyroid Hormone Receptor BetaTranscript NameElongation Factor Alpha�Heat Shock Protein Elongation Factor Alpha�Heat Shock Protein Transcript Name40S Ribosomal Protein S12�ArginaseBetacateninElongation Factor Alpha�Heat Shock Protein Transforming Growth Factor Beta40S ribosomal protein S12�C Reactive ProteinlikeElongation Factor Alpha�Eukaryotic Translation Initiation Factor 3D�Transforming Growth Factor BetaApolipoprotein Elongation Factor Alpha�Ribosomal Protein L8�Superoxide DismutaseThyroid Hormone Receptor BetaThyroid Hormone Receptor BetaTranscript Name40S ribosomal protein S12�ArginaseElongation Factor Alpha�40S ribosomal protein S12�ArginaseC Reactive ProteinlikeElongation Factor Alpha�Ribosomal Protein L8�Transforming Growth Factor BetaApolipoprotein Aryl Hydrocarbon ReceptorElongation Factor Alpha�Heat Shock Protein Ribosomal Protein L8� Hrpvalue Hrpvalue Dayspvalue DayspvalueZ scoreZ scoreZ scoreZ scorePLOS ONE 101371journalpone0236104 August FixativesRNALater vs NBFRNALater vs NBFRNALater vs PGRNALater vs PGFixativesRNALater vs PGRNALater vs NBFRNALater vs ZFRNALater vs ZFFixativesRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGPG vs NBFFixativesRNALater vs NBFRNALater vs NBFRNALater vs NBFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs ZFRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGRNALater vs PGContinued PLOS ONE 0cEffects of tissue fixation on nucleic acids from microdissected tissue samples of a teleostTable ContinuedAryl Hydrocarbon ReceptorEstrogen Receptor Beta Hepcidin Thyroid Hormone Receptor BetaType II Iodothyronine DeiodinaseAryl Hydrocarbon ReceptorHepcidin Thyroid Hormone Receptor BetaType II Iodothyronine DeiodinasePG vs NBFPG vs NBFPG vs NBFPG vs NBFPG vs NBFPG vs ZFPG vs ZFPG vs ZFPG vs ZFSignificant differences pvalue � in transcript abundance between RNALater1 samples and samples fixed in neutral buffered formalin NBF ZFix1 ZF and PAXgene PG� Indicates housekeeping transcripts101371journalpone0236104t002Table KruskalWallis nonparametric ANOVA results Significant differences in transcript abundance betweenfixation times for each fixativeTranscript Name40S ribosomal protein S12�ZFixpvalueCytochrome P450 family subfamily AElongation Factor Alpha�Eukaryotic Translation Initiation Factor 3D�Ribosomal Protein L8�TataBox Binding ProteinThyroid Hormone Receptor BetaTransforming Growth Factor BetaTranscript Name40S ribosomal protein S12�Cytochrome P450 family subfamily ABeta

Thyroid_Cancer

Primary squamous cell carcinoma ofis anextremely rare aggressive malignancy with a poor prognosis However almost noreportthus far has investigated the microvasculature of ThyPSCC imaged usingcontrastenhanced ultrasoundthe thyroid ThyPSCCCase Report A 59yearold male patient presented to our hospital with progressivelyworsening hoarse voice symptoms for days and was diagnosed with left unilateralvocal fold palsy Ultrasonography revealed a solitary marked hypoechoic thyroid nodulewith an unclear boundary in the inferior part of the left lobe Color Doppler flow imagingshowed a poor blood flow signalinside this nodule Contrastenhanced ultrasoundimages showed a persistent low peak enhancement of the nodule from its periphery to itscenter The timeintensity curve displayed a washin time of s a time to peak of s apeak signal intensity of and a washout time of s for the thyroid tumor Finallyleft hemithyroidectomy of the thyroid tumor was performed and histopathologic andimmunohistochemical evaluations confirmed the diagnosis of ThyPSCC Postoperativelythe patient received a combination therapy of chemotherapy radiotherapy and targetedtherapy but the patient died months after surgeryPrimary squamous cell carcinoma ofConclusionthe thyroid is a rare butaggressive malignancy of the thyroid Herein we reported a case of ThyPSCC and itsultrasonography and pathologic findingsKeywords thyroid cancer thyroid nodules TNs thyroid ultrasound US primary squamous cell carcinomacontrast enhanced ultrasound CEUSINTRODUCTIONPrimary squamous cell carcinoma of the thyroid ThyPSCC is a rare thyroid malignancy withhigh aggressiveness and poor prognosis comprising ˆ¼“ of all primary thyroid carcinomas“ Owing to the rapidly progressing and highly invasive nature of the malignancy patients withThyPSCC often present at an advanced stage and are difficult to diagnose in the early stage becauseof its rare incidence and lack of typical imaging findings Thyroid ultrasonography and fineneedle aspiration biopsy FNAB are the diagnostic tools ofchoice for evaluating patients with suspected thyroid nodules Contrastenhanced ultrasoundCEUS as a relatively novel US technique is used to investigate the microvasculature of thyroidnodules and improve the diagnostic accuracy of thyroid nodules accompanied by the use of ThyroidEdited byChristoph ReinersUniversity HospitalW¼rzburg GermanyReviewed byPasqualino MalandrinoUniversity of Catania ItalyDaniela PasqualiUniversity of Campania LuigiVanvitelli ItalyCorrespondenceChengcheng NiuniuchengchengcsueducnSpecialty sectionThis was submitted toCancer Endocrinologya section of the journalFrontiers in EndocrinologyReceived February Accepted June Published August CitationChen S Peng Q Zhang Q and Niu C ContrastEnhanced Ultrasoundof Primary Squamous Cell Carcinomaof the Thyroid A Case ReportFront Endocrinol 103389fendo202000512Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaImaging Reporting and Data Systems for ultrasonographicfeatures “ However very few published studies havereported the use of ultrasonography for ThyPSCC To ourknowledge this is the first case describing the CEUS findingsof ThyPSCCreached its peak [time to peak TTP] at s with a peakintensity of Then the nodule slowly declined until allthe microbubbles washed out at s Figures 1CD Based onits malignant conventional ultrasound features and the poormicrovasculature revealed by CEUS we inferred that the nodulewas a malignant tumorCASE REPORTA 59yearold male patient presented to our hospital withprogressively worsening hoarse voice symptoms for daysand was diagnosed with left unilateral vocal fold palsy Ahighresolution ultrasound instrument Siemens Acuson S3000Mountain View CA USA equipped with a to 9MHz linearprobe was used Thyroid ultrasonography revealed a solitary — — 26cm3 marked hypoechoic thyroid nodule with anunclear boundary in the inferior part of the left lobe AThis nodule exhibited many malignant ultrasound featuressuch as solid components hypoechogenicity and microlobulatedmargins Color Doppler flow imaging CDFI showed poorblood flow signals in the nodule B Contrastenhancedultrasound was performed with a bolus intravenous injectionof mL of SonoVue Bracco Milan Italy followed by mLof saline Contrast pulse sequencing technology was used andthe timeintensity curves TICs of the nodule were calculatedThe nodule began to be slowly enhanced from the peripheryto the center at s washin time and the enhancementAfterneckthepositronultrasonographyemissiontomography“computed tomography was carried for evaluatingthesituation of distant metastases Positron emissiontomography“computed tomography showed a mass withincreased glucose metabolism in the inferior part of the leftthyroid lobe A which indicated it as a malignantmass whereas there was no evidence of lymph nodes metastasisand distant metastases Then ultrasonographyguided FNABwas performed for the left thyroid mass immediately Cytologicexamination by fineneedle aspiration FNA revealed sheets oftumor cells with giant deepstained nuclei Bethesda categoryV B Finally a left hemithyroidectomy of the thyroidtumor was undertaken The lower edge of the tumor reachedthe upper mediastinum and the depth of the tumor invadedthe esophagus and trachea which could not be completelyremoved According to the eighth edition of the AmericanJoint Committee on CancerTumor Lymph Node MetastasisTNM staging system the patient was in TNM stage IIIT4a N0 M0 Histopathological examination of hematoxylinand eosin staining showed that a carcinoma in the inferiorFIGURE Ultrasonography images of primary squamous cell carcinoma of the thyroid A Longitudinal grayscale sonography revealed a solid marked hypoechoicthyroid nodule in the inferior part of the left lobe B Color Doppler flow imaging showed a poor blood flow signal inside this nodule C Contrastenhanced ultrasoundimage showed a persistent low peak enhancement of the nodule at s D Timeintensity curves displayed the washin time of s TTP of s peak signalintensity of and washout time of s for the thyroid tumorFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaFIGURE A A positron emission tomography“computed tomography scan showed increased 18Ffluorodeoxyglucose metabolism in the left neck mass BPreoperative fineneedle aspiration cytology of the mass demonstrated a few sheets of malignantlooking tumor cells with giant deep stained nuclei hematoxylin andeosin magnification — FIGURE Hematoxylin and eosin staining of primary squamous cell carcinoma of the thyroid A magnification — B magnification — C magnification — D magnification — part of the thyroid lobe A had no obvious palisadearrangementintercellular bridges or keratinization with acancer pearl Figures 3B“D Immunohistochemically tumorcells were positive for cytokeratin CK19 Acytokeratin and CK56 B epithelial membraneantigen EMA C p40 D p63 Aand Ki67 B and negative for thyroglobulinTG C and thyroid transcription factor TTF1D In view of these findings the tumor was diagnosedas poorly diï¬erentiated ThyPSCC Postoperatively the patientreceived two cycles of chemotherapy with docetaxelcisplatinintensitymodulated radiotherapy and nimotuzumabtargetedtherapy However the patient died months after surgeryDISCUSSIONPrimary squamous cell carcinoma of the thyroid is a thyroidmalignancy with extremely rare incidence and the clinicaldiagnosis and treatment guidelines for this disease have noconsensus The biological behavior of ThyPSCC is aggressiveFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaFIGURE Immunohistochemical staining of primary squamous cell carcinoma of the thyroid magnification — Immunohistochemical staining for A CK19 BCK56 C EMA D p40 all of which were deeply stained positiveFIGURE Immunohistochemical staining of primary squamous cell carcinoma of the thyroid magnification — Immunohistochemical staining for A p63 B Ki C TG D TTF1 and p63 was deeply stain positive Ki67 proliferation index was TG and TTF1 did not stain negativeFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell Carcinomaand the prognosis is poor with a median overall survival of “ months which depends on the diï¬erent tumor grades Yang et al using the Surveillance Epidemiology and End ResultsProgram database reported that poorly diï¬erentiated tumrade occupied the highest percentages of all graded tumors andthe median survival was months which is similar to the survivaltime in our case Highfrequency ultrasound as the basic imaging modality inthe diagnosis of thyroid nodules has found gradually increasingdiï¬erentiated thyroid cancers over recent years Theultrasonography imaging findings of ThyPSCC have seldombeen published Regarding the ultrasonography findings Chenet al reported that ThyPSCC presented as a thyroid masswith eggshell calcification peripheral soft tissue with a blurredmargin and minimal vascular signals on CDFI sonographyIn the case of Jang et al ThyPSCC presented as a largewelldefined lobulated heterogeneously hypoechoic mass withdiï¬use microcalcifications on ultrasonography Kondo et al reported that a welldiï¬erentiated ThyPSCC showed acystic hypoechoic mass with a smooth margin and rapidlygrew with margin change blurring in year In our case thispoorly diï¬erentiated ThyPSCC presented as a solitary markedhypoechoic thyroid mass with an irregular margin and unclearboundary with a normal thyroid The irregular margin andunclear boundary with normal thyroid corresponded to tumorinvasion with adjacent tissue infiltration which is consistentwith the findings during the operation that tumor invasion withthe esophagus cannot be completely removed Poor blood flowsignals on CDFI sonography and persistent hypoenhancement onCEUS of the mass are consistent with squamous cell carcinomawhich has no obvious vascularity on pathologic examinationMany studies have investigated the application of CEUS toimprove the diagnostic accuracy of thyroid nodules despiteits usage in ThyPSCC being scarce Zhang et al foundthat highcircularequal enhancement indicated benign thyroidnodules and low enhancement indicated malignant thyroidnodules Ma et al investigated whether incomplete noring or heterogeneous enhancement later washin time andlow peak intensity on CEUS were independent risk factorsin predicting malignantthyroid nodules Deng et al detected that papillary thyroid carcinomas PTCs exhibited lowenhancement a lower peak signal intensity and a lower areaunder the curve AUC than peripheral thyroid parenchyma onCEUS In our study the TICs of CEUS for ThyPSCCshowed a washin time of s a TTP of s a peak signalintensity as low as and a washout time of s Thisis similar to the results of PTCs with a slow washin time alower peak signal intensity and a lower AUC as in previousreports To our knowledge no reports on CEUS imagingfindings of ThyPSCC have appeared in the Englishlanguageliterature According to Jang et al ThyPSCC showed a largeheterogeneously enhancing thyroid mass with a large centralnonenhancing portion on enhanced CT which correspondedwell with the squamous cell carcinoma portion with a necroticportion in pathologic staining Because of the rapid growth ofsquamous tumor cells relatively few interstitial blood vessels intumors were related to the low peak signal intensity and low AUCon CEUSisusefulstainingWith increasing malignancy in squamous cell carcinoma thetypical squamous cell carcinoma findings of intercellular bridgesand keratinized cancer pearl can decrease or disappearImmunohistochemicalin diagnosingprimary thyroid cancer In this case positivity for CK56and EMA and negativity for TTF1 and TG expressionpredicted squamous cell carcinoma derivation and excludedthe possibility ofthese common tumors Furtherpositivity for p63 and Ki67 expression as poor prognosticmarkers was associated with its poorly diï¬erentiated tumrade CONCLUSIONPrimary squamous cell carcinoma of the thyroid is an extremelyrare tumor and very few studies describe its ultrasonographicimaging findings It is difficult to establish a clinical guidelinefor diagnosis Our case presents the CEUS features of ThyPSCCindicating that the TICs of ThyPSCC are similar to the enhancingparameters of PTCs with a slow washin time a lower peak signalintensity and a lower AUCDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studies involving human participants were reviewed andapproved by the Ethics Committee of Second Xiangya HospitalCentral South University China The patientsparticipantsprovided their written informed consentto participate inthis study Written informed consent was obtained from theindividuals for the publication of any potentially identifiableimages or data included in this AUTHOR CONTRIBUTIONSAll authors listed have made a substantial direct and intellectualcontribution to the work and approved it for publicationFUNDINGof ChinaThis project was funded by the National Natural ScienceFoundationProvincialNatural Science Foundation of China 2018JJ2575 andHunan Provincial Health Commission Research FoundationProject B2019166 HunanFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alREFERENCES Yang S Li C Shi X Ma B Xu W Jiang H et al Primary squamous cellcarcinoma in the thyroid gland a populationbased analysis using the SEERdatabase World J Surg “ 101007s00268019049062 Limberg J Ullmann TM Stefanova D Finnerty BM Beninato T Fahey TJet al Prognostic characteristics of primary squamous cell carcinoma of thethyroid a national cancer database analysis World J Surg “ 101007s00268019050985Thyroid Primary Squamous Cell Carcinomacontrastenhanced ultrasound Ultrasound Med Biol “ 1016jultrasmedbio201810020 Casella C Ministrini S Galani A Mastriale F Cappelli C Portolani NThe new TNM staging system for thyroid cancer and the risk of diseasedownstaging Front Endocrinol 103389fendo201800541 Zhang Y Zhou P Tian SM Zhao YF Li JL Li L Usefulness of combineduse of contrastenhanced ultrasound and TIRADS classification for thediï¬erentiation of benign from malignant lesions of thyroid nodules EurRadiol “ 101007s003300164508y Koyama S Fujiwara K Nosaka K Fukuhara T Morisaki T MiyakeN et al Immunohistochemical features of primary pure squamous cellcarcinoma in the thyroid an autopsy case Case Rep Oncol “ Zhang YZ Xu T Gong HY Li CY Ye XH Lin HJ et al Application ofhighresolution ultrasound realtime elastography and contrastenhancedultrasound in diï¬erentiating solid thyroid nodules Medicine95e5329 101097MD00000000000053290000579220161108000016 Wang SS Ye DX Wang B Xie C The expressions of keratins andP63 in primary squamous cell carcinoma ofthe thyroid gland anapplication of raman spectroscopy Onco Targets Ther “ 102147OTTS229436 Chen CY Tseng HS Lee CH Chan PW Primary squamous cellcarcinoma of the thyroid gland with eggshell calcification sonographicand computed tomographic findings J Ultrasound Med “ 107863jum201029111667 Yasumatsu R Sato M Uchi R Nakano T Hashimoto K Kogo R et al Thetreatment and outcome analysis of primary squamous cell carcinoma of thethyroid Auris Nasus Larynx “ 101016janl201707009 Kao NH Tan CS H Koh AJ The utility of immunohistochemistry indiï¬erentiating metastatic primary squamous cell carcinoma of the thyroidfrom a primary lung squamous cell carcinoma Case Rep Endocrinol “ Jang JY Kwon KW Kim SW Youn I Primary squamouscarcinoma ofandtomographic“ 1014366usg13022cellrecurrence ultrasonographicthyroid gland with localUltrasonographycomputedfindings Raggio B Barrcarcinomacell 1031486toj180002J Ghandour Z Friedlander P Primary squamousof“thyroid OchsnertheJ Kondo T Matsuyoshi A Matsuyoshi H Goto R Ono K Honda Y et alA case of primary thyroid squamous cell cancer transformation frombenign tumour associated with chronic thyroiditis BMJ Case Rep 2009bcr1020081137 101136bcr1020081137 Ma JJ Ding H Xu BH Xu C Song LJ Huang BJ et al Diagnosticand contrastmalignant101089thy201performances ofenhancedultrasonographythyroid nodules Thyroid“ color dopplergrayscalepredictingfindingsvariousin Deng J Zhou P Tian SM Zhang L Liofefficacydiagnosticofradiationdiï¬erentiating9e90674 101371journalpone0090674PONED1330329imagingthyroidandnodulescontrastenhancedimpulseforcesolidfocalJL Qian Y ComparisonacousticinuseultrasoundcombinedPLoS ONEtheir Haugen BR Alexander EK Bible KC Doherty GM Mandel SJ Nikiforov YEet al American thyroid association management guidelines for adultpatients with thyroid nodules and diï¬erentiated thyroid cancer the Americanthyroid association guidelines task force on thyroid nodules and diï¬erentiatedthyroid cancer Thyroid “ 101089thy20150020 Tessler FN Middleton WD Grant EG Hoang JK Berland LL Teefey SAet al ACR thyroid imaging reporting and data system TIRADS whitepaper of the ACR TIRADS committee J Am Coll Radiol “ 101016jjacr201701046 Kwak JY Han KH Yoon JH Moon HJ Son EJ Park SH et al Thyroidimaging reporting and data system for US features of nodules a step inestablishing better stratification of cancer risk Radiology “ 101148radiol11110206radiol11110206 Peng Q Niu C Zhang Q Zhang M Chen S Mummified thyroid nodulesconventional and contrastenhanced ultrasound features J Ultrasound Med “ 101002jum14712 Peng Q Niu C Zhang M Chen S Sonographic characteristics ofpapillary thyroid carcinoma with coexistent hashimoto™sthyroiditisconventional ultrasound acoustic radiation force impulse imaging and Struller F Senne M Falch C Kirschniak A Konigsrainer A Mullerthe thyroid case report andS Primary squamous cell carcinoma ofsystematic review of the literature Int J Surg Case Rep “ 101016jijscr201706011 Wang W Ouyang Q Meng C Jing L Li X Treatment optimization andprognostic considerations for primary squamous cell carcinoma of thethyroid Gland Surg “ 1021037gs20191107Conflict 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 Chen Peng Zhang and Niu This is an openaccess distributed under the terms of the Creative Commons Attribution License CC BYThe use distribution or reproduction in other forums is permitted provided theoriginal authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'

Thyroid_Cancer

"Insulin shares a limited physiological concentration range with other endocrine hormones Not onlytoo low but also too high systemic insulin levels are detrimental for body functionsMain body The physiological function and clinical relevance of insulin are usually seen in association with its rolein maintaining glucose homeostasis However insulin is an anabolic hormone which stimulates a large number ofcellular responses Not only too low but also excess insulin concentrations are detrimental to the physiologicalbalance Although the glucoregulatory activity of insulin is mitigated during hyperinsulinemia by dampening theefficiency of insulin signaling œinsulin resistance this is not the case for most other hormonal actions of insulinincluding the promotion of protein synthesis de novo lipogenesis and cell proliferation the inhibition of lipolysisof autophagydependent cellular turnover and of nuclear factor E2related factor2 Nrf2dependent antioxidativeand other defense mechanisms Hence there is no general insulin resistance but selective impairment of insulinsignaling which causes less glucose uptake from the blood and reduced activation of endothelial NO synthaseeNOS Because of the largely unrestricted insulin signaling hyperinsulinemia increases the risk of obesity type diabetes and cardiovascular disease and decreases health span and life expectancy In epidemiological studieshighdose insulin therapy is associated with an increased risk of cardiovascular disease Randomized controlled trialsof insulin treatment did not observe any effect on disease risk but these trials only studied low insulin doses up to IUday Proof for a causal link between elevated insulin levels and cardiovascular disease risk comes fromMendelian randomization studies comparing individuals with genetically controlled low or high insulin productionConclusions The detrimental actions of prolonged high insulin concentrations seen also in cell culture argue infavor of a lifestyle that limits circadian insulin levels The health risks associated with hyperinsulinemia may haveimplications for treatment regimens used in type diabetesKeywords Hyperinsulinemia Insulin resistance Nrf2 Autophagy eNOS Obesity Type diabetes mellitusInflammation Oxidative stress Cardiovascular morbidity and mortalityBackgroundMost endocrine hormones exhibit a window of optimalphysiological concentrations with compromised function of the anism at levels below or above that rangeFor instance subnormal levels of thyroid hormone define the clinical condition of hypothyroidism above normalrepresent hyperthyroidism which usuallyrequires therapy Addison™s disease is characterized bylevels Correspondence kerstinkempfwdgzde2WestGerman Centre of Diabetes and Health Duesseldorf Catholic HospitalGroup Hohensandweg Duesseldorf GermanyFull list of author information is available at the end of the insufficient cortisol production while excess synthesis isseen in Cushing syndromeFor insulin we argue here that not only hypoinsulinemiabut also hyperinsulinemia is detrimental to body functionsHypoinsulinemia causes insulindeficient diabetes and thehormonal actions of insulin are necessary for the life of complex anisms [] On the other hand permanently elevatedlevels of insulin may cause disturbance of normal cellularphysiology and an function We describe the molecularbasis of these undesired insulin actions and consequences ofhyperinsulinemia for healthrelevant endpoints such as obesity or cardiovascular diseases 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 0cKolb BMC Medicine Page of transformingproteins345trisphosphateMain textInsulin signaling pathwaysBinding of insulin to its cognate cell surfacebound receptor causes a conformational change which initiatesa cascade of signaling events Autophosphorylation bythe insulin receptor tyrosine kinase is accompanied bytyrosine phosphorylation of receptor substrates suchas insulin receptor substrate IRS and Src homology domaincontainingSHCproteins Phosphorylation of IRS allows binding ofphosphatidylinositol3kinase PI3K and synthesis ofphosphatidylinositolPIP3which eventually leads to the phosphorylation and activation of the serinethreoninespecific protein kinaseB AKT Upon activation AKT interacts with severalsubstrates which mediate anabolic effects of insulinthese include glucose uptake glycogen synthesis denovo lipogenesis and protein synthesis [] Additionalpathways triggered by the activated insulin receptorcomprise phosphorylation of SHC followed by activathe Rat sarcoma Ras“rapidly acceleratedtion offibrosarcoma Raf“mitogenactivated protein kinasesignalregulated kinasekinaseERK pathway Theamitogenactivated kinase promoting cell proliferationand further cellular activities including protein synthesis [] Another pathway triggered by the engaged insulin receptor involves activation of NADPH oxidase and subsequent hydrogen peroxidemediated inhibition of phosphatase and tensin homolog PTENwhich is an important negative regulator of PI3Ksignaling [] Fig terminal kinase ERK isMEK“extracellularInsulin secretionInsulin secretion by pancreatic islet cells responds tothe level of circulating nutrients such as glucose aminoacids and free fatty acids Sweeteners may further increase carbohydrateinduced insulin secretion A largenumber of endogenous factors contribute to the regulation of cell activity either stimulatory inhibitory orboth contextdependent These include hormones neurotransmitters and immune mediators [“] Insulin isessential for maintaining glucose homeostasis primarilyby facilitating the postmeal uptake of glucose intomuscle and fat cells via translocation of the glucosetransporter [] In the absence of dietary glucose supply and after depletion of glycogen stores glucose in circulation primarily comes from gluconeogenesis in theliver If circulating insulin levels are below the concentrations required for stimulating glucose uptake fromthe blood endogenous stores of fat and protein must beused for energy production For the maintenance of lifein the fasting state circulating insulin levels range between approx and pmoll “ percentile asdetermined for healthy adult persons in the NationalHealth and Nutrition Examination Survey NHANES[] In response to meals with varying carbohydratecontent insulin levels may rise to the range of approx“ pmoll []Insulin promotes obesityAlmost years ago insulin injections were one of theoptions of therapy in nondiabetic persons suffering fromundernutrition in the context of various diseases Insulindoses were in the range of those applied in type Fig Metabolic signaling of insulin is anabolic Insulin signaling through the insulin receptor engages several pathways and results in ananabolic state of metabolism The canonical pathway via phosphokinases PI3K and AKTPKB promotes glucose uptake and glycogen and lipidsyntheses whereas lipolysis is inhibited in adipocytes as well as hepatic gluconeogenesis In addition AKT kinases activate mTORC1 whichsupports de novo lipogenesis and protein synthesis The insulin signaling pathway via SHC and the MAP kinases MEK and ERK promotes cellproliferation and protein synthesis Another insulin signaling pathway involves NOX4 and the inhibition of PTEN an inhibitor of the PI3KAKT pathway 0cKolb BMC Medicine Page of diabetes and led to increased appetite and weight gain[] Indeed one major function of insulin as an anabolic hormone is to favor energy storage over usageThis is reflected by the finding that insulin infusion mUkgmin significantly inhibits lipolysis in the skeletalmuscle about and even more effective in adiposetissue about [] Doubling fasting insulin levelssuffices to inhibit lipolysis by approx and to promote lipogenesis for both mean insulin concentrationfor effect EC50 of approx pmoll [] At thisinsulin level gluconeogenesis is still ongoing For halfmaximal inhibition of gluconeogenesis insulin concentrations must rise to approx pmoll in arterial circulation In order to stimulate glucose uptake to halfmaximum insulin levels must rise to even higher levelsapprox ten times the fasting insulin concentrations “ percentiles for stimulating glucose uptake approx“ pmoll [] Thus a modest rise doubling offasting insulin levels will already substantially inhibit lipolysis and promote lipogenesis while gluconeogenesis isnot yet inhibited Since such small increases of systemicinsulin concentrations are enough for favoring adipogenesis fasting and diurnal insulin levels are a determinantof obesity risk Indeed several data support the obesitypromoting role of insulin for a detailed review see []Fig These include epidemiological studies which foundhigh fasting insulin levels and concomitant insulin resistance in children and adolescents to be associatedwith higher weight gain in later years [] Studies inadults are less consistent [] Pharmaceutical interventions that lower insulin secretion such as treatment withdiazoxide or octreotide led to significant body weightloss [“] This fits with the observation that insulintherapy promotes weight gain [] One probable reasonis that insulin levels in the high normal range are closeto EC50 concentrations for inhibition of lipolysis []In mice modest lowering of circulating insulin concentrations by genetic manipulation ofinsulin genescaused resistance to weight gain despite a highfat diet[] Decreasing insulin gene expression in adult micevia partial gene ablation reversed dietinduced obesity[] In men the Hph1 œT polymorphism in the insulingene region was found to be associated with higher fasting insulin levels and a more rapid weight gain in obesepersons[] A Mendelian randomization analysisshowed that persons with genetically determined higherinsulin secretion to oral glucose exhibited a higher bodymass index BMI [] supporting a causal relationshipbetween insulin and obesity riskTaken together moderate to high normal levels of insulin in metabolic healthy persons appear to be a riskfactor for the development of obesitytransientElevated insulin concentrations impair cellularfunctions”insulin œtoxicityThere is ample evidence thatincreases ofmetabolic or immune mediator levels are benign physiological responses to biochemical challenges such as therise of systemic glucose or cytokines following mealsHowever chronic elevations of such mediators evenwhen modest in amplitude are usually detrimental tocellular functions [] In the case of glucose the termglucose toxicity was coined to describe this phenomenon[] Prolonged conditions of elevated glucose concentrations cause dysfunction of numerous cell types in thebody including beta cells neurons and the endothelium via several pathways including increased oxidative stress and activation of the sorbitol pathway [“] As described below there seems to be a similardetrimental outcome oflongterm elevated insulinconcentrations on cellular functions a correspondingterm would be insulin toxicityFig Insulin promotes obesity Several independent types of observations support the conclusion that insulin promotes adipogenesis andobesity For details see description in the general text 0cKolb BMC Medicine Page of When cells are exposed to continuously elevated insulin levels there is a partial downregulation of insulin signaling The resulting œinsulin resistance is not primarilydue to less insulin receptor expression on the cell surface but due to impaired insulin signal transduction as aresult of receptor dysfunction In response to prolongedhyperinsulinemia there is diminished autophosphorylation of the insulin receptor compared to that observedafter shortterm exposure to insulin and subsequentsteps of the PI3K“AKT signaling pathway are affected[ ] Consequently in muscle and fat cells there isless AKTstimulated translocation of GLUT to the cellsurface Fig Thus insulin resistance can be seen as aprotective mechanism for preventing excess activation ofglucose transport from the blood despite chronically elevated insulin levels for maintaining glucose homeostasisin vivo and for mitigating metabolic and oxidative stressdue to excess glucose influx [“] Limiting glucoseexportfrom the blood does not necessarily requiredampening of insulin signaling During the early weeksof feeding with a high caloric diet mice show decreasedinsulindependent glucose uptake despite unperturbedinsulinstimulated AKT phosphorylation [ ] Fig An interesting aspect is that the partitioning of insulinreceptor isoforms A and B and of hybrid insulininsulinlike growth factor1 receptors among cell types maycontribute to insulin resistance in some tissues but thepathophysiological relevance is unknown []The phenomenon of insulin toxicity partly arises fromthe fact that there are additional cellular responses to elevated insulin levels which are not toned down duringrole ofinsulin resistance Fig These comprise the upregulation of protein synthesis and the accumulation of ubiquitinated or otherwise modified proteins probably dueto insufficient degradation of these polypeptides [] Amajorinsulin signaling via the canonicalmitogenactivated protein MAP kinase pathway Ras“MEK“ERK as well as via activation of NADPH oxidase has been observed [] Even some AKTdependentpathways do not appear to be suppressed by insulin resistance such as de novo lipogenesis in hepatocytes orthe upregulation of mechanistic target of rapamycincomplex mTORC1 [“] Enhanced activity ofmTORC1 leads to increased protein synthesis and to deteriorated cell functions largely because of suppressedautophagy []Hence chronic exposure of cells to high ambient insulin concentrations causes an imbalance of cellular responses because of the downregulation of some insulinsignaling pathways œinsulin resistance but not ofothers The resulting functional state of cells is characterized by an unbalanced anabolic activity of insulin favoring protein synthesis while suppressing autophagyThe latter inhibits autophagic removal and turnover ofproteins and lipids which favors cell senescence [] Inshortterm experiments of exposure to high insulinlevels a protective cellular stress response is observedthe unfolded protein response probably due to the accumulation of derivatized proteins in the absence ofenough disposal In experimentally induced or diabetesassociated chronic insulin resistance and hyperinsulinemiathesuch a protectivestressresponse ofFig Signaling of insulin during insulin resistance During insulin resistance signaling through AKT kinases is partially impaired Not all AKTdependent pathways are affected as well as other signaling pathways indicating that insulin resistance is selective Therefore hyperinsulinemiain the presence of insulin resistance promotes anabolic cell activities via the MEK“ERK pathway and via mTORC1 Although the PI3KAKT pathwayis impaired during insulin resistance and provides only insufficient translocation of GLUT4 for glucose uptake and deficient activation of eNOSthere appears to be a normal activation of mTORC1 In addition to the anabolic consequences of signaling via the MEKERK pathway depicted inthe figure there is enhanced expression of ET1 and PAI1 not shown as well as inhibition of autophagy and of the nuclear factor Nrf2 whichcompromises cell constituent turnover and cell defense mechanisms to radical stress respectively Hyperinsulinemia downregulates glucoseuptake not only via dampening the PI3KAKT pathway œinsulin resistance but also via as yet unknown other pathways 0cKolb BMC Medicine Page of endoplasmic reticulum to high insulin levels is diminished or absent []Another activity of insulin is the suppression of transcription of the nuclear factor Nrf2 via induction of heterogeneous ribonucleoproteins F and K [] Nrf2 is thecentral regulator ofthe protective response of cellsagainst oxidative and other types of electrophile stress[] Suppression of Nrf2 expression is expected to impair the antioxidant and cytoprotective defense capacityof cells Insulin signaling required for Nrf2 inhibition occurs via the MAP kinase pathway and thus is not mitigated by insulin resistance [] Fig It therefore canbe assumed that hyperinsulinemia increases the susceptibility of cells against oxidative or other electrophilestress caused by environmental insults Prolonged exposure of cells to high insulin concentrations can thereforebe regarded as toxic Indeed exposure to nmoll insulin has been found to cause DNA damage in a numberof cell types including human lymphocytes [ ] Atthe only concentration tested nmoll insulin impairs oxygen radical defense and sensitizes apoptosispathways in human islets [] In the brain of micehyperinsulinemia impairs electrophysiological functionsof neurons and protein turnover causing a transition toa senescent cell state and an accompanying cognitive decline [] The direct toxic property of insulin deservesfurther studyChronically elevated insulin concentrations impair bodyfunctionsLongevityThe above list of detrimental cellular responses to highambientinsulin concentrations suggests concomitantfunctional impairments at the level of the anism Thisfits with the observed impact of insulin on longevityStudies in nonvertebrate model systems such as thenematode Caenorhabditis elegans or the fruit fly Drosophila melanogaster find that moderate to high insulinactivity shortens lifespan [ ] A consistent findingfrom mouse model studies is that decreased signaling ofanabolic hormones like insulin insulinlike growth factor or growth hormone results in a prolonged lifespan[] Disruption of the insulinreceptor substrate genecaused insulinresistance with defects in insulin signaling[] and led to an extension of lifespan by “ []A knockout of the insulin receptor in adipose tissue ofmice resulted in an increase of lifespan [] Disruption of the Ins1 gene and one of the two mouse Ins2alleles lowered insulin levels by “ Ins2ˆ’ miceversus Ins2 controls in aged female mice without altering circulating insulinlike growth factorIGF1levels These aged experimental mice exhibited lowerfasting glucose improved insulin sensitivity and “lifespan extension across[]two different dietsConcomitantly the proteome and transcriptome indicated a profile associated with healthy aging An important aspect is that this study selectively addressed insulinOther interventions for promoting longevity or extending healthspan such as caloric restriction not only lowercircadian insulin levels but several additional hormonesincluding IGF1 are also affected []InsulinIGF1 and hybrid insulinIGF1 receptorsshare signaling via PI3K and AKT The subsequent activation of the protein kinase mTORC1 is a major pathway for supporting somatic growth protein synthesisand fertility while impeding autophagy and lifespanSuppression of mTOR signaling by treatment with rapamycin prolongs life in model anisms and mice []In humans hyperinsulinemia in pre type diabetes isassociated with increased mTORC1 activity which mayhave a negative impact on beta cell survival healthspanand longevity []In the Leiden Longevity Studyfollowup of nonagenarians for years showed a strongassociation of low insulin and glucose levels with healthyaging []Since both IGF1 and insulin employ PI3K and AKTfor signal transduction it is difficult to disentangle thecontribution of insulin versus IGF1 to the modulationof longevity In animal models selective downregulationof circulating insulin levels improved the lifespan ofmice and in elderly persons of the Leiden LongevityStudy only insulin and glucose but not IGF1 consistently met all four predefined criteria of healthy aging[ ] Therefore it may be concluded that low circulating insulin concentrations are not only a marker oflongevity but are causally involved in promoting healthspan or lifespan extensionDetrimental combination of hyperinsulinemia with insulinresistanceInsulin resistance is defined as an attenuated effect of insulin on blood glucose homeostasis primarily by less efficient export of glucose from the blood into skeletalmuscle adipose and liver tissue Permanently elevatedinsulin concentrations in the blood are often consideredas an attempt to overcome insulin resistance Indeed induction of insulin resistance by genetic disruption of insulin signaling as well as by increased growth hormonelevels or an inflammatory milieu causes hyperinsulinemia [“] The opposite causality is of more relevanceHyperinsulinemia during insulin infusion in humansleads to systemic insulin resistance [] while in vitrohigh ambient insulin concentrations cause an increase ininsulin resistance in isolated adipocytes [] A summaryanalysis of nine studies in rodents and seven trials inhumans confirmed that the first detectable change in thefasting state after feeding a high caloric diet for severaldays is an increase of basal insulin concentrations but 0cKolb BMC Medicine Page of not of blood glucose concentrations or insulin resistance[] Both increased secretion of insulin by ß cells anddecreased insulin clearance in the liver contribute to elevated insulin levels postmeal the latter being of primaryimportance in the case of carbohydraterich food []functionincluding relaxation ofThe combination of hyperinsulinemia and insulin resistance appears to promote hypertension and atherogenesis Fig One important molecule for maintainingvesselthe arterialsmooth muscle layeris nitric oxide NO which isgenerated by endothelial NO synthase eNOS Insulinincreases NO production via posttranslational modification of eNOS via PI3KAKT activity howeverthismechanism is suppressed during insulin resistance [] Decreased local NO production impairs arterialsmooth muscle relaxation and concomitant vasodilatation An important factor in this context is the calciumion homeostasis of vascular smooth muscle cells Underphysiological conditions insulin promotes both calciuminflux into the cytoplasm of smooth muscle cells via several ion channels including Ltype and storeoperatedCa2 channels and counterregulatory NOmediated efflux of Ca2 and K ions which prevents calcium ioninduced myosin lightchain phosphorylation andFig Hyperinsulinemia insulin resistance and cardiovasculardisease High insulin concentrations in the blood may occur due togenetic predisposition overnutrition or highdose insulin treatmentof type diabetes Hyperinsulinemia induces œinsulin resistance as adefense response to maintain glucose homeostasis Converselyinsulin resistance may be directly induced such as by growthhormone or proinflammatory cytokines Hyperinsulinemia andinsulin resistance enhance the risk of cardiovascular disease byinducing endothelial dysfunction suppression of endothelial nitricoxide synthase eNOS and activation and promotion of calcium ioninflux into smooth muscle cells resulting in increased vascular toneenhanced reabsorption of sodium ions in renal tubules adhesion ofmacrophages to the vessel wall and development of arterial lesionswith increased lipoprotein lipase activity and cardiovascular diseaseconcomitant vascular contractility During insulin resistance NO production is impaired while the supportiveeffect of insulin on calcium ion influx via PI3K deltaand possibly the MEK“ERK pathway and vasoconstriction is still present Fig [ ]At the same time insulin signals through the mitogenactivated protein MAP kinase pathway to upregulatethe expression of endothelin1 ET1 plasminogen activator inhibitor1 PAI1 adhesion molecules and proinflammatory cytokines [ ] The reninangiotensinsystem is activated in the context of endothelial dysfunction and contributes together with decreased NO production and increased ET1 secretion to vascularstiffening and upregulation of vascular tone [“] Inthe absence of hyperinsulinemiainsulin resistance thelower insulin levels exert less potential proatherogenicactivities which are counteracted by insulinstimulatedlocal NO production [ ]Elevated insulin levels also increase the risk of hypertension by enhancing renal reabsorption of sodium ionsby several transport systems in different segments of thenephron Fig Signaling of insulin occurs via insulinreceptor substrate IRS2 and is not suppressed duringinsulin resistance while signaling via IRS1 for counterregulatory mechanisms including local NO production isimpaired [ ] These detrimental actions may be mitigated during chronic hyperinsulinemiainsulin resistance [] However a metaanalysis of prospectiveepidemiological studies showed that the pooled relativerisk of hypertension was when comparing the highest to the lowest category of fasting insulin levels and for comparing highest to lowest selective insulinresistance categories calculated as homeostasis modelassessment of insulin resistance HOMAIR []As a consequence of endothelial dysfunction duringprolonged treatment with insulin arterial lesions rich inlipids are formed [] The progression of early fattystreak lesions to plaques is accompanied by the adhesionand proinflammatory activity of macrophages whicheventually develop into foam cells This process is drivenby endothelial and macrophage lipoprotein lipase activity as demonstrated by the observation of less atherosclerosis in mice with inactivated lipoprotein lipase gene[“] Lipoprotein lipase activity in macrophages isenhanced with higher insulin levels in vivo but there isno direct stimulatory effect of insulin on isolated macrophages []The concern that hyperinsulinemia might promote arterial disease in diabetic persons developed in the late1960s due to the steady increase of incidences of atherosclerosis in diabetic persons despite improved glycemiaand decreased risk of ketosis due to insulin therapy []Since then a wealth of data supports the observationthatis ainsulin resistance and hyperinsulinemia 0cKolb BMC Medicine Page of marker of increased risk of cardiovascular disease in thegeneral population and in patients with diabetes [] Although observational studies suggested an approximatelylinear relation between the severity of hyperglycemiaand vascular damage severallarge randomized controlled trials have shown that intense glycemic controlper se does not decrease the risk of macrovascularcardiovascular events [] indeed insulin therapy may evenincrease the risk [ ] However these trials werenot randomized for insulin treatment and treatment ofCVD risk factors was not kept similar between patientsubgroups In the United Kingdom Prospective DiabetesStudy UKPDS hyperinsulinemia and insulin resistancewere not mitigated by insulin treatment and fastingplasma insulin levels even rose [] By contrastinUKPDS and other trials [ “] oral treatmentwith the biguanide metformin reduced the risk of cardiovascular events and in parallel decreased insulin resistance and hyperinsulinemiaIn epidemiological studies of type diabetesit hasbeen consistently observed that the addition of insulin tothe treatment regimen or the intensification of insulintreatment result in a higher rate of cardiovascular events[“] Fig Indeed it has been shown that therisk increases with increasing insulin dosage [ ]These epidemiological studies may suffer from residualFig Hazard ratio of insulin medication versus different reference medications Shown are adjusted hazard ratios HR for each study with confidence interval Moderate insulin exposure high insulin exposure moderate insulin dose to units per day §high insulin dose units per day 0cKolb BMC Medicine Page of confounding since it is difficult to account for the possibly more advanced disease stage of patients receivinginsulin A higher rate of hypoglycemic events may be anadditional confounder However covariates consideredin the statistical analyses cover a broad range of potential risk factors from different categories SupplementTable Large randomized controlled trials such asUKPDS [] or the Outcome Reduction With InitialGlargine Intervention ORIGIN Trial [] did not observe an increased incidence of cardiovascular diseasewith insulin therapy but these trials focused on lowdose insulin therapy of up to a median of IUday or IUkgday respectively Similar randomized trials ofhigherdose insulin therapy as typicalfor realworldconditions have not been conducted Recent studies ofrealworld clinical settings report mean daily basal insulin doses of close to IUkg in the Canadian REALITY Study for insulinexperienced patients with type diabetes [] and of IUkg in a physician survey inNew York [] In the European multicentre EUTREAT Study mean baseline insulin doses were between and U per day depending on the type of insulin therapy regimen applied [] It can be concludedthat under realworld conditions the majority of insulinexperienced patients with type diabetes receive higherinsulin doses per day than those tried in UKPDS orORIGINIn the absence of randomized controlled trials aMendelian randomization is an appropriate approach oftesting for a causal relationship in humans Mendelianrandomization studies made use of the finding that somegenotypes are associated with high or low fasting insulinlevels When comparing individuals carrying ‰¥ allelesthat raise fasting insulin levels with those exhibiting genetically determined low fasting insulin levels an increasedrisk of elevated blood pressure cardiovascular disease andtype diabetes was observed [] In two large recentMendelian randomization studies a genetic profile predicting high insulin levels in the blood after adjustmentfor BMI was also associated with increased systolic bloodpressure and risk of myocardial infarction []ConclusionsAs discussed aboveinsulin signaling engages at leastthree different pathways and modifies a large number ofcellular responses Table Transient elevations of systemic insulin concentrations are physiological responsesto dietary stimuli or other challenges such as environmental toxins [] In case of prolonged upregulationof insulin levels such as in response to overnutritionglucose homeostasis is maintained by mitigating insulinsignaling via PI3KAKT for glucose export from theblood into tissues Consequently insulin resistance hasbeen considered as a defense response in order to avoidTable Key messagescid129 Insulin employs at least three different pathways of signal transductionOne pathway involves phosphorylation steps via IRS“PI3K“AKT anotheris the MAP kinases Ras“MEP“ERK and third leads to the activation ofNOX4cid129 Insulin resistance is selective because it partially mitigates the PI3KAKTpathway for limiting glucose uptake and eNOS activation despitehyperinsulinemia but many other hormonal actions of insulin are notsuppressedcid129 Signaling via mTOR and the MEPERK pathway causes suppression ofautophagy and NRF2 leading to deficient turnover and impaired celldefensecid129 Moderate to high normal insulin levels inhibit lipolysis and promotelipogenesisobesitycid129 Insulin resistance and hyperinsulinemia are interdependent Dietinduced hyperinsulinemia precedes insulin resistancecid129 In epidemiological studies insulin therapy of type diabetes isassociated with a higher risk of cardiovascular events or deathcid129 Randomized trials of insulin therapy and associated risks only studieddosages up to IUdaycid129 Mendelian randomization studies found that genetically determinedhigh insulin levels lead to cardiovascular diseasecid129 Suppression of hyperinsulinemia and concomitant œinsulin resistanceprovides substantial health benefitshypoglycemia [] However other hormonal actions ofinsulin via the MAP kinase MEKERK pathway and inpart via PI3KAKT are no

Thyroid_Cancer

Decreased expression of the thyroid hormone‘inactivating enzyme type deiodinase is associated with lower survival rates in breast canceriuri Martin Goemann1 Vicente Rodrigues Marczyk15 Mariana Recamonde‘Mendoza23 Simone Magagnin Wajner15 Marcia Silveira Graudenz45 Ana Luiza Maia thyroid hormones tHs are critical regulators of cellular processes while changes in their levels impact all the hallmarks of cancer Disturbed expression of type deiodinase DIO3 the main tH‘inactivating enzyme occurs in several human neoplasms and has been associated with adverse outcomes Here we investigated the patterns of DIO3 expression and its prognostic significance in breast cancer DIO3 expression was evaluated by immunohistochemistry in a primary cohort of patients with breast cancer and validated in a second cohort using RnA sequencing data from the TCGA database DNA methylation data were obtained from the same database DIO3 expression was present in normal and tumoral breast tissue Low levels of DIO3 expression were associated with increased mortality in the primary cohort Accordingly low DIO3 mRnA levels were associated with an increased risk of death in a multivariate model in the validation cohort DnA methylation analysis revealed that the DIO3 gene promoter is hypermethylated in tumors when compared to normal tissue In DIO3 is expressed in normal and tumoral breast tissue while decreased expression relates to poor overall survival in breast cancer patients Finally loss of DIO3 expression is associated with hypermethylation of the gene promoter and might have therapeutic implicationsBreast cancer is the most common cancer in women worldwide accounting for more than two million new cancer cases and of all cancerrelated deaths in women in Despite remarkable advances in the treatment of breast cancer in recent decades not all patients benefit from current therapeutic options and thus will experience relapse23 Genomic tests improve the clinical prediction of patient outcomes and determine the necessity of adjuvant chemotherapy with endocrine therapy34 However it is a highly heterogeneous disease that is diverse in its behavior and responsiveness to the different modalities of treatment56 Breast cancer is characterized based on receptor and gene expression profiles that together with the classic clinicopathological variables guide the treatment and estimate the risk of recurrence34 Gene expression profiling studies have established at least four molecularly distinct types of breast cancer that can be expanded to the œintrinsic subtypes luminal A LumA luminal B LumB HER2enriched basallike and normallike7“Numerous studies have established thyroid hormones THs as critical regulators of multiple cellular processes in normal and tumor cells10 They contribute to cellular proliferation and differentiation during development and adulthood and are finetuned for tissuespecific control1011 Clinical studies associate TH levels with breast 1Thyroid Unit Endocrine Division Hospital de Cl­nicas de Porto Alegre Rua Ramiro Barcelos Porto Alegre RS CEP Brasil 2Institute of Informatics Universidade Federal Do Rio Grande Do Sul Porto Alegre Brazil 3Bioinformatics Core Hospital de Cl­nicas de Porto Alegre Porto Alegre Brazil 4Department of Pathology Hospital de Cl­nicas de Porto Alegre Porto Alegre Brazil 5Faculdade de Medicina Universidade Federal Do Rio Grande Do Sul Porto Alegre Brazil email almaiaufrgsbrScientific RepoRtS 101038s41598020708924Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Patterns of expression of DIO3 in breast samples Immunostaining was performed as described in Materials and Methods From left to right A normal glandular breast tissue B breast carcinoma with low expression overall intensity C breast carcinoma with moderate expression overall intensity and D breast cancinoma with high expression overall intensity of DIO3 protein evaluated through immunohistochemistry The staining intensity level is used to calculate the Hscore combined with the percentage of positive cells see œMethodscancer risk and mortality1213 while in a0vitro models demonstrate the effect of THs on breast cancer cell proliferation apoptosis and migration14“ T4 promotes cell proliferation through the αv3 integrin receptor14 while the proliferative effects of T3 depend at least partially on the presence of estrogen receptors in breast cancer cells1718 Clinically however the effects of THs on specific histopathological and molecular subtypes of breast cancer are still unclear1920Modulation of THs concentrations is orchestrated by a group of selenoproteins called iodothyronine deiodinases which can activate and inactivate thyroid hormones21 Briefly the type deiodinase DIO1 catalyzes both activation and inactivation of thyroxine T4 generating triiodothyronine T3 and reverse triiodothyronine rT3 respectively22 Type deiodinase DIO2 acts locally converting the prohormone T4 into the active T3 Meanwhile type deiodinase DIO3 is the main THinactivating enzyme by degrading T4 and T3 to inactive metabolites rT3 and diiodothyronine respectively21 The DIO3 gene is found in the DLK1DIO3 genomic region which is located on human chromosome 14q3223 DIO3 gene is subject to genomic imprinting an uncommon epigenetic phenomenon that results in the preferential expression of one of the alleles paternal allele in the case2425 DIO3 gene expression is increased in several tissues during embryogenesis but it decreases in most tissues in adulthood2627 Notably DIO3 is expressed in normal and pathological hyperproliferative conditions where it has been implicated in cell proliferation and differentiation20252628 In particular studies have demonstrated that the local control of THs signaling provided by the regulation of DIO3 activity is associated with cancer development progression and recurrence28“ We have previously reported that DIO3 mRNA and activity levels are increased in papillary thyroid cancer PTC which are associated with larger tumor size and the presence of lymph node and distant metastasis at diagnosis30 Others have described hyperexpression of this enzyme in basal cell carcinoma BCC where it modulates intracellular T3 concentrations and thus contributes to the cell tumorigenic potential31 DIO3 exerts a similar function in colon cancer which suggests that attenuation of the TH signal is part of the oncogenic process at least in some types of cancer28Considering the implied role of the DIO3 gene in human neoplasms and the potential effect of TH in breast carcinogenesis13“ we investigated the expression patterns of DIO3 in normal breast tissue and breast cancer Here we demonstrate that DIO3 is expressed in normal breast tissue and breast cancer tissue In breast cancer reduced DIO3 expression is associated with decreased overall survival Interestingly loss of DIO3 expression might be explained at least partially by gene promoter hypermethylationResultsDIO3 in normal breast and fibroadenoma DIO3 immunohistochemistry staining was detected in all samples of normal breast tissue N at an overall moderate intensity Hscore ± DIO3 staining was predominantly cytoplasmatic and more pronounced in the apical extremity in luminal cells in both ducts and acini of the breast Fig a01A DIO3 was markedly positive in myoepithelial cells Fig a01A bottom Benign fibroadenoma lesions N were also positive for DIO3 staining with an intensity comparable to healthy tissue Hscore ± vs ± P Scientific RepoRtS 101038s41598020708924Vol1234567890wwwnaturecomscientificreports 0cCharacteristicMedian age at diagnosis range”yearsTumor size in the largest dimension”mmMedian IQRMean ± SDEstrogen receptor”no PositiveNegativeMissingProgesterone receptor”no PositiveNegativeMissingHER2 status”no PositiveNegativeMissingHistological type of tumor”no Invasive Ductal Carcinoma IDCInvasive Lobular Carcinoma ILCDuctal Carcinoma in a0situ DCISClinicalpathological subtype”no Luminal ALuminal BHER2Triple NegativeNon classifiedLymph node metastasis”no YesNoDistant metastasis”no YesNoTumor staging”no Stage IIIStage IIIIVMissingPretreatment hypothyroidism”no Posttreatment hypothyroidism”no Followup mean ± SD”monthsAllcause mortality”no Mean survival months CIPrimary cohort N Validation cohort N “ “ “ ± AJCC NANA PAM50 ” “ NANA “ “Table Baseline characteristics of patients with breast cancer included in the primary cohort and in the validation cohort NA not available IQR interquatile range SD standard deviation HER2 human epidermal growth factor receptor2 AJCC American Joint Committee on Cancer Classified by the AJCC staging system Classified by PAM50 data available for patientsDIO3 protein in breast cancer the primary cohort To study DIO3 expression in breast cancer we analyzed a cohort of patients who had been seen at our institution primary cohort N and validated the results in the TCGABRCA cohort validation cohort N The clinicopathological characteristics of the patients from both cohorts are summarized in Table a0Patterns of DIO3 staining evaluated through immunohistochemistry in breast cancer samples are shown in Fig a01B“D DIO3 staining in FFPE breast cancer tissues was positive in samples of invasive ductal carcinoma IDC with a mean Hscore of ± When evaluating invasive lobular carcinoma ILC only of samples was positive for DIO3 Hscore A sample of ductal carcinoma in a0situ DCIS was also positive for DIO3 expression Hscore A graph comparing the Hscore for DIO3 in nonmalignant tissues and malignant breast cancer types is presented in Fig a02A Mean DIO3 Hscores of primary tumors were similar to the nontumoral tissues with a marginal decrease in DIO3 seen in invasive lobular carcinoma ILC P Scientific RepoRtS 101038s41598020708924Vol0123456789wwwnaturecomscientificreports 0cType of tissuetumorANormalbreastDFibroadenomaIDCILCLymph node statusPNSerocSHerocSHEstrogen receptor statusHER2 statusCPNSpositivenegativeTNM stagingFPNSPNSpositivenegativeDistant metastasisPNSBEerocSHerocSHerocSHerocSHnegativepositiveabsencepresenceIIIIIIIVGLog Rank p0012liavvrusfoytilibaborPDIO3 positiveDIO3 negativePatients at riskDIO3 posDIO3 negMonthsFigure a0 DIO3 staining and clinicopathological characteristics of patients with breast cancer in the primary cohort A“F Box plots of DIO3 staining in breast tissue samples evaluated through immunohistochemistry and quantified by HScore Samples were divided according to clinicopathological data as follows A type of tissue analyzed B ER status C HER2 status D lymph node status E distant metastasis and F TNM anatomic staging G Kaplan“Meier plot of overall survival in patients with the presence gray or absence black of DIO3 staining in breast cancer evaluated through immunohistochemistry ER estrogen receptor HER2 human epidermal growth factor receptor2 IDC invasive ductal carcinoma ILC invasive lobular carcinoma NS not significant P The mean Hscore of invasive ductal carcinoma was similar to that of normal tissue P No differences were observed between the molecular subtypes of breast cancer P data not shown There was no difference in the Hscore between tumors with ERpositive and ERnegative status P Fig a02B or between tumors with HER2positive and HER2negative status P Fig a02C Among the primary tumors there was no significant correlation between Hscore and Ki67 levels P or between Hscore and histological tumor grade P We found no association of DIO3 positivity negative or positive with tumor size P The mean Hscore in primary tumors of patients without nodal metastases was similar to that observed in patients with lymph node metastasis P Similarly Hscores of primary tumors of patients with distant metastasis did no differ from those without distant metastasis P Fig a02DE There were no differences on DIO3 Hscores when comparing patients with stage III vs stage IIIIV disease P Fig a02F We obtained both primary and lymph node tissues from patients In this subset of patients DIO3 staining was comparable between paired primary tumor and lymph node metastasis P Table a0 shows the variables associated with an increased risk of death in the primary cohort univariate analysis We observed that negative DIO3 staining was associated with poor prognosis HR CI to P Therefore additional studies were performed using Kaplan“Meier analysis and the logrank Scientific RepoRtS 101038s41598020708924Vol1234567890wwwnaturecomscientificreports 0cVariableAge at diagnosis yearsTumor size mmLymph node metastasis pos vs negDistant metastasis pos vs negER status pos vs negP status pos vs negHER2 positivity pos vs negTNM staging IIIIV vs IIIDIO3 status neg vs posHR CI “ “ “ “ “ “ “ “ “P valueTable Univariate Cox regression analysis of overall survival in breast cancer patients in the primary cohort HR hazard ratio CI confidence interval ER estrogen receptor P progesterone HER2 human epidermal growth factor receptor2test Patients with negative DIO3 staining had a worse overall survival than those with positive DIO3 staining The mean overall survival was a0months CI to in the DIO3negative group and a0months CI to in the DIO3positive group Fig a02G logrank P DIO3 mRNA in breast cancer patients validation cohort It has been previously demonstrated that DIO3 protein levels and activity correlate with DIO3 mRNA levels in different contexts303233 Therefore to validate differences of DIO3 expression among patients with breast cancer we analyzed DIO3 mRNA expression in a second cohort using available gene expression data from the TCGABRCA study In this second population DIO3 expression was found to be reduced in primary solid tumors N compared to that observed in normal breast samples N logFC adjusted P value Fig a03A even when the comparison was made only with matched normal tissues logFC adjusted P value Fig a03B The majority of tumor subtypes with the exception of normallike tumors classified according to PAM50 classification system showed reduced DIO3 expression compared to normal tissue Fig a03C On the other hand DIO3 expression was increased in ERpositive samples compared to that in ERnegative samples logFC P Fig a03D There was no significant difference when comparing DIO3 expression between patients with or without lymph node disease logFC adjusted P value or distant metastasis logFC adjusted P value Fig a03E Decreased DIO3 mRNA expression was observed in all tumor stages compared to that seen in normal tissue P However no differences were found between the different tumor stages Fig a03F Interestingly lower DIO3 expression was associated with greater tumor size P and ER negativity P We then evaluated the prognostic value of DIO3 mRNA expression for patient survival We considered patients as having high DIO3 expression when their logCPM values were above the median and as having low DIO3 expression when their logCPM values were below the median Low DIO3 expression was associated with reduced survival with an HR of CI to P in the univariate model Table a0 Additional analysis using a multivariate model adjusted for all variables with a P in the univariate analysis demonstrated that low DIO3 was an independent prognostic factor for death HR IC to P Table a0 Fig a04A The estimated overall survival rate at five years in the Kaplan“Meier analysis was CI to in the high DIO3 group and CI to in the low DIO3 group Fig a04AIn the subgroup analysis of patients with advanced disease stage IV those with low DIO3 expression had reduced overall survival compared to patients with high DIO3 expression P Fig a04B Notably low DIO3 expression was associated with worse overall survival among patients with ERpositive tumors P but not among those with ERnegative tumors P Supplementary Fig a0Methylation of DIO3 gene promoter To further investigate possible factors that could lead to decreased DIO3 expression in breast cancer we performed DNA methylation analysis of a subgroup of patients from TCGABRCA database from whom DNA methylation data were available N Our analysis demonstrated that global DNA methylation levels of breast cancer samples were similar to those of healthy breast tissues Fig a05A However the methylation levels of CpG sites in the DIO3 gene region were increased compared to those from healthy tissue Fig a05B P Figure a0 details the CpG sites that are hypermethylated within the DIO3 gene region The first kbp of ² flanking region red are known to be extremely G C rich of the sequence and this region is highly conserved between mouse and human genome34 Promoter region a0bp of the ² flanking region is composed of several promoter elements Fig a05C enhanced including a TATA box two CAAT boxes and CG rich regions35 We observed a significant increase in DNA methylation levels in CpG sites that are located both at the promoter region and in the ² flanking kbp conserved region of the gene Fig a05CDScientific RepoRtS 101038s41598020708924Vol0123456789wwwnaturecomscientificreports 0cFigure a0 The relationship between DIO3 mRNA expression and clinicopathological parameters in breast cancer samples of patients from the TCGABRCA cohort expressed in Log2 counts per million voomtransformed Comparative expression demonstrates that DIO3 mRNA is decreased in tumoral tissue when compared to normal tissue when analyzing A all samples or B only matched samples C All tumor subtypes have decreased expression of DIO3 mRNA when compared to normal tissue with the exception of normallike tumors compared to normal tissue DIO3 mRNA levels were also reduced in basallike tumors when compared to luminal A logFC adjusted P value and in luminal B when compared to luminal A subtypes logFC adjusted P value and D DIO3 expression is increased in ERpositive samples when compared to ERnegative samples E DIO3 expression is similar in patients with or without metastasis F When samples were separated according to tumor staging all tumor stages had decreased DIO3 expression when compared to normal tissue but there was no difference in expression between the stages ER estrogen receptor Adjusted P value in comparison to normal tissueVariablesAge at diagnosis yearsTumor size ‰¥ a0cm vs ‰¤ a0cmLymph node pos vs negDistant metastasis pos vs negE2 status pos vs negP status pos vs negHER2 positivity pos vs negTNM staging IIIIV vs IIIDIO3 status low vs highUnivariate analysisHR CI “ “ “ ““ “ “ “ “P value Multivariate analysisHR CI “ “ “ “ “ “P value “Table Univariate and multivariate Cox regression and for overall survival in the validation cohort HR hazard ratio CI confidence interval ER estrogen receptor P progesterone HER2 human epidermal growth factor receptor2 All variables with P were included in the multivariate model TNM is not included as it is derived from variables already present in the modelDiscussionDisruption of the iodothyronine deiodinases expression leads to changes in TH concentrations which might contribute to cancer development and progression by impacting virtually all the hallmarks of cancer10 Here we demonstrate that the THinactivating enzyme DIO3 is expressed in normal breast tissue and that its expression is highly prevalent in breast cancer More interestingly our results demonstrated that low DIO3 expression Scientific RepoRtS 101038s41598020708924Vol1234567890wwwnaturecomscientificreports 0cAOverall SurvivalP BOverall Survival Stage IV patientsHighLowDIO3DIO3groupgroupP0011liavvrusfoytilibaborPliavvrusHighLowDIO3DIO3groupgroupHR for death IC to foytilibaborPPatients at riskHigh DIO3Low DIO3MonthsPatients at riskHigh DIO3Low DIO3MonthsFigure a0 Kaplan“Meier estimates of overall survival in patients of the TCGABRCA cohort according to DIO3 mRNA expression Patients were grouped according to the median of DIO3 expression in the population as presenting high DIO3 expression gray lines or low DIO3 expression black lines Plot A shows the overall survival in the entire cohort Plot B refers only to patients with stage IV disease HR hazard ratio CI confidence intervalwas an independent prognostic factor for reduced overall survival in two different populations of patients with breast cancerData on the expression of iodothyronine deiodinases in human breast tissue are scarce Low levels of DIO1 were reported in normal and lactating tissues but DIO2 and DIO3 have not been analyzed thus far36 Here we show that DIO3 is expressed at both the mRNA and protein levels in normal human breast tissue Expression of DIO3 mRNA has been previously described in breast cancer cell lines MCF7 and MDAMB231 cells DIO3 mRNA was found to be upregulated in MCF7 cells and downregulated in MDAMB231 cells when compared to the nontumoral cell line MCF10A cells DIO3mediated T3 deiodination also occurs in MCF7 cells In these cells DIO3 expression is to regulated by retinoids but not by estradiol37“ These findings are consistent with the presence of DIO3 in other tissues of ectodermal origin such as the skin and the nervous system4041The role of thyroid hormone metabolism on human tumorigenesis has been largely debated10 In breast cancer previous studies showed that higher levels of the thyroid hormone receptor alpha were an independent prognostic factor for increased overall survival42 More recently high levels of the thyroid hormone receptor beta in breast tumors were also associated with increased breast cancerspecific survival43In basal cell carcinomas BCC for instance a DIO3mediated decrease in T3 levels relates to increased cell proliferation31 Similarly in colon cancer cells DIO3 knockdown and consequent increases in T3 levels are associated with reduced cell proliferation and induction of differentiation44 High levels of DIO3 expression in primary PTC tumors were associated with advanced disease at the diagnosis30 Some data indeed suggest that T3 can contribute to tumor growth in breast cancer cells in a0vitro17 while a microenvironment with low T3 levels could facilitate invasiveness and dedifferentiation However in agreement with our data in breast cancer similar levels of DIO3 mRNA are observed in glioblastoma and liver carcinomas as compared to respective normal tissues45 These differences could be attributed to the tissue embryological origin since the tissues of ectodermal origin seem to maintain DIO3 expression during adulthood while DIO3 gene is subject to imprinting in other tissues Loss of DIO3 expression was associated with tumor aggressiveness in colon cancer and also in thyroid cancer DIO3 expression is present in papillary and follicular subtypes but not in the most aggressive and dedifferentiated anaplastic subtype30 Taken together these results indicate that although expression of the enzyme is often upregulated in the neoplastic tissue compared to normal tissue loss of DIO3 expression is a common hallmark of dedifferentiation in the neoplastic process which might confer its prognostic significance Alternatively the distinct pattern of expression could be the result of DIO3 regulation or related to the cancertype specific methylation signatureAlthough this was an exploratory study our results point to a prognostic role for DIO3 expression in breast cancer In a primary cohort of patients with breast cancer negative DIO3 staining in the primary tumor was associated with significantly worse prognosis HR CI to P when compared to patients who were DIO3positive More interesting in the second cohort low DIO3 expression was an independent prognostic factor for death in a model adjusted for age tumor size lymph node and distant metastasis estrogen and progesterone status HR IC “ P The prognostic role of DIO3 expression was particularly relevant in the subgroup of patients with advanced diseaseIntriguingly the difference in survival between groups with distinct DIO3 expression was limited to ERpositive patients Previous studies indicate the existence of a crosstalk between estrogen and THdependent regulatory pathways in breast cancer14174647 which might be a potential explanation T3 regulates cell cycle progression and proliferation in breast cancer cells in a0vitro by a common mechanism involving ER and T3 receptormediated pathways46 Moreover T4 can phosphorylate nuclear ERalpha in MCF7 cells via a MAPKdependent pathway promoting proliferation14 Therefore loss of DIO3 expression and the consequent increase in intracellular T3 levels could be specifically detrimental to tumors that express ER as our results suggest Contributing to this Scientific RepoRtS 101038s41598020708924Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Panel A demonstrates mean global DNA methylation levels values in breast cancer tissue compared to healthy breast tissue Panel B demonstrates that the mean DNA methylation of DIO3 gene region is increased in tumor tissue when compared to normal tissue P Panel C is a schematic representation of the location of DIO3 gene in chromosome and the regions that were evaluated by CpG probes The promoter region is composed by several promoter elements including a TATA box two CAAT boxes and CG rich regions C enhanced Significant hypermethylation in several CpG sites is observed in the promoter region of the gene Panel D presents mean values of CpG sites mapped in DIO3 gene region comparing normal and tumoral tissueinterplay previous studies have demonstrated that estrogen progesterone and their receptors regulate DIO3 activity in rat uteri and decidua4849 Therefore we cannot rule out that in the breast DIO3 expression depends partially on the presence of functional estrogen and progesterone receptorsScientific RepoRtS 101038s41598020708924Vol1234567890wwwnaturecomscientificreports 0cThe DIO3 gene is subject to genomic imprinting an uncommon epigenetic phenomenon that results in the preferential expression of one allele the paternal allele in this case2425 The disturbed expression of genes and miRNAs or altered hypermethylation patterns of the DLK1DIO3 genomic region is involved in the pathogenesis of different types of cancer50“ Thus we hypothesized that the loss of DIO3 expression in breast tumors could be a consequence of gene hypermethylation in the tumoral context Indeed our results show that while the mean global methylation in breast tumors is comparable to that of normal tissue the DIO3 genomic region especially its promoter region is significantly hypermethylated in tumors Fig a05C enhanced These findings might explain at least in part the reduced DIO3 expression in breast cancer Of interest the DIO3 gene was also found to be hypermethylated in Bcell Tcell and myeloid malignancies and lung cancer5152Our study has some limitations The absence of data on DIO3 enzymatic activity limits the assumption that the decreases of DIO3 levels cause alterations in intracellular TH homeostasis Alternatively changes in DIO3 expression could simply represent a consequence of broader epigenetic modifications in the tumoral context It is also important to consider that complete clinical data on patient thyroid status was not available which could interfere with deiodinase expression54 Therefore the complex changes on deiodinases and the overall effect on intracellular TH status are still unclear in breast cancer Additionally our analysis is limited to two populations using two different methodology and despite robust supporting data results should be confirmed in other cohortsIn the results of this study demonstrate DIO3 expression in breast tissue and breast cancer Importantly low DIO3 expression is associated with reduced overall survival suggesting that DIO3 might have a prognostic role in this disease Reduced DIO3 expression in breast cancer can be explained at least in part by gene hypermethylation Due to its potential to modulate thyroid hormone intracellular levels and interplay with estrogen metabolism in breast cancer the DIO3 expression might have therapeutic implicationsMethodsPatients and tissues primary cohort Neoplastic tissue from patients diagnosed with breast cancer was retrospectively collected from a consecutive series of unselected patients in the pathology department of Hospital de Cl­nicas de Porto Alegre Tissue samples of the normal breast N and fibroadenomas N were also obtained Histopathological reports containing information on tumor type grade and immunohistochemistry were retrieved clinical data were retrospectively reviewed in medical records Tumors were histologically classified according to the 8th edition of the American Joint Committee on Cancer AJCC staging system56 All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional andor national research committee The study was reviewed and approved by the Institutional Review Board and Research Ethics Committee from the Hospital de Cl­nicas de Porto Alegre with a waiver of informed consent Protocol number Immunohistochemistry studies and DIO3 staining assessment DIO3 protein expression was evaluated by immunohistochemical studies on 6mm sections of formalinfixed paraffinembedded FFPE tissue blocks from normal breast tissues fibroadenomas and primary breast cancers The immunohistochemical technique consists of tissue deparaffinization and rehydration antigenic recovery inactivation of endogenous peroxidase and blockage of unspecific reactions Samples were incubated overnight at a temperature of a0°C with an antiDIO3 rabbit polyclonal antibody Abcam Cambridge UK at a dilution of followed by subsequent incubation with a biotinylated secondary antibody a streptavidin“HRP conjugate LSAB Dako Carpinteria CA USA and diaminobenzidine tetrahydrochloride Kit DAB Dako The slides were examined using an Olympus BX51 microscope The QCapture Pro software Qimaging Surrey BC Canada was used to capture the images DIO3 staining was evaluated by an experienced pathologist blinded to the molecular profile and TNM staging The immunohistochemical results of DIO3 staining were assessed dichotomously negative or positive and semiquantitatively using the Hscore method as described previously5758 The Hscore combines the percentage of positive cells and staining intensity level weak moderate strong and is calculated using the following formula [ — cells — cells — cells ] with results ranging from to Positive epidermis and placenta and epidermal nevus and negative connective and adipose tissue internal controls were assessed for all the evaluated cases Samples from the primary cohort were classified concerning the presence or absence of these receptors and the level of Ki67 expression into the following groups Luminal A LumA luminal B LumB triple negative and HER2 A Ki67 index cut point of was defined to distinguish HER2 negative lumB from lumA tumors5960Differential gene expression and methylation analysis For the validation cohort RNA sequencing RNASeq RSEM gene expression data from The Cancer Genome Atlas TCGA breast cancer BRCA study were obtained from the Genomic Data Commons GDC Data Portal gdcporta lcninihgov using the TCGAbiolinks RBioconductor package61 Raw expression signals for primary solid tumor samples N and solid normal tissue samples N were normalized and analyzed for differential expression of DIO3 using the limmavoom pipeline from the limma RBioconductor package62 P values were adjusted for multiple comparisons using the false discovery rate FDR procedure of Benjamini and Hochberg63 Clinicopathological information for TCGABRCA samples was downloaded through TCGAbiolinks and the Broad GDAC Firehose gdacbread insti tute merged level clinical data For tumors of the TCGABRCA cohort data retrieved from PAM50 classification were used to define tumor subtype classification64 Overall survival OS was estimated by the Kaplan“Meier method and compared by the logrank test using functions provided by TCGABiolinks For the methylation analysis we used the TCGAbiolinks RBioconductor package30 to obtain and analyze Illumina a0K methylation and clinical data for samples from the TCGABRCA study includScientific RepoRtS 101038s41598020708924V

Thyroid_Cancer

Protocol Study protocol for the use of photobiomodulation with red or infrared LED on waist circumference reduction a randomised double blind clinical trialMarcelo Marreira Lidiane Rocha Mota Daniela F¡tima Teixeira Silva Christiane Pavani To cite Marreira a0M Rocha Mota a0L Silva a0DFT et a0al Study protocol for the use of photobiomodulation with red or infrared LED on waist circumference reduction a randomised double blind clinical trial BMJ 202010e036684 101136bmj 2019036684 –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 December Revised July Accepted July Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJBiophotonics Applied to Health Sciences Universidade Nove de Julho Sao Paulo BrazilCorrespondence toDr Christiane Pavani chrispavani gmail comIntroduction The search for non invasive procedures to reduce localised adiposity in aesthetics clinics has recently been increasing In this context procedures such as cryolipolysis ultracavitation photobiomodulation PBM and other techniques have been proposed Some studies have shown that PBM can be used in body contouring However there is no standardisation of the protocol More than that as in other techniques for reducing adipose tissue the availability of triacylglycerol may affect the lipid profile in the blood bringing consequences to the general health of an individual This work will aim to compare the light wavelengths when using PBM as a technique for reducing the abdominal waist circumference while also evaluating the efficacy of the method Changes in the lipid profile in the blood with a long term follow up will also be appraisedMethods and analysis This will be a controlled randomised double blind single centred clinical trial patients will be recruited at the Nove de Julho University Brazil and then divided into three groups Group A”RED PBM Group B”INFRARED PBM Group C”PLACEBO Sham treatment The treatments will consist of eight sessions two times a week for weeks At each session the participants will receive minutes PBM using a radiant exposure of Jcm2 with an abdominal strap containing LED clusters with devices each following the indication of randomisation All of the groups will receive min of Aussie Current at kHz modulated at Hz “ mA The main outcome of this study will be waist circumference reduction The secondary variables will be anthropometric data lipid profile liver function and adipose tissue thickness changes in the local microcirculation and the quality of life and self esteem The analyses will be performed at four stages of the research D0 end of the eighth session D30 days after the last session FU15 days after the last session FU90 and days after the last session FU180Ethics and dissemination The Ethics Committee of the Nove de Julho University Brazil approved the modified version of this project under No on June This study is not yet recruiting The results obtained Strengths and limitations of this study –º The use of the same dosimetry at different wavelengths will allow for a real comparison between red and infrared as being the most suitable wavelength for body contouring –º Analyses of body contouring will be performed by non invasive methods –º The waist circumference measurement will not discriminate the factors underlying the volume modifications –º The habits of the participants such as diet and exercise routines may affect the results –º Gender may affect the results and be dependent on the number of participants of each gender These differences may not be considered by the statistical analysiswill be published in a peer reviewed journal in the related fieldTrial registration number Brazilian Registry of Clinical Trials”ReBec RBR 9bwxcxINTRODUCTIONFat storage is intended to protect the human body in cases of prolonged fasting intense physical activity and temperature regulation Once freed from these situations fat is stored unnecessarily putting the individual at the risk of health problems together with a greater pr sity for pathologies such as systemic arterial hypertension diabetes mellitus metabolic syndrome and even some types of neoplasms1“Another type of negative impact that is related to excessive fat storage is body dissatisfaction This naturally leads to a decrease in the individual™s self esteem4 Studies have shown that aesthetic treatments significantly increase a patient™s quality of life They Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access are associated with improved self esteem6“ There are some traditional surgical methods for the reduction of abdominal adiposity However the methods are invasive techniques which may present a high number of complications such as bruising seroma pain perforated ans and viscera as well as with an increased risk of deep vein thrombosis10“ The demand for minimally invasive procedures that are aimed at reducing abdominal fat has increased by about while the demand for surgical procedures has decreased by around Among the minimally invasive techniques one can mention low level laser therapy which has recently also been called photobiomodulation PBM PBM has many novel advantages when compared with traditional techniques such as surgical procedures since it can guarantee the preservation of the noble adjacent structures such as the nerves the blood vessels and the skin15 PBM has been widely studied for several applications due to its important biochemical cellular consequences and its few side effects16 Some manuscripts have described erythema and oedema as the main side effects of PBM but importantly these side effects may have been higher as a result of the patient using any drug that increased photosensitivitySome other studies have shown that PBM can be used in body contouring18“ Sadly there is no standardisation of the protocol The treatments vary in terms of the number of sessions “ their frequency “ times per week and wavelength nm nm nm nm while other dosimetry information such as irradiance Wcm2 and radiant exposure Jcm2 are frequently not mentioned Recently Croghan and coworkers showed that two times a week was the best frequency when compared with one or three times a week This was in terms of improving the patients™ quality of life and body satisfaction as well as their weight waist circumference body mass index BMI and body fat mass reduction18 However more studies are needed in order to standardise the wavelength the dosimetry and the application time as well as the durability of the results achievedOne of the proposed mechanisms for a PBM effect in adipose tissue is the formation of transient pores in the adipocyte membranes thus allowing for the lipids to escape15 Adipocyte apoptosis activation has also been proposed The production of reactive oxygen species is also possible due to the action of PBM and this is related to the mitochondrial activation on account of the radiation absorption by the cytochrome c oxidase molecules This is followed by an increased ATP synthesis and with an increased cyclic adenosine monophosphate messenger which can trigger the activation of the lipases that perform the hydrolysis of the triglycerides into fatty acids and glycerol23Some reports have affirmed that the results obtained by the use of PBM for reducing waist circumference are modest and that the reduction is temporary which deserves much greater attention from researchers for a better understanding of this factor These effects may be associated with the mechanisms of action and the dosimetric parameters being used24 When taken to the tissues the free fatty acids are used as an energy source during beta oxidation for the production of ATP In some literature reports PBM is associated with aerobic or resistance exercise while other reports have mentioned waist and arm circumference reductions with the use of PBM displaying an absence of diet restrictions or exercise requirements25“ It is also reported that the amount of fat mobilised during a PBM session is similar to the amount that is consumed during a meal in such a way that it can be absorbed by normal body energy requirements andor exercise routine while at the same time the risk of atherosclerosis is not increased by the treatment30 On the other hand if not consumed these fatty acids may be re esterified and redistributed throughout the body30 causing no final changes in waist circumference Since neuromuscular electrical stimulation increases energy expenditure in a similar way to that associated with exercise the protocol will be complemented with the Aussie current application31As for other techniques for reducing the adipose tissue the availability of triacylglycerol may affect the lipid profile in the blood bringing consequences for the general health of the individual Some studies have shown that these important treatments may affect the serum lipid levels while others affirm that there are no changes in the serum lipid levels32 Given these scenarios this work will aim to compare the different light wavelengths when using PBM as a technique for the reduction of abdominal waist circumference while at the same time evaluating the efficacy of the method and by following the changes in the lipid profile in the blood as well as with reviewing the long term follow upMETHODSStudy designThis will be a controlled randomised double blind single centred clinical trial designed in accordance with the criteria as established by the Standard Protocol Items Recommendations for Interventional Trials It will be conducted at the Nove de Julho University located in the city of S£o Paulo Brazil The recruitment will be performed from September to November through the university website Thus the selection of sites includes urban locations the city of S£o Paulo and its neighbourhoods After verbal and written explanations regarding the procedures the risks and the benefits by MM a coauthor of this protocol those individuals who agree to participate in the study will sign an informed consent form Based on an anamnesis questionnaire the researchers will check if the participants meet the inclusionexclusion criteria The anamnesis questionnaire will include identification data anthropometric data clinical history and daily living habits especially dietary intake physical activity assessments and menstrual period appraisals Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cSince dietary intake and physical activity may have direct effects on the results at each evaluation before the treatment and the follow up sessions days of food records and physical activity levels will be measured The enrolment period will be extended until the sample size is reachedPatient and public involvement statementThe patients andor the public will not be involved in the design the recruitment or in the conduct of the studyInclusion criteriaThis study will include men and women aged “ years with a BMI of between and kgm2 normotrophic and overweight together with hyperplasia of the abdominal fat tissue abdominal skin folds higher than mm Those who agree to participate in this research will sign an informed consent form see online supplementary file Exclusion criteriaThe following people will be excluded from this survey those participants who are undergoing aesthetic treatments to reduce waist circumference those who have been previously submitted to abdominoplasty or liposuction surgeries those who are on a diet in order to reduce their measurements those people who engage in a physical activity more than two times a week those who are using or have taken drugs or food supplements in last days in order to reduce their measurements and their weight which may affect their lipid metabolism appetite or nutrients absorption those who have been submitted previously to oophorectomy those with signs andor symptoms of climacteric at the menopause pregnant or lactating women those participants who are not regular in attending the sessions those participants who present metabolic dysfunctions diabetes and thyroid disorders cardiovascular problems hypertension cardiac insufficiency arrhythmia thrombosis pacemaker use respiratory issues asthma chronic obstructive pulmonary disease haematological disturbances anaemia renal non alcoholic fatty liver disease dermatological or digestive disorders gastritis ulcers those with a history of oncological pathology those with cognitive deficitsSample calculationIn order to calculate the sample size a study showing the therapeutical effects of PBM when associated with aerobic plus resistance training was used26 The researchers used the highest and the lowest abdominal circumference values as well as the SD of the measurements The highest and lowest abdominal circumference values for the PBM group were and respectively and the highest SD of the measurements was with being the number of intervention groups in the study The effect size hence was calculated when using these values as described belowˆ† biggerˆ’smaller σˆšn2 ˆ’ ˆš accessWhen using the effect size value as calculated above the sample size was calculated using GPower software V3192 Dusseldorf Germany Two way Analysis of Variance ANOVA was used for the interactions within and between the groups in order to evaluate the differences between the three groups studied as well as for the five evaluations during the treatments and the follow up The test power was α005 The sample size was calculated on participantsRandomisationThe randomisation will be performed by DFTS a coauthor of this work who is not directly linked to the treatments or the evaluation of the participants by using the Excel program Microsoft USA The participants will be randomised into blocks of and into groups designated as A B and C Opaque envelopes will be identified by sequence numbers and they will receive a paper containing the information about which treatment will be performed on the participant™s abdomen according to the draw The sealed envelopes will be safely kept with the researcher who generated the randomisation DFTS Before the beginning of the procedures the researcher responsible for the procedure LRM a coauthor of this protocol will receive each envelope and proceed with the treatment as indicated according to its allocation group A team of undergraduate students previously trained and prepared is going to be part of the research group and for the treatment or evaluation of the participants This study will be a double blind study since the participants will not be aware of the group in which heshe is participating only the researcher who will perform the procedure will know The data collection and analyses will be performed by another researcher MM a coauthor of this study who will also be unaware of the allocationsInterventionThe abdomen of the participants will be cleaned by using a neutral cleansing soap They will receive eye protection using goggles for safety This will also help with the blindness of the study PBM will be applied when using abdominal straps as developed by Cosmedical Mau¡ S£o Paulo Brazil following the parameters as described in table The abdomen strap will be covered with a sheet and that will also help with the blindness of the study All of the participants will receive min of PBM with an abdominal strap containing LED clusters with devices following the indication of the randomisation being per group Group A”RED ± nm Group B”INFRARED ± nm Group C”PLACEBO The treatment will consist of eight sessions that will occur two times a week totalling month of treatments The placebo group will use a strap with no light emission but it will emit the same sounds like that of the active device In order to increase the oxidation of the free fatty acids the participants will receive min of Aussie Current at kHz modulated at Hz “ mA for min Tensor Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access Table Dosimetry for the studyParameterRed LEDContinuousCentre wavelength nmSpectral width nmOperating modeAverage radiant power”one LED mWAperture diameter”one LED cmPower density at aperture mWcm2Beam spot size at target”one LED cm2Total number of LEDsArea irradiated cm2Irradiance at target mWcm2 Exposure duration sRadiant exposure Jcm2Energy density at aperture Jcm2Radiant energy kJApplication techniqueNumber and frequency of treatment sessionsContact—week for a month a total of sessionsInfrared LEDContinuousContact—week for a montha total of sessionsDGM Electronica Santo Andr© S£o Paulo Brazil33 None of the PBM devices will significantly increase the temperature at the target area causing no burns or skin damage No modifications in the intervention will be performed for any reason However the participants who withdraw their consent or the ones who are not assiduous to the sessions will be removed from the studyThe dosimetric parameters that will be used in this protocol as presented in table were measured andor calculated The centre wavelength and the spectral width of the devices were measured by a Spectrophotometer USB2000XR1 Ocean Optics Florida USA The radiant power of one LED was measured by a Power Meter FieldMaxII TO Coherent Santa Clara California USA The abdominal straps will be composed of LED clusters having LEDs each totalling LEDs units and distributed in a cm— cm area cm2 each cm2 total see figure The effective irradiated area will be cm2 times the beam spot size at the target The irradiance at the target was determined by the ratio between the average radiant power mW and the beam spot site at the target cm2 The radiant exposure was determined by multiplying the irradiance at the target mWcm2 by the exposure duration s The radiant energy was calculated by multiplying the average radiant power of one LED mW by the total number of LEDs and by the exposure duration sFigure Photobiomodulation PBM application PBM device off A and on B patient receiving the experimental protocol in dorsal decubitus min per session C and DOutcomesThe main outcome of this study will be waist circumference reduction The secondary variables will be anthropometric data lipid profile liver function and adipose tissue thickness as measured by ultrasound changes in the local microcirculation quality of life and self esteem The participants will be evaluated at the same time of the day at all times throughout the studyThe anthropometric data that will be collected will be body weight height and BMI skin fold thickness and bioimpedance Blood will be collected for analyses of the lipid profile total cholesterol high density lipoprotein HDL Low density lipoprotein LDL triglycerides and liver function serum glutamic oxaloacetic transaminase SGOT serum glutamic pyruvic transaminase SGPT All of this will be processed and analysed at SCS Medicina Diagn³stica S£o Caetano do Sul Brazil a partner laboratory The analyses will be performed at four stages of the research D0 end of the eighth session D30 days after the last session FU15 days after the last session FU90 and days after the last session FU180Abdominal ultrasound will be performed to assess the fat layer thickness before and after the treatments For the recording of the local temperature a technique that is widely used is infrared thermography using a Compact Thermal Camera C2 FLIR Systems Oregon USA The thermal camera by means of infrared emission from the body or from the material analysed has the ability to calculate the temperature of a given surface It is possible through this method that the study will infer the changes in local microcirculation34The quality of life questionnaire ˜The WHO Quality of Life WHOQOL BREF™ as well as the Body Shape Questionnaire ˜BSQ34 Self Image Scale™ will be used for the participants These questionnaires have been translated and submitted to cross cultural adaption into Brazilian Portuguese37 The Brazilian Portuguese version of these questionnaires will be applied by MM The questionnaires will take around min to be completed The quality of life and the self image questionnaires will be applied at D0”and again at the end of the last session D30 The flow chart of the study is presented in figure Adverse events will be collected during the treatment sessions and they will be reported to the regulatory agency and again Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cRecruitment recruitmen t accessEvaluated for eligibility Elected patients n174 Anamnesis application of the quality of life and selfesteem questionnaire anthropometry blood collection ultrasonography Randomised n Allocation Group A LED with devices ± nm with Wcm2 Group B LED with devices ± nm with Wcm2 Group C Sham Each session minutes being measured at the end of each session waist circumference and IRT Total Sessions Analysis at the end of treatment Application of the quality of life and selfesteem questionnaire anthropometry blood collection IRT ultrasonography FollowUp days Anthropometry and blood collection days Anthropometry and blood collection days Anthropometry blood collection ultrasonography Figure Flow chart describing the study design the sample composition and the experimental protocol IRT infrared thermographyat the final publication of the results Since the participants are enrolled and randomised the investigators will make efforts to keep the participants together during the follow up by making phone calls emailWhatsApp contact with the patients and with relevant instructions regarding healthcare and beautyAs a strategy to improve adherence at each session the participant will schedule the next visit and receive a card with some instructions regarding preparation for the evaluation day and the appointment date of the next visit When a participant misses a session heshe will receive a phone call in order to reschedule the missed sessionData analysis planThe data that will be collected from this study will only be administered by the principal investigators the authors of this document Since the study will be of short duration and with known minimal risks this trial will not need a formal data monitoring committee After the data collection the data will be anised using Microsoft Office Excel by DFTS coauthor of this protocol and then stored on ˜a protected by password™ computer at the university The data will be analysed by descriptive and inferential statistics and then compiled into tables andor graphs using SPSS V240 For testing the normality of the data the Shapiro Wilk test will be performed If the data show a non parametric behaviour a mathematical function will be used in order to normalise the data Two way ANOVA tests followed by the Bonferroni post test will be performed in order to compare the treatments along with the time points being evaluated Some parameters Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access may affect the results of the therapy and they are going to be analysed as co variables for example skin phototype by the Fitzpatrick scale the stage of the menstrual cycle total cholesterol and triglycerides altered or not α005 will be considered the level of significance for all of the tests used Since this trial is part of a PhD thesis study an interim analysis will be performed for the qualification examination and this can be used at trial adaptations such as for a sample size re estimation or for stopping the trial The trial protocol and the full study report will be fully available at the end of the study after the manuscript of the results has been published At the end of the study the participants from the placebo group who received the treatment will experience no adverse effects and they will have received the most effective treatmentDISCUSSIONStudies have shown that lasers used in PBM typically operate at powers of mW or less They can produce energy in the visible spectrum wavelengths “ nm and near the infrared regions “ nm Light penetration in the soft tissues is known to be directly related to wavelength that is the longer the wavelength the greater the penetration The reports on PBM for reducing local adiposity include the use of green nm red and nm and infrared and nm However there is no comparison available regarding the best wavelength for this purpose18“ Based on the localisation of fat tissue more profound when compared with epidermis and dermis this study will choose red and infrared light for the comparisons The use of the same dosimetry at these different wavelengths will allow for the evaluation of the most suitable wavelength for body contouringSince the waist circumference measurements will not discriminate against the factors underlying the volume modifications a placebo group will be included This will allow for the measurement of the differences in waist circumference due to daily habits or hormonal variations as in the menstrual cycle of women The measurements of the skin folds and bioimpedance will complement the evaluation in terms of body fat At each evaluation before the treatment and the follow up sessions days of food records and physical activity levels will be measuredGender may also affect the results Sexual dimorphism in adipogenesis is already known as well as sex hormones in the white adipose tissue function and in adipose metabolism39“ If the sample consists of a huge difference in men and women this factor cannot be considered in the statistical analysisThe development of a non invasive protocol for PBM together with an Aussie current for the reduction of adiposity may present an important novel tool for the reduction of health risk problems as well as for increasing an individual™s self esteemETHICS AND DISSEMINATIONThe Ethics Committee of the Nove de Julho University S£o Paulo Brazil approved the modified version of this project and the Patient Informed Consent Form under No on June according to the guidelines of the Brazilian National Ethics Committee The protocol of this study has already been registered in the Brazilian Registry of Clinical Trials being first registered on November and modified on August providing full public access to the protocol information including all items from the WHO Trial Registration Data Set MM and DFTS will be the data curators with the data stored on ˜a protected by password™ computer at the university The results acquired within this project will be presented in conferences and published in a journal in the related field The authorship of the results paper and the conference abstracts will include the authors of this protocol together with other researchers who may contribute to the procedures or to the analysis of the data Any modifications of this protocol will require a formal amendment and they will be approved by the Ethics Committee of the Nove de Julho University The modifications will be properly reported and justified in the manuscript for the publication of the results The main results obtained will be sent to the participants by mailAcknowledgements The authors would like to thank the Nove de Julho University UNINOVE S£o Paulo Brazil for the availability of its laboratories the company Cosmedical for the development of the equipment for PBM and the SCS Medicina Diagn³stica Laboratory S£o Caetano do Sul Brazil for their partnership in the analyses of the laboratory testsContributors MM LR M DFTS and CP designed the study MM and LR M will conduct the experiments and will be making the data acquisitions DFTS and CP will perform data analysis and interpretation MM and LR M drafted the work while CP and DFTS revised it critically for important intellectual content All of the authors approved the final version of the manuscriptFunding The authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorsCompeting interests None declaredPatient consent for publication ObtainedProvenance and peer review Not commissioned externally peer reviewed 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 a0http creativecommons licenses by nc ORCID iDsDaniela F¡tima Teixeira a0Silva http orcid Christiane a0Pavani http orcid REFERENCES Silva Figueiredo P Carla Inada A Marcelino G et a0al Fatty acids consumption the role metabolic aspects involved in obesity and its associated disorders Nutrients “ Landecho MF Tuero C Valent­ V et a0al Relevance of leptin and other adipokines in obesity associated cardiovascular risk Nutrients “ Kong Y Zhang S Wu R et a0al New insights into different adipokines in linking the pathophysiology of obesity and psoriasis Lipids Health Dis “ Jim©nez Flores P Jim©nez Cruz A Bacardi Gasc³n M Body image dissatisfaction in children and adolescents a systematic review Nutr Hosp “ Weinberger N A Kersting A Riedel Heller SG et a0al Body Dissatisfaction in individuals with obesity compared to normal weight Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cindividuals a systematic review and meta analysis Obes Facts “ Kouris A Platsidaki E Christodoulou C et a0al Patients™ self esteem before and after chemical peeling procedure J Cosmet Laser Ther “ Stundzaite Barsauskiene G Tutkuviene J Barkus A et a0al Facial perception self esteem and psychosocial well being in patients after nasal surgery due to trauma cancer and aesthetic needs cluster analysis of multiple interrelations Ann Hum Biol “ Ribeiro F Steiner D Quality of life before and after cosmetic procedures on the face a cross sectional study in a public service J Cosmet Dermatol “ Bensoussan J C Bolton MA Pi S et a0al Quality of life before and after cosmetic surgery CNS Spectr “ Appleton SE Ngan A Kent B et a0al Risk factors influencing transfusion rates in DIEP flap breast reconstruction Plast Reconstr Surg “ Lievain L Aktouf A Auquit Auckbur I et a0al [Abdominoplasty complications particularities of the post bariatric patients within a patients series] Ann Chir Plast Esthet “ Sterodimas A Boriani F Nicaretta B et a0al Revision Abdominoplasty with truncal Liposculpting a year experience Aesthetic Plast Surg “ Al Dujaili Z Karcher C Henry M et a0al Fat reduction complications and management J Am Acad Dermatol “ Krueger N Mai SV Luebberding S et a0al Cryolipolysis for noninvasive body contouring clinical efficacy and patient satisfaction Clin Cosmet Investig Dermatol “ Neira R Arroyave J Ramirez H et a0al Fat liquefaction effect of low level laser energy on adipose tissue Plast Reconstr Surg “ Karu T Mitochondrial mechanisms of photobiomodulation in context of new data about multiple roles of ATP Photomed Laser Surg “ Feng J Zhang Y Xing D Low power laser irradiation LPLI promotes VEGF expression and vascular endothelial cell proliferation through the activation of ERKSp1 pathway Cell Signal “ Croghan IT Hurt RT Schroeder DR et a0al Low level laser therapy for weight reduction a randomized pilot study Lasers Med Sci “ Thornfeldt CR Thaxton PM Horn

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

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

LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathwaysXue Wu123 XiaoFeng Li123 Qian Wu4 RuiQi Ma123 Jiang Qian123 Rui Zhang123·Basic Research·1Department of Ophthalmology Eye ENT Hospital of Fudan University Shanghai China2NHC Key Laboratory of Myopia Fudan University Shanghai China 3Laboratory of Myopia Chinese Academy of Medical Sciences Shanghai China4Department of Pathology West China Hospital Sichuan University Chengdu Sichuan Province ChinaCofirst authors Xue Wu and XiaoFeng LiCorrespondence to Rui Zhang Department of Ophthalmology Eye ENT Hospital of Fudan University Fen Yang Road Shanghai China zhangrui936163comReceived Accepted Our research suggests that SNHG15 may play a vital role as a potential marker in UM that predicts poor prognosis Besides GSEA indicates the underlying signaling pathways enriched differentially in SNHG15 high expression phenotype KEYWORDS SNHG15 uveal melanoma the Cancer Genome Atlas pathology prognosis Gene Set Enrichment Analysis1018240ijo20200804Citation Wu X Li XF Wu Q Ma RQ Qian J Zhang R LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathways Int J Ophthalmol Abstract— AIM To evaluate the role of long noncoding RNA lncRNA SNHG15 and its potential pathways in uveal melanoma UM METHODS The SNHG15 mRNA expression level and corresponding clinicopathological characteristics of patients with UM were obtained from the Cancer Genome Atlas TCGA database and further analyzed The SPSS statistical software package was used for statistical analyses To investigate the potential function of SNHG15 in UM we conducted indepth research on Gene Set Enrichment Analysis GSEA— RESULTS The univariate analysis revealed that the age tumor diameter pathological type extrascleral extension cancer status and high expression of SNHG15 were statistical risk factors for death from all causes The multivariate analysis suggested that the mRNA expression level of SNHG15 was an independent risk factor for death from all causes as was age and pathological type KaplanMeier survival analysis confirmed that UM patients with high SNHG15 expression might have a poor prognosis In addition SNHG15 was significantly differentially expressed in the different groups of tumor pathologic stage metastasis and living status Besides the logistic regression analysis indicated that high SNHG15 expression group in UM was significantly associated with cancer status pathologic stage metastasis and living status Moreover the GSEA indicated the potential pathways regulated by SNHG15 in UM INTRODUCTIONU veal melanoma UM the most common intraocular cancer in adult worldwide[] is a malignant tumor that originates in melanocytes of the choroid plexus ciliary body and iris of the eye At present despite definitive radiotherapy or removal of the primary lesion numerous patients eventually develop metastases and subsequently prognosis is significantly poor[] In addition UM tends to metastasize to liver through hematogenous pathway a distant site relative to their origins in the eye[] There is an incubation period between the enucleation of the primary tumor and the emergence of metastasis which can range from a few months to several decades[] Despite the advancement of UM management there are currently no effective therapy once the metastases occurred[] Therefore close followup and further research on the pathogenesis and novel makers exploration of UM are of great significance for accurate diagnosis appropriate therapy and prognosis prediction Long noncoding RNA lncRNA is a class of noncoding transcripts with a length of larger than nucleotides[] which has been involved widely in biological processes of different cancers including cell cycle apoptosis cell differentiation[] In the development of UM lncRNA is also reported to play a vital role in cell cycle cell proliferation apoptosis invasion and autophagy[] For example silencing of lncRNA PVT1 prevents the development of UM by impairing microRNA173pdependent MDM2 upregulation[] ZNNT1 can suppress Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomathe progression of UM by inducing the expression of crucial autophagy gene[] The lncRNA RHPN1AS1 facilitates the tumorigenesis of UM by influencing cell proliferation and migration[] However the study of vital lncRNAs in UM still remains to be exploredSNHG15 a novel lncRNA located on chromosome 7p13[] is identified to play a key role in many types of human tumors such as osteosarcoma[] papillary thyroid carcinoma[] pancreatic ductal adenocarcinoma[] colorectal carcinoma[] hepatocellular carcinoma[] prostate cancer[] and breast cancer[] To our knowledge the potential impact of SNHG15 on the tumorigenesis of UM seems unclear recently Thus the purpose of this study was to evaluate the pivotal role of SNHG15 in the progression of UM In addition the relationship between SNHG15 expression and clinicopathologic characteristics in UM was preliminarily demonstrated To explore the underlying mechanisms of the biological pathways involved in UM we conducted a research on Gene Set Enrichment Analysis GSEA MATERIALS AND METHODSEthical Approval The study protocol was approved by the Ethics Committee of the Eye ENT Hospital of Fudan University and all procedures were complied with the principles of the Declaration of Helsinki All datasets of our present study were downloaded from an database TCGA so there was no written informed consent from participantsRNASequencing Patient Data and Bioinformatics Analysis The RNASeq gene expression level and clinicopathological characteristics including cases were obtained from the official website of the Cancer Genome Atlas TCGA UM project portalgdccancergov Patients with UM were classified as two groups based on the median SNHG15 expression level cutoff value794 FPKM Finally patients with UM were retained and their clinicopathological characteristics were further analyzed including the detailed information of age gender tumor diameter thickness pathological type extrascleral extension cancer status pathological stage metastasis living status SNHG15 expressionGene Set Enrichment Analysis GSEA is a common bioanalysis used to interpret and analyze microarray and other similar data and to speculate related pathways that can significantly enrich regulatory genes[] Through TCGA UM project we obtained the RNASeq gene expression level of UM patients And the analysis was conducted using GSEA v30 software In this study according to the association with SNHG15 expression the ordered gene list was generated firstly by GSEA Subsequently GSEA was conducted to clarify statistically significant differences between the two groups with high and low SNHG15 expression A total of permutations were performed The SNHG15 expression level was identified as a phenotype label The related pathways statistically enriched in each phenotype were selected with the nominal P005 and an false discovery rate FDR Statistical Analysis The SPSS statistical software package SPSS Inc USA was used for statistical analyses Both the univariate and multivariate analyses using Cox analysis were performed to demonstrate independent prognostic biomarkers for UM patients The survival curve was generated by conducting KaplanMeier method To compare the significant differences in overall survival OS the logrank test was conducted The plot chart was performed to visualize the difference of SNHG15 expression level for diverse variables through Graphpad The relationship between the SNHG15 expression and clinicopathological characteristics were analyzed using logistic regression The median value of SNHG15 expression was selected as the cutoff value P005 was considered statistically significantRESULTSPatient Characteristics The records of primary UM with both RNASeq gene expression level and clinicopathological characteristics were obtained from TCGA database The mean age of UM patients was years old including males and females The mean value of tumor diameter and thickness were and mm respectively In our study cohort the pathological type of UM included epithelioid cell dominant type and spindle cell dominant type of tumors were epithelioid cell dominant and were spindle cell dominant There were cases without extrascleral extension and cases with extrascleral extension The cancer status included tumorfree cases and cases with tumor Pathologic stage II was found in cases and stage IIIIV in cases And of cases had metastases of cases had no metastases Of cases cases died of all causes Survival Outcomes and Multivariate Analysis Prognostic factors of UM were analyzed using univariate and multivariate Cox regression The univariate analysis suggested that high SNHG15 expression was a risk factor for death from all causes Other clinicopathologic variables related to poor prognosis included age tumor diameter pathological type extrascleral extension cancer status Table In a multivariate analysis SNHG15 was an independent risk factor for death from all causes as was age and pathological typeSNHG15 Expression Associated with Clinical Pathological Characteristics A total of UM cases with SNHG15 expression data and clinicopathologic characteristics were analyzed from TCGA KaplanMeier survival analysis 0cTable Prognostic parameters in UM were analyzed using univariate and multivariate Cox regressionDeath from all causesParametersnmeanUnivariate analysisPHR95CIAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelioid cell dominantSpindle cell dominantExtrascleral extensionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVSNHG15HighLowUM Uveal melanomaMultivariate analysisPHR95CIdemonstrated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group Figure 1A P005 As shown in Figure 1B1D SNHG15 was statistically differentially expressed in diverse groups of the tumor pathologic stage stage II vs IIIIV P00257 metastasis P00071 living status P00017 To clarify the clinicopathologic impact of SNHG15 we also used logistic regression and concluded that the SNHG15 expression based on median value of FPKM as a categorical variable was statistically related to clinicopathologic features Table High SNHG15 expression was significantly related to cancer status pathologic stage metastasis living status in UM all P005 Table These results demonstrated that UM with high SNHG15 expression were prone to progress to cancer status of survival with tumor a more advanced stage metastasis and poor living status when compared to the low SNHG15 expression group However there was no statistically significant difference in age gender tumor diameter thickness pathological type extrascleral invasionMain Enriched Pathways in UM Tissues with High SNHG15 Expression To explore the SNHG15related potential signaling pathways activated in UM GSEA was performed In the current study based on the association with SNHG15 expression the gene list was generated firstly Figure The SNHG15 expression was associated with clinical pathological characteristics A Patients with high SNHG15 expression had a shorter OS when compared with the low SNHG15 expression group P002 BD The expression of SNHG15 was statistically different in diverse groups of the tumor pathologic stage P00257 metastasis P00071 living status P00017 aP005 bP001by GSEA To clarify the statistically significant differences between high and low SNHG15 expression groups GSEA was Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaTable Association between SNHG15 expression and clinicopathologic variables using logistic regressionParametersAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelialNonepithelialExtrascleral invasionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVMetastasesNoYesLiving statusAliveDeadnmeanSNHG15 expressionLowHighPOR95CINESNominal PvalESTable Enriched pathways for differential SNHG15 expression in UMNameSpliceosomeCell cyclePyrimidine metabolismDNA replicationNucleotide excision repairRNA degradationHomologous recombinationMismatch repairUM Uveal melanoma ES Enrichment score NES Normal enrichment score FDR False discovery rateFDR Qvalconducted subsequently The results indicated that there were significant differences in spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair among patients with high SNHG15 expression phenotype Figure Table DISCUSSIONAccumulating evidences indicate that SNHG15 plays a dual role in the tumorigenesis and development of different tumors[] Previously SNHG15 has been demonstrated as a carcinogenic lncRNA which is usually upregulated in tumor tissues compared with normal tissues[] It exerts 0cFigure Enrichment plots from GSEA Spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair are enriched significantly in SNHG15 high expression phenotypean oncogenic effect via various epigenetic mechanisms[] For example it can suppress the expression of miR3383p and facilitate the proliferation of colorectal cancer cells[] It plays a carcinogenic role by affecting miR3383pFKBP1A axis in prostate cancer[] It can also enhance hepatocellular carcinoma progression by negative regulation of miR1413p[] However there are reports that SNHG15 has a tumor suppressive effect suggesting that low SNHG15 expression is related to poor prognosis in thyroid cancer and upregulating expression of SNHG15 can significantly suppress cell proliferation[] At present the impact of SNHG15 on UM is still unclear Therefore vital roles and potential biological mechanism of SNHG15 in UM needs to be elucidated In this study we revealed that high SNHG15 expression was related to clinicopathologic features in UM Through RNASeq gene expression level and clinicopathological characteristics obtained from the TCGA UM project we analyzed the relationship among SNHG15 expression clinicopathological features and prognosis of UM The univariate analysis demonstrated that SNHG15 expression level age tumor diameter pathological type extrascleral extension and cancer status were risk factors for death from all causes The multivariate analysis suggested that high SNHG15 expression along with age and pathological type was an independent risk factor for death from all causes Therefore the results demonstrated that high SNHG15 expression was an independent predictor of poor prognosis in UM through univariate and multivariate analysis KaplanMeier survival analysis also indicated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group in UM In addition an analysis was conducted to further explore the relationship between SNHG15 and clinicopathological features The SNHG15 expression was statistically different in diverse groups of the tumor pathologic stage metastasis and living status Besides high SNHG15 expression based on median expression value of FPKM in UM was associated with cancer status of survival with tumor advanced pathologic stage metastasis and living status It demonstrated that high SNHG15 expression in UM was strongly related to poor prognosis The mechanisms of SNHG15 dysregulation in malignant tumors are quite complex and are far from being completely understood Previous studies have suggested that SNHG15 is involved in diverse pathological and physiological processes of many tumors through their abnormal expressions including cell proliferation invasion migration and autophagy[] To explore the biological mechanism of SNHG15 in UM GSEA was conducted It indicated that spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair were all enriched differentially in SNHG15 high expression phenotype Alternative splicing is essential for gene regulation and abnormal splicing plays a vital role in inactivating tumor suppressor genes or activating oncogenes[] SNHG15 may have an impact on the invasion Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaand migration of UM cells by affecting spliceosomal related factors The abnormal cell proliferation of tumor is related to the lack of checkpoint control over the cell cycle which is the basis of genetic instability[] Evidence shows that the lack of homologous recombination may facilitate the disturbance of cell cycle the instability and accumulated mutations of genome during the progression and development[] Mismatch repair proteins have an significant role in DNA hypermethylation alteration and tumorigenesis[] SNHG15 is closely related to DNA replication and mismatch repair demonstrating that SNHG15 may promote the occurrence of UM by affecting DNA replication and DNA mismatch repair It indicated that SNHG15 may be identified as a novel marker of diagnosis therapeutic and prognosis prediction in UM However the related mechanism needs to be further elucidated This research also has some limitations The most important one is the limited number of patients and time of followup In addition some patient characteristics such as ciliary body involvement were not completely recorded in the database In fact ciliary body involvement plays a critical role in UM[]In conclusion this study aims to demonstrate the vital role of SNHG15 in UM and the potential relationship between SNHG15 expression and clinical parameters SNHG15 expression may be a valuable biomarker for poor survival in UM Moreover we have preliminarily explored the crucial pathway associated with SNHG15 in UM However further experimental validation is needed to be performed for clarifying the significant impact of SNHG15 And it is of great significance to further identify its independent prognostic value in a largescale standardized researches on UMACKNOWLEDGEMENTSFoundations Supported by the National Natural Science Foundation of China No81970835 No81800867 Conflicts of Interest Wu X None Li XF None Wu Q None Ma RQ None Qian J None Zhang R NoneREFERENCES van Raamsdonk CD Griewank KG Crosby MB Garrido MC Vemula S Wiesner T Obenauf AC Wackernagel W Green G Bouvier N Sozen MM Baimukanova G Roy R Heguy A Dolgalev I Khanin R Busam K Speicher MR O€™Brien J Bastian BC Mutations in GNA11 in uveal melanoma N Engl J Med Carvajal RD Sosman JA Quevedo JF Milhem MM Joshua AM Kudchadkar RR Linette GP Gajewski TF Lutzky J Lawson DH Lao CD Flynn PJ Albertini MR Sato T Lewis K Doyle A Ancell K Panageas KS Bluth M Hedvat C Erinjeri J Ambrosini G Marr B Abramson DH Dickson MA Wolchok JD Chapman PB Schwartz GK Effect of selumetinib vs chemotherapy on progressionfree survival in uveal melanoma a randomized clinical trial JAMA Shain AH Bagger MM Yu R Chang D Liu SS Vemula S Weier JF Wadt K Heegaard S Bastian BC Kiilgaard JF The genetic evolution of metastatic uveal melanoma Nat Genet Bagger M SmidtNielsen I Andersen MK Jensen PK Heegaard S Andersen KK Friis S Kiilgaard JF Longterm metastatic risk after biopsy of posterior uveal melanoma Ophthalmology Kujala E Mäkitie T Kivelä T Very longterm prognosis of patients with malignant uveal melanoma Invest Ophthalmol Vis Sci Chandran SS Somerville RPT Yang JC Sherry RM Klebanoff CA Goff SL Wunderlich JR Danforth DN Zlott D Paria BC Sabesan AC Srivastava AK Xi LQ Pham TH Raffeld M White DE Toomey MA Rosenberg SA Kammula US Treatment of metastatic uveal melanoma with adoptive transfer of tumourinfiltrating lymphocytes a singlecentre twostage singlearm phase study Lancet Oncol Mendell JT Targeting a long noncoding RNA in breast cancer N Engl J Med Lan Y Xiao XW He ZC Luo Y Wu CF Li L Song X Long noncoding RNA OCC1 suppresses cell growth through destabilizing HuR protein in colorectal cancer Nucleic Acids Res Cao CH Sun JY Zhang DY Guo XJ Xie LW Li X Wu DH Liu L The long intergenic noncoding RNA UFC1 a target of microRNA 34a interacts with the mRNA stabilizing protein HuR to increase levels of βcatenin in HCC cells Gastroenterology 20151482415426e18 Wang P Xue YQ Han YM Lin L Wu C Xu S Jiang ZP Xu JF Liu QY Cao XT The STAT3binding long noncoding RNA lncDC controls human dendritic cell differentiation Science Zheng XL Tang HW Zhao XF Sun YM Jiang YF Liu YH Long noncoding RNA FTH1P3 facilitates uveal melanoma cell growth and invasion through miR2245p PLoS One 20171211e0184746 Lu QK Zhao N Zha GP Wang HY Tong QH Xin SH LncRNA HOXA11AS exerts oncogenic functions by repressing p21 and miR in uveal melanoma DNA Cell Biol Lu LN Yu XY Zhang LL Ding X Pan H Wen XY Xu SQ Xing Y Fan JY Ge SF Zhang H Jia RB Fan XQ The long noncoding RNA RHPN1AS1 promotes uveal melanoma progression Int J Mol Sci Wu S Chen H Han N Zhang CX Yan HT Long noncoding RNA PVT1 silencing prevents the development of uveal melanoma by impairing MicroRNA173pdependent MDM2 upregulation Invest Ophthalmol Vis Sci Li P He J Yang Z Ge SF Zhang H Zhong Q Fan XQ ZNNT1 long noncoding RNA induces autophagy to inhibit tumorigenesis of uveal melanoma by regulating key autophagy gene expression Autophagy Dong YZ Meng XM Li GS Long noncoding RNA SNHG15 indicates poor prognosis of nonsmall cell lung cancer and promotes 0ccell proliferation and invasion Eur Rev Med Pharmacol Sci SNHG15 serves as an oncogene and predicts poor prognosis in epithelial ovarian cancer Onco Targets Ther Liu K Hou Y Liu YK Zheng J LncRNA SNHG15 contributes to proliferation invasion and autophagy in osteosarcoma cells by sponging miR141 J Biomed Sci Wu DM Wang S Wen X Han XR Wang YJ Shen M Fan SH Zhang ZF Shan Q Li MQ Hu B Lu J Chen GQ Zheng YL LncRNA SNHG15 acts as a ceRNA to regulate YAP1Hippo signaling pathway by sponging miR200a3p in papillary thyroid carcinoma Cell Death Dis Guo XB Yin HS Wang JY Evaluating the diagnostic and prognostic value of long noncoding RNA SNHG15 in pancreatic ductal adenocarcinoma Eur Rev Med Pharmacol Sci Sun XT Bai Y Yang C Hu SY Hou ZL Wang GX Long noncoding RNA SNHG15 enhances the development of colorectal carcinoma via functioning as a ceRNA through miR141SIRT1Wntβcatenin axis Artif Cells Nanomed Biotechnol Ye JF Tan LD Fu Y Xu HJ Wen LJ Deng Y Liu K LncRNA SNHG15 promotes hepatocellular carcinoma progression by sponging miR1413p J Cell Biochem Zhang JH Wei HW Yang HG Long noncoding RNA SNHG15 a potential prognostic biomarker for hepatocellular carcinoma Eur Rev Med Pharmacol Sci Zhang YL Zhang DH Lv J Wang S Zhang Q LncRNA SNHG15 Acts as an oncogene in prostate cancer by regulating miR3383pFKBP1A axis Gene Kong QL Qiu M Long noncoding RNA SNHG15 promotes human breast cancer proliferation migration and invasion by sponging miR2113p Biochem Biophys Res Commun Wang TQ Sun HB Bao Y En R Tian YJ Zhao W Jia LZ POM121 overexpression is related to a poor prognosis in colorectal cancer Expert Rev Mol Diagn Shuai Y Ma ZH Lu JW Feng JF LncRNA SNHG15 a new budding star in human cancers Cell Prolif 2020531e12716 Qu C Dai CM Guo YH Qin R Liu JB Long noncoding RNA Ma YW Xue YX Liu XB Qu CB Cai H Wang P Li ZQ Li Z Liu YH SNHG15 affects the growth of glioma microvascular endothelial cells by negatively regulating miR153 Oncol Rep Li M Bian ZH Jin GY Zhang J Yao SR Feng YY Wang X Yin Y Fei BJ You QJ Huang ZH LncRNASNHG15 enhances cell proliferation in colorectal cancer by inhibiting miR3383p Cancer Med Liu YC Li JL Li F Li M Shao Y Wu LP SNHG15 functions as a tumor suppressor in thyroid cancer J Cell Biochem Liu YC Li JL Li M Li F Shao Y Wu LP microRNA5105p promotes thyroid cancer cell proliferation migration and invasion through suppressing SNHG15 J Cell Biochem Li YW Guo HY Jin CJ Qiu CP Gao M Zhang L Liu ZJ Kong BH Spliceosomeassociated factor CTNNBL1 promotes proliferation and invasion in ovarian cancer Exp Cell Res Williams GH Stoeber K The cell cycle and cancer J Pathol Yu B Ding YM Liao XF Wang CH Wang B Chen XY Overexpression of PARPBP correlates with tumor progression and poor prognosis in hepatocellular carcinoma Dig Dis Sci Maiuri AR Peng M Podicheti R Sriramkumar S Kamplain CM Rusch DB DeStefano Shields CE Sears CL O€™Hagan HM Mismatch repair proteins initiate epigenetic alterations during inflammationdriven tumorigenesis Cancer Res Berry D Seider M Stinnett S Mruthyunjaya P Schefler AC Ocular Oncology Study Consortium Relationship of clinical features and baseline tumor size with gene expression profile status in uveal melanoma a Multiinstitutional study Retina Jiang ZM Yu FH Li M Upregulation of BCL2 kD proteininteracting protein BNIP3 is predictive of unfavorable prognosis in uveal melanoma Med Sci Monit Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0c'

Thyroid_Cancer

Millions of people are suffering from cancers but accurate early diagnosis and effectivetreatment are stilllong noncoding RNAslncRNAs have been proven to play an important role in diseases especially cancersThese lncRNAs execute their functions by regulating gene expression Thereforeidentifying lncRNAs which are related to cancers could help researchers gain a deeperunderstanding of cancer mechanisms and help them find treatment options A largenumber of relationships between lncRNAs and cancers have been verified by biologicalexperiments which give us a chance to use computational methods to identifycancerrelated lncRNAs In this paper we applied the convolutional neural network CNNto identify cancerrelated lncRNAs by lncRNA™s target genes and their tissue expressionspecificity Since lncRNA regulates target gene expression and it has been reportedto have tissue expression specificity their target genes and expression in differenttissues were used as features of lncRNAs Then the deep belief network DBN wasused to unsupervised encode features of lncRNAs Finally CNN was used to predictcancerrelated lncRNAs based on known relationships between lncRNAs and cancersFor each type of cancer we built a CNN model to predict its related lncRNAs Weidentified more related lncRNAs for kinds of cancers Tencross validation has beenused to prove the performance of our method The results showed that our method isbetter than several previous methods with area under the curve AUC and areaunder the precision“recall curve AUPR To verify the accuracy of our results casestudies have been doneKeywords long noncoding RNA lncRNA cancer convolutional neural network CNN deep belief network DBNmachine learningINTRODUCTIONFour to nine percent of the sequences™ transcription are long noncoding RNAs lncRNAs inmammalian genomes Canzio Ji lncRNA was regarded as the noise ofgenome transcription and did not have biological functions at first However an increasing numberof studies have reported that lncRNA is widely Robinson involved in chromosomeEdited byLei DengCentral South University ChinaReviewed byHao LinUniversity of Electronic Science andTechnology of China ChinaInner Mongolia University ChinaJuan WangCorrespondenceNan Dudunan05aliyuncomGanfeng Xiexiegfaliyuncom These authors share first authorshipSpecialty sectionThis was submitted toMolecular Medicinea section of the journalFrontiers in Cell and DevelopmentalBiologyReceived June Accepted June Published August CitationLiu Z Zhang Y Han X Li C Yang XGao J Xie G and Du N Identifying CancerRelated lncRNAsBased on a Convolutional NeuralNetwork Front Cell Dev Biol 103389fcell202000637Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsgenomicimprintingchromatin modificationsilencingtranscriptional activationinterference andnuclear transport Cheng 2018a Recently it has beenproven to be associated with many kinds of cancerstranscriptionalThe secondary structure spliced form and subcellularlocalization of most lncRNAs are conserved Karner which is very important for lncRNA to execute functionsHowever compared to the functions of microRNAs miRNAsand proteins the function oflncRNA is more difficult todetermine According to the position of lncRNA in the genomerelative to proteincoding genes it can be divided into five typessense antisense bidirectional intronic and intergenicMany researchers have found lncRNAs play an important rolein cancers Avgeris Cheng 2018b Zhao and neurodegenerative diseases Peng and Zhao as other biological molecules Zhang T Bai Cheng 2019a Liang Although manyresearchers have verified many associations between lncRNAsand cancers by biological experiments compared with ourknowledge about diseaserelated genes we still do not knowenough about diseaserelated lncRNAs Considering the timeand money cost of finding diseaserelated lncRNAs more andmore researchers tend to use computational methods to identifydiseaserelated lncRNAs These methods could be divided intothree categories machine learning methods network methodsand other methodsMachine learning methods build models based on thesimilarities of diseases orlncRNAs and their biologicalcharacteristics Cheng Cheng 2019b Zeng Zou Lan developed thelncRNA“disease association prediction LDAP which is amethod based on bagging support vector machine SVM toidentify lncRNA“disease associations They used similarities oflncRNAs and diseases as the features Yu developedcollaborative filtering naive Bayesian classifier CFNBC based onnaive Bayesian They integrated miRNA“lncRNA associationsmiRNA“disease associations and lncRNA“disease associationsto infer more lncRNA“disease associations Considering thediscriminative contributions of the similarity association andinteraction relationships among lncRNAs disease and miRNAsXuan 2019a developed a dual convolutional neuralnetwork CNN with attention mechanisms to predict diseaserelated lncRNAsNetwork methods are the most common way to identifyassociations between diseases and lncRNAs nowadays Gu Yu Zhang J Kuang Wang L Liu Thiskind of method would build one or multiple networks toinfer new information Wang L built a lncRNA“miRNA“disease interactive network and used their novel methodœLDLMD to predict associations between lncRNAs and diseasesSumathipala used a multilevel network topologywhich includes lncRNA“protein protein“protein interactionprotein“disease relationship to use network diï¬usion algorithmto predict diseaserelated lncRNAs The graph convolutionalnetwork GCN and CNN were used on a lncRNA“miRNA“disease network by Xuan 2019b Deng builtlncRNA similarity network disease similarity network miRNAsimilarity network and their associations Then they calculatedthe metapath and feature vector for each lncRNA“disease pair inthe heterogeneous information networkOther methods may borrow the feature extraction methodor similarity conjecture of network methods but the core ofthis method is matrix decomposition or matrix completionLu developed the geometric matrix completionlncRNA“disease association GMCLDA which is a methodbased on geometric matrix completion They calculated diseasesimilarity based on Disease Ontology DO and calculatedthe Gaussian interaction profile kernel similarity for lncRNAsThen they inferred diseaserelated lncRNAs based on theassociation patterns among functionally similar lncRNAs andsimilar diseases Wang Y proposed a weightedmatrix factorization to capture the interintraassociationsbetween diï¬erent types of nodes Then they approximated thelncRNA“disease association matrix using the optimized matricesand weights to predict diseaserelated lncRNAs Localityconstrained linear coding label propagation Latent DirichletAllocation LLCLPLDA was developed by Xie Firstly localconstraint features of lncRNAs and diseases wereextracted by localityconstrained linear coding LLC Thenthey predicted diseaserelated lncRNAs by label propagationLP strategyHowever previous methods did not consider the regulatingtarget gene expression of lncRNA which is an important functionof lncRNA and plays an important role in associations betweenlncRNAs and diseases In addition deep learning methods arean important tool and have shown their power in bioinformaticsChen Lv Wei Wu Zhao 2019abc Therefore in this paper we used thisinformation as features of lncRNA In addition the expressionof lncRNA in diï¬erent tissues were also used as the featuresof lncRNA Then the deep belief network DBN was used toencode and the CNN was used to classifyMETHODSFeature ExtractionTissue Expression Specificity of Long NoncodingRNACompared with proteincoding geneslncRNA shows strongtissue specificity The specificity of lncRNAs in diï¬erent kindsof tissues and cell types has been proven by many biologicalexperiments The diï¬erent expression also plays an importantrole in essential cellular processes Sasaki testedthe expression of lncRNAs in diï¬erent tissues and found lncRNAs exhibited tissuespecific expression and oflncRNAs were only expressed in one discrete tissue Thereforethe expression of lncRNAs in diï¬erent tissues were used asthe featuresWe obtained the expression of lncRNAs in diï¬erenttissues which included adipose adrenal breast colon heartkidney liver lung lymph node ovary placenta prostate testisand thyroidTherefore the dimension of each lncRNA™s expression featureis ˆ— Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsTherefore the dimension of each lncRNA™s target gene featureis ˆ— Deep Belief NetworkThe DBN can eï¬ectively learn complex dependencies betweenvariables Zhao 2019d The DBN contains many layers ofhidden variables which can eï¬ectively learn the internal featurerepresentation of the data and can also be used as an eï¬ectivenonlinear dimensionality reduction methodWhen the observable variables are known the joint posteriorprobabilities of the hidden variables are no longer independentof each other so it is difficult to accurately estimate the posteriorprobabilities of all hidden variables The posterior probability ofearly DBN is generally approximated by Monte Carlo methodbut its efficiency is relatively low which makes its parameterlearning difficult In order to eï¬ectively train the DBN weconvert the sigmoid belief network of each layer to a restrictedBoltzmann machine RBM The advantage of this is that theposterior probabilities of the hidden variables are independentof each other which makes it easy to sample In this way theDBN can be regarded as being stacked from top to bottom bymultiple RBMs and the hidden layer of the Lth RBM is used asthe observable layer of the L 1th RBM Further the DBN canbe trained quickly by layerbylayer training that is starting fromthe bottom layer and training only one layer at a time until thelast layer The specific layerbylayer training process is to trainthe RBM of each layer in turn from bottom to top Assuming wehave trained the RBM in the first L1 layer we can calculate theconditional probability of the bottomup hidden variablesphihiˆ’ σ bi Wihiˆ’where bi is the bias of ith layer of RBM Wi is the connectionweight hi is the ith layer of RBMThe process of training DBN is as followsFIGURE The number of target genes for each long noncoding RNAlncRNAFIGURE The distribution of the number of target genes lncRNA longnoncoding RNAreverseTarget Gene of Long Noncoding RNAQuantitativechainreaction qRTPCR and Western blot were used to testthe diï¬erentexpression genes after knocking down oroverexpressing lncRNAstranscriptasepolymeraseWe obtained target genes of lncRNA from LncRNA2TargetInput train dataset ˆvn learning rate λJiang As we can see in Figure there are kinds of lncRNAsOne lncRNA has more than target genes Then we drawthe distribution of the number of target genes correspondingto lncRNAAsshown in Figure most ofthe target genes arecorresponding to less than five lncRNAs Therefore if we usedthem to be the features of lncRNAs the features would be sparseTherefore we only select the most common target genes to bethe features The genes which are corresponding to more thanfive lncRNAs were selected as the features of lncRNAs There are kinds of genes Then we need to encode these genesF [G1 G2 · · · G45]where G1 denotes the first gene of these genes and F denotesthe feature of lncRNA For each lncRNA if G1 is the target geneof it then G1 otherwise G1 Output weight matrix Wl bias al and blFor l 1LInitialization Wi al bi Sample from train dataset ˆh0For i lˆ’Sample hi based on phi ˆhiˆ’EndSet hi1as the train sample to train lth layer ofRBMEndSince the dimension of expression feature and target genefeature are diï¬erent we should reduce the dimension of targetgene feature and make it the same as the expression feature™sTherefore in this paper two layers of RBM were used to builda DBN modelThe number of nodes oftheand respectively Sigmoid function was used astwo layers was theFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alactivation functionσ x eˆ’xTherefore the dimension of final features is ˆ— F cid20 G1 G2 · · · G13E1 E2 · · · E13 cid21A Method to Identify CancerRelated lncRNAsConvolutional Neural NetworkThe power of CNN in dealing with bioinformatic problems hasbeen proven by many researchers We selected CNN as theclassifier based on two reasons The dimension of features is ˆ— which can be regarded as an image The outstandingperformance of CNN in image classificationThere are five layers in our CNN model The structure of CNNis shown as Table where G1 G2 · · · G13 denotes target gene feature after DBNand E1 E2 · · · E13 denotes the expression of lncRNAs in diï¬erent tissuesTABLE The structure of convolutional neural network CNNLayersParameterConvolutional layerPooling layerConvolutional layerPooling layerFully connected layerOutputFilter kernel size Activation function tanhpool size Activation function tanhFilter kernel size Activation function tanhpool size Activation function tanhUnits Activation function tanhUnits Activation function sigmoidWork FrameFigure shows the work frame of our method œDBN“CNNThere are three steps of our methods Firstly we should extractfeatures of lncRNAs There are two parts of features expressionfeature and target gene feature Then DBN was used to encodethe target gene feature After encoding the two kinds of featureswere combined together Finally CNN was used to classifyRESULTSData DescriptionThe known associations between lncRNA and diseases wereobtained from LncRNADisease database Bao Wetotally obtained kinds of cancerrelated lncRNAs The numberof their corresponding lncRNAs is shown as Figure As shown in Figure People™s understanding of cancerrelated lncRNAs varies widely We have known more than lncRNAs for some cancers but few lncRNAs are known for somecancers To better build our model we only selected cancerswhich have more than related lncRNAs Therefore kindsof cancers were selectedFIGURE Work frame of deep belief network DBN“convolutional neural network CNN lncRNA long noncoding RNAFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsFIGURE The number of long noncoding RNAs lncRNAs for each cancerTABLE The performance of deep belief network DBN“convolutional neuralnetwork CNN in cancersCancerArea undercurve AUCArea under precisioncurve AUPRCervical cancerBreast cancerColorectal cancerStomach cancerUrinary bladder cancerLung cancerOvarian cancerThyroid cancerProstate cancerLiver cancerPancreatic cancerOvarian epithelial cancerGallbladder cancerEndometrial cancerColon cancerEsophageal cancerThetargetgenes oflncRNAs were obtained fromLncRNA2Target database We have discussed about this insection Target Gene of Long Noncoding RNAFIGURE The receiver operating characteristic ROC curves of the threemethods DBN deep belief network CNN convolutional neural network PCAprincipal component analysisFIGURE The area under the precision“recall curve AUPR of the threemethods DBN deep belief network CNN convolutional neural network PCAprincipal component analysisThe expression oftissues wasobtained from NONCODEV5 Zhao We only usedhuman datalncRNAs in diï¬erentThe Performance of Deep BeliefNetwork“Convolutional Neural NetworkWe did 10cross validation on each cancer Area under the curveAUC Cheng Dao Zhang and areaunder the precision“recall curve AUPR were used to evaluatethe performance of DBN“CNN The results are shown in Table As we can see in Table the performance of DBN“CNN isquite diï¬erent in diï¬erent cancers This may be caused by thediï¬erent sample sizes The average AUC is and AUPR is Comparison ExperimentsTo verify the superior of DBN“CNN we compared it with similarmethods Since the main function of DBN is to reduce dimensionprincipal component analysis PCA has the same functionTherefore instead of using DBN to encode we used PCA thistime and CNN was used to classify the features after PCA We callthis method PCA“CNN In addition we also used the deep neuralnetwork DNN to replace CNN so this comparison method wascalled DBN“DNNWe used these three methods to test on cancers andsummarized the results to get a final AUC and AUPR for eachmethod The receiver operating characteristic ROC curves areshown in Figure As shown in Figure the blue curve denotes the results ofDBN“CNN The red and black curves denote PCA“CNN andDBN“DNN respectively As we can see DBN“CNN performedbest among these three methods The AUC of DBN“CNN is which is better than and for PCA“CNN andDBN“DNN respectivelyFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsAs shown in Figure the AUPR of DBN“CNN is the highestwith the least standard errorCase StudyLiu found down syndrome cell adhesion molecule antisense RNA DSCAMAS1 is associated with breast cancerby constructing two suppression subtracted cDNA librariesMartensUzunova reported the associationbetween H19 and bladder cancer They also pointed out that H19could be the biomarker of bladder cancerShi measured the expression level of lncRNAsLoc554202 in breast cancer tissues and found that Loc554202was significantly increased compared with normal control andassociated with advanced pathologic stage and tumor sizeCONCLUSIONSIncreasing evidence has shown the relationship between lncRNAsand cancers lncRNAs could be the biomarkers to help diagnosecancer and also help researchers understand the mechanismof cancers Compared with people™s knowledge of diseaserelated protein coding genes we knew few about diseaserelated lncRNAs However the biological experiments for findingdiseaserelated lncRNAs are timeconsuming and expensiveTherefore in this paper we proposed a novel method foridentifying cancerrelated lncRNAs We called this methodœDBN“CNN which is a fusion of DBN and CNN Two kindsof features were used based on the biological background SincelncRNAs have tissuespecific expression and the expression ofcancer tissues is diï¬erent from normal tissues the expressionoftissues could provide importantin diï¬erentlncRNAsREFERENCESAvgeris M Tsilimantou A Levis P K Tokas T Sideris D C StravodimosK Loss of GAS5 tumour suppressor lncRNA an independentmolecular cancer biomarker for shortterm relapse and progression in bladdercancer patients Br J Cancer “ 101038s4141601803206Bai Y Dai X Ye T Zhang P Yan X Gong X PlncRNADBa repository of plant lncRNAs and lncRNARBP protein interactions CurrBioinform “ Bao Z Yang Z Huang Z Zhou Y Cui Q and Dong D LncRNADisease an updated database of long noncoding RNAassociateddiseases Nucleic Acids Res D1034“D1037 101093nargky905Canzio D Nwakeze C L Horta A Rajkumar S M Coï¬ey E L Duï¬y EE Antisense lncRNA transcription mediates DNA demethylationto drive stochastic protocadherin α promoter choice Cell “653e15 101016jcell201903008Chen X Shi W and Deng L Prediction of disease comorbidity usinghetesim scores based on multiple heterogeneous networks Curr Gene Ther “ Cheng L Computational and biological methods for gene therapy CurrGene Ther Cheng L Hu Y Sun J Zhou M and Jiang Q 2018a DincRNA afor exploring disease“comprehensive webbased bioinformaticsassociations 101093bioinformaticsbty002ncRNA functionBioinformaticstoolkitandCheng L Jiang Y Ju H Sun J Peng J Zhou M 2018busingcrossontologyInfAcrOntsimilaritiescalculatingtermtheirexecutelncRNAsinformation for us to identify cancerrelated lncRNAs Inadditionregulation function byinteracting with their target genes Therefore the target genesof lncRNAs can also be the features of lncRNAs To encode thefeatures DBN was used to reduce the dimension Finally CNNwas used to identify real cancerrelated lncRNAs based on thefinal featureTo verify the eï¬ectiveness of our method we comparedDBN“CNN with PCA“CNN and DBN“DNN since PCA canalso reduce the dimension of features and DNN can also doclassification The results showed that DBN“CNN performedbest Finally case studies have been done to verify the accuracy ofour results We found potential lncRNAs for kinds of cancerswhich can be a kind of guidance for researchers finding novelcancerrelated lncRNAsDATA AVAILABILITY STATEMENTThe datasets presented in this study can be found in onlinerepositoryrepositoriesrepositories Theandnumbersbethesupplementary materialaccessionnamesfoundcantheofinAUTHOR CONTRIBUTIONSND and GX designed the research ZL performed the researchand wrote the manuscript YZ and XH acquired the dataand reviewed and edited the manuscript CL XY and JGanalyzed the data All authors reviewed the manuscript andprovided commentsinformation flow by a random walk BMC Genomics 19Suppl 101186s1286401743386Cheng L Yang H Zhao H Pei X Shi H Sun J 2019a MetSigDisa manually curated resource for the metabolic signatures of diseases BriefBioinform “ 101093bibbbx103Cheng L Zhao H Wang P Zhou W Luo M Li T 2019bComputational Methods for identifying similar diseases molecular therapyNucleic Acids “ 101016jomtn201909019Dao F Y Lv H Zulfiqar H Yang H Su W Gao H Acomputational platform to identify origins of replication sites in eukaryotesBrief Bioinform 101093bibbbaa017 [Epub ahead of print]Deng L Li W and Zhang J LDAH2V Exploring metapaths acrossmultiple networks for lncRNAdisease association prediction IEEEACMTransac Comput Biol Bioinform 101109TCBB20192946257 [Epubahead of print]Gu C Liao B Li X Cai L Li Z Li K Global network randomwalk for predicting potential human lncRNAdisease associations Sci Rep 101038s4159801712763zJiJ TangJ Xia KJandJiang Rtumorigenesis microenvironment CurrBioinformLncRNA in“Jiang Q Wang J Wu X Ma R Zhang T Jin S LncRNA2Targeta database for diï¬erentially expressed genes after lncRNA knockdown oroverexpression Nucleic Acids Res D193“D196 101093nargku1173Karner H Webb CH Carmona S Liu Y Lin B Erhard M Functional conservation of lncRNA JPX despite sequence and structuraldivergence J Mol Biol “ 101016jjmb201909002Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsKuang L Zhao H Wang L Xuan Z and Pei T A novel approachbased on point cut set to predict associations of diseases and LncRNAs CurrBioinform “ Lan W Li M Zhao K Liu J Wu FX Pan Y LDAP a webserver for lncRNAdisease association prediction Bioinformatics “ 101093bioinformaticsbtw639Liang C Changlu Q He Z Tongze F and Xue Z gutMDisorder acomprehensive database for dysbiosis of the gut microbiota in disorders andinterventions Nucleic Acids Res Liu D Rudland P Sibson D and Barraclough R Identification ofmRNAs diï¬erentiallyexpressed between benign and malignant breast tumourcells Br J Cancer “ 101038sjbjc6600456Liu X Hong Z Liu J Lin Y Alfonso RP Zou Q Computational methods for identifying the critical nodes in biologicalnetworks Brief Bioinform “ 101093bibbbz011Lu C Yang M Li M Li Y Wu F and Wang J Predicting humanlncRNAdisease associations based on geometric matrix completion IEEE JBiomed Health Inform 101109JBHI20192958389 [Epub ahead of print] Protein function predictionto deep learning Proteomics 19e1900119Lv Z B Ao C Y and Zou Qfrom traditionalclassifier 101002pmic201900119MartensUzunova E S Böttcher R Croce C M Jenster G Visakorpi T andCalin G A Long noncoding RNA in prostate bladder and kidneycancer Eur Urol “ 101016jeururo201312003Peng J and Zhao T Reduction in TOM1 expression exacerbatesAlzheimer™s disease Proc Natl Acad Sci USA “ 101073pnas1917589117Robinson E K Covarrubias S and Carpenter S The how and why oflncRNA function an innate immune perspective Biochim Biophys Acta GeneRegul Mech 101016jbbagrm2019194419Sasaki Y T Sano MIdeue T Kin T Asai K and Hirose T Identification and characterization of human noncoding RNAs withtissuespecific expression Biochem Biophys Res Commun “ 101016jbbrc200704034Sumathipala M Maiorino E Weiss S T and Sharma AShi Y Lu J Zhou J Tan X He Y Ding J Long noncodingRNA Loc554202 regulates proliferation and migration in breast cancer cellsBiochem Biophys Res Commun “ 101016jbbrc201402144Network diï¬usion approach to predictlncRNA disease associationsusing multitype biological networks LION Front Physiol 103389fphys201900888Wang L Xuan Z Zhou S Kuang L and Pei T A novel modelassociations based on the LncRNA“for predicting LncRNAdiseaseMiRNAdisease interactive network Curr BioinformWang Y Yu G Wang J Fu G Guo M and Domeniconi C Weightedmatrix factorization on multirelational data for LncRNAdisease associationprediction Methods “ 101016jymeth201906015Wei L Su R Wang B Li X Zou Q and Gao X Integrationof deep feature representations and handcrafted featuresto improvethe prediction of N 6methyladenosine sites Neurocomputing “ 101016jneucom201804082Wu B Zhang H Lin L Wang H Gao Y Zhao L A similarity searching system for biological phenotype images using deepconvolutional encoderdecoder architecture Curr Bioinform “ Xie G Huang S Luo Y Ma L Lin Z and Sun Y LLCLPLDA a novelmodel for predicting lncRNA“disease associations Mol Genet Genomics “ 101007s00438019015908Xuan P Cao Y Zhang T Kong R and Zhang Z2019a Dualconvolutional neural networks with attention mechanisms based methodfor predicting diseaserelated lncRNA genes Front Genet 103389fgene201900416Xuan P Pan S Zhang T Liu Y and Sun H 2019b Graph convolutionalnetwork and convolutional neural network based method for predictinglncRNAdisease associations Cells 103390cells8091012Yu G Fu G Lu C Ren Y and Wang J BRWLDA birandomwalks for predicting lncRNAdisease associations Oncotarget “ 1018632oncotarget19588Yu J Xuan Z Feng X Zou Q and Wang L A novel collaborativefiltering model for LncRNAdisease association prediction based on the NaïveBayesian classifier BMC Bioinform 101186s1285901929850Zeng X X Wang W Deng G S Bing J X and Zou Q Prediction ofpotential diseaseassociated microRNAs by using neural networks Mol TherNucleic Acids “ 101016jomtn201904010Zhangand Deng LJ Zhang Z Chen ZIntegratinglncRNAdisease associationIEEEACM Transac Comput Biol Bioinform “multiple heterogeneous networks for novelinference 101109TCBB20172701379Zhang T Tan P Wang L Jin N Li Y Zhang L RNALocate aresource for RNA subcellular localizations Nucleic Acids Res D135“D138 101093nargkw728Zhang Z M Tan J X Wang F Dao F Y Zhang Z Y and LinH Early diagnosis of hepatocellular carcinoma using machinelearning method Front Bioeng Biotechnol 103389fbioe2020Zhao T Cheng L Zang T and Hu Y 2019a Peptidemajor histocompatibilitycomplex class I binding prediction based on deep learning with novel featureFront Genet 103389fgene201901191and Cheng LIdentifyingAlzheimer™s diseaserelated proteins by LRRGD BMC Bioinform 101186s1285901931247Zhao T Hu Y Zang T2019bZhao T Hu Y Zang T and Cheng L MRTFB regulates the expressionof NOMO1 in colon Proc Natl Acad Sci USA 101073pnas2000499117Zhao T Hu Y Zang T and Wang Y 2019c Integrate GWAS eQTLand mQTL Data to Identify Alzheimer™s diseaserelated genes Front Genet 103389fgene201901021Zhao T Wang D Hu Y Zhang N Zang T and Wang Y 2019d IdentifyingAlzheimer™s diseaserelated miRNA based on semiclustering Curr Gene Ther “ Zhao Y Li H Fang S Kang Y Wu W Hao Y NONCODE an informative and valuable data source of long noncoding RNAs NucleicAcids Res D203“D208 101093nargkv1252Zou Q Xing P Wei L and Liu B Gene2vec gene subsequenceembedding for prediction of mammalian N6methyladenosine sites frommRNA RNA “ 101261rna069112118Conflict 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 Liu Zhang Han Li Yang Gao Xie and Du This is an openaccess distributed under the terms of the Creative Commons Attribution License CCBY The use distribution or reproduction in other forums is permitted providedthe original authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0c'

Thyroid_Cancer

"Nonsmall cell lung cancer is the most common cause of cancer death worldwide highlighting the need fornovel therapeutic concepts In particular there is still a lack of treatment strategies for the group of elderly and frail patientswho are frequently not capable of receiving standard therapy regimens Despite comprising the majority of lung cancerpatients this group is underrepresented in clinical trials This applies also to elderly and frail patients suffering fromunresectable stage III NSCLC who are unfit for chemotherapy and therefore cannot receive the standard therapycomprising of radiochemotherapy and the recently approved subsequent durvalumab consolidation therapy These patientsoften receive radiotherapy only which raises the concern of undertreatment The TRADEhypo trial aims at optimizingtreatment of this patient group by combining radiotherapy with concomitant durvalumab administration therebyemploying the immunepromoting effects of radiotherapy and determining safety feasibility and efficacy of this treatmentMethods design In this prospective phase II clinical trial durvalumab therapy will be combined with either conventionallyfractionated CONgroup or hypofractionated HYPOgroup thoracic radiotherapy A safety stopandgo leadin phase willassess safety of hypofractionated radiotherapy with respect to severe pneumonitis in small patient cohorts before ing fullenrollment Tumor tissue blood and stool samples will be collected before and during the study period to investigate theimmunological mechanisms responsible for checkpoint inhibitor efficacy and immunepromoting effects of radiotherapyContinued on next page Correspondence FarastukBozmehrmeduniheidelbergde1Department of Thoracic Oncology Thoraxklinik at University Hospital ofHeidelberg R¶ntgenstraŸe Heidelberg Germany2Translational Lung Research Center Heidelberg TLRCH Member of theGerman Center for Lung Research DZL Im Neuenheimer Feld Heidelberg GermanyFull 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 0cBozmehr BMC Cancer Page of Continued from previous pageDiscussion Preclinical data suggests that irradiationinduced immunogenicity can be further increased if applied in ahypofractionated setting potentially boosting the expected synergistic effect with immune checkpoint inhibition in restoringthe immune antitumor response If proven safe and efficient a hypofractionated radiation schedule can provide a considerablymore practicable option for the patient Taking into consideration the intend to develop a combination treatment strategy thatcan be made available to patients soon after proving to be efficient and the potentially elevated toxicity of a hypofractionatedradiotherapy approach this trial was designed as a twotrialsinone design An accompanying translational research program isplanned striving to gain insights into the tumorhost biology and to identify suitable biomarkers to predict therapy responseTrial registration Clinicaltrialsgov NCT04351256 Registered April EudraCT “ Registered October Keywords Nonsmall cell lung cancer NSCLC Radioimmunotherapy Immune checkpoint inhibition AntiPDL1 monoclonalantibody Hypofractionated radiation Geriatric risk profileBackgroundLung cancer is the most common cause of cancer deathworldwide with nonsmall celllung cancer NSCLCrepresenting “ of cases [ ] Improving therapeutic strategies is thus of imminent importance especially considering elderly and frail patients With a medianage of about years at diagnosis lung cancer clearly is adisease of the elderly yet this group is underrepresentedin clinical trials and these patients are frequently not capable of receiving standard treatment protocols due to ageand tobaccoassociated comorbidities [“]attracting both immunocompetentIn recent years the advent of immunotherapy has pavedthe way for novel therapeutic concepts including the combination of radiotherapy with immune checkpoint inhibitioneg Programmed cell death ligand PD1 PDL1 Thisapproach is of particular interest as it utilizes synergistic effects While immune checkpoint inhibitors can restore thepatients™ antitumor immunity through T cell activationradiotherapy may further boostimmunemediated anticancer mechanisms by exposing tumorassociated antigensand byantigenpresenting cells and tumoricidal effector cells [ ] Indeedfor patients with unresectable stage III NSCLC the PACIFICtrial has revealed a profound clinical benefit treatment withthe antiPDL1 monoclonal antibody durvalumab after chemoradiotherapy with remarkably low toxicities [ ] As aresult sequential treatment with durvalumab after chemoradiotherapy has become the new standard treatment for locally advanced unresectable NSCLC However about ofpatients do not receive chemotherapeutic agents presumablydue to significantly higher rates of age and comorbidityrelated adverse events AE under chemoradiotherapy [] Thus elderly and frail patients often receive radiotherapyalone raising the serious concern of undertreatment and theneed for new therapeutic concepts [ ]Considering the immunepromoting effects of radiotherapy a combination with durvalumab therapy mayimprove response rates in these potentially undertreatedpatients Moreoverif applied early concomitant localdatathatsuggestradiotherapy with systemic immunotherapy may particularly increase control of distant micrometastases Preclinicalirradiationinducedimmunogenicity can even be further increased if appliedin a hypofractionated setting with single doses ‰¥ GrayGy in line with a radiation dosedependent abscopal[“] While a hypofractionated radiationeffectschedule is also considerably shorter and more convenient for the patient safety of concurrent immunoradiotherapy is a concern as both therapy modalities maycause severe pneumonitisIn this prospective phase II clinical trial we thereforeaim to determine feasibility and treatment efficacy ofdurvalumab treatment combined with thoracic radiotherapy TRT in previously untreated NSCLC stage IIIpatients unable to receive radiochemotherapy Strivingto develop a combination treatment strategy thatifproven safe and efficient can be quickly made availableto patients a twotrialsinone design was chosen thatcombines durvalumab with either conventionally fractionated CONgroup or hypofractionated thoracicradiotherapy HYPOgroup This study not only aims toincrease the efficacy of radiotherapy by utilizing theimmunesensitizing effects elicited by PDL1 inhibitionbut will also provide biomaterials that will be analyzedwith respect to immunological mechanisms responsiblefor checkpoint inhibitor efficacy and immunepromotingeffects of radiotherapy as well as potential biomarkersMethodsdesignStudy designThe TRADEhypo trialis a prospective randomized label multicenter phase II trial with a safety stopandgo leadin phase Fig During the leadin phasepatients in the HYPOgroup who will receive durvalumab in combination with hypofractionated thoracicradiotherapy will be closely evaluated with regard totoxicity defined as pneumonitis ‰¥ grade within 0cBozmehr BMC Cancer Page of Fig Study design of the TRADEhypo trial Patients will be enrolled according to eligibility criteria and treated with either a hypofractionated TRTregimen HYPOgroup or conventionally fractionated TRT CONgroup in combination with durvalumab For the HYPOgroup a safety stopandgophase with a design precedes full enrollment Whenever this arm is for recruitment patients will be allocated to this arm until the cohort isclosed whenever HYPOarm is closed for Stop Go decision evaluation based on the toxicity assessment of this regimen weeks after the end of TRTpatients are allocated to the CONarm When the study proceeds to expansion phase patients will be allocated to treatment arms by randomizationusing œbiased coin algorithm An efficacy interim analysis will be performed after patients have been enrolled in each armweeks after radiotherapy in small cohorts n beforeproceeding with full enrollment into this arm Fig respectrelated biomarkersto treatmentinduced changes and immuneStudy settingThe TRADEhypo trial will recruit patients from participating centers across Germany over a period of months Start of recruitment was planned for April but was delayed to May due to the Covid19pandemic A full list of sites can be obtained at clinicaltrialsgov NCT04351256Study objectivesThe primary objective of this study is to evaluate safetyand tolerability of conventionally fractionated CONgroup and hypofractionated HYPOgroup TRT incombination with durvalumab in patients with unresectable stage III NSCLC unfit for chemotherapy Moreoverefficacies of the two modes of radiotherapy will be evaluated with respect to response rates Further parameterswill be determined in order to assess efficacy safety andquality of life QoL in both treatment arms by recordingincidence and severity of adverse events AEs as well asspecific laboratory abnormalitiesExploratory endpoints include assessment of vulnerability and analyses of tumor tissue blood and stoolsamples that are collected during the clinical trial withCharacteristics of participantsA total of patients will be included into this studyPatients potentially eligible for trialinclusion will beapproached and asked to participate as they present inthe clinic Before a patient™s participation in the clinicalstudy the investigator must obtain written informedconsentEach participant must be eligible regarding all inclusion and exclusion criteria set for this trial Table Key inclusion criteria include diagnosis of unresectablestage III NSCLC and nonfeasibility of sequential chemoradiotherapy due to increased oxygenindependentvulnerability as reflected by fulfilling at least one of thefollowing criteria i Performance status Eastern Cooperative Oncology Group [ECOG] scale ii ECOG and Charlson comorbidity index CCI ‰¥ or iii age ‰¥ Moreover participants must have at least one measurable site of disease as defined by RECIST as wellas adequate bone marrow hepatic and renal functionPatients having received prior immunotherapy other investigational agents or thoracic radiotherapy within thepast years will be excluded from the study Additionally participants must not have an active or recent history of a known or suspected autoimmune disease or 0cBozmehr BMC Cancer Page of Fig Cohort design of the safety stopandgo leadin phase HYPOgroup The safety leadin phase follows a design in order to carefullyevaluate the toxicity of the treatment in the HYPOgroup with respect of the occurrence of a grade pneumonitis œevent within weeksafter the end of TRT Two events in the first six patients two events in the first or two events in the first patients will result in terminationof the HYPOgroup œStop If no event is observed within the first two safety cohorts ie the first patients the HYPOarm will be ed forfull enrollment with close toxicity assessment with respect to pneumonitis grade and terminated as soon as two events are reported withinthe subsequent six patients œGo Full enrollment in the HYPOarm will only take place if the criteria for the nontoxicity scenario are met ie ‰¤ event in n patients œGoany other medical condition conflicting with the studyinterventions and not have used immunosuppressivemedication For a full list of the inclusion and exclusioncriteria see Table TreatmentDurvalumabPatients will be enrolled based on the inˆ’ exclusion criteria Two treatment groups will be evaluated Onegroup will receive durvalumab immunotherapy combined with conventionally fractionated TRT CONgroup and the other one with hypofractionated TRTHYPOgroup In both groups durvalumab will be administered intravenously at a fixed dose of mg onday and every weeks thereafter for a maximum of months maximum doses last dose at week untilconfirmed disease progressioninacceptable toxicitywithdrawal of consent or end of the study Fig andTable RadiotherapyAll patients are subjected to preparation of individualpositioning devices and CTbased planning Motionmanagement may comprise either 4DCT or midbreathing CT image acquisition Further imaging modalitiessuch as FDGPETCT may be used when deemed necessary Gross tumor volumes GTV will be contouredand expanded by adequate clinical CTV and planningPTV safety margins in order to account for subclinicaldisease and positioning errors No elective nodal irradiation will be performed As for ans at risk bothlungs spinal cord heart and esophagus must be contoured In the HYPOarm no more than of œbothlungs minus GTV should receive Gy in the CONarm no more than of œboth lungs minus GTVshould receive GyIn the HYPOarm fractions of Gy will be administered total dose Gy corresponding to GyBED αβ In the CONarm fractions of Gywill be administered total dose Gy corresponding to GyBED αβ Dose deviations of ± are acceptable when clinically indicated Radiotherapy isscheduled to start within h after the first administration of durvalumab Dose prescription will follow international reports ICRU and Both 3Dconformal and modulated photon radiation techniquessuch as IMRT and VMATRapdArc are acceptable Allparticipating institution are encouraged to perform regular if no daily positioning control using either portalimaging or onboardCT devicesStudy proceduresIn order to minimize the risk exposure of patients whensubjected to the hypofractionated radiation regimen a 0cBozmehr BMC Cancer Page of Table Complete list of inclusion and exclusion criteriaInclusion criteriacid129 Fullyinformed written consent and locally required authorization obtained from the patient legal representative prior to performing any protocolrelated procedures including screening evaluationscid129 Age ‰¥ yearscid129 Histologically documented diagnosis of unresectable stage III NSCLCcid129 Nonfeasibility of sequential chemoˆ’radiotherapy as determined by the site™s multidisciplinary tumor board if there is no tumor board then thisdecision will be made by the investigator in consultation with a radiation oncologist if the investigator is not a radiation oncologist or by the investigator in consultation with an oncologist if the investigator is not an oncologistcid129 Fulfills at least one of the following criteria—‹ ECOG —‹ ECOG and CCI ‰¥ —‹ Age ‰¥ yearscid129 Must have a life expectancy of at least weekscid129 FEV1 ‰¥ of predictedcid129 DLCO ‰¥ of predictedcid129 FVC or VC ‰¥ of predictedcid129 At least one measurable site of disease as defined by RECIST criteriacid129 Adequate bone marrow renal and hepatic functioncid129 Female patients with reproductive potential must have a negative urine or serum pregnancy test within days prior to start of trialcid129 Evidence of postmenopausal status or negative urinary or serum pregnancy test for female premenopausal patientscid129 The patient is willing and able to comply with the protocol for the duration of the study including hospital visits for treatment and scheduledfollowup visits and examinationsExclusion criteriacid129 Concurrent enrollment in another clinical study or enrollment within days prior to first dose of treatment unless it is an observational noninterventional clinical study or during the followup period of an interventional studycid129 Prior immunotherapy or use of other investigational agentscid129 History or current radiology suggestive of interstitial lung diseasecid129 Oxygendependent medical conditioncid129 Any concurrent chemotherapy investigational product IP biologic or hormonal therapy for cancer treatmentcid129 Prior thoracic radiotherapy within the past years before the first dose of study drugcid129 Major surgery within weeks prior to enrollment into the study patients must have recovered from effects of any major surgery Local nonmajorsurgery for palliative intent is acceptablecid129 Active or prior documented autoimmune or inflammatory disorders with the following exceptions Patients with vitiligo or alopecia patients withhypothyroidism stable on hormone replacement or any chronic skin condition that does not require systemic therapy Patients without activedisease in the last years may be included but only after consultation with the study physiciancid129 Active uncontrolled inflammatory bowel disease Patients in stable remission for more than year may be includedcid129 Uncontrolled intercurrent illness including but not limited to ongoing or active infection symptomatic congestive heart failure uncontrolledhypertension unstable angina pectoris uncontrolled cardiac arrhythmia interstitial lung disease gastrointestinal conditions associated with diarrheaor psychiatric illnesssocial situations that would limit compliance with study requirement substantially increase risk of incurring AEs or compromisethe ability of the patient to give written informed consentcid129 History of another primary malignancy except for a malignancy that has been treated with curative intent and was not active for ‰¥ years beforethe first dose of IP and of low potential risk for recurrence or adequately treated nonmelanoma skin cancer or lentigo maligna without evidence ofdisease or adequately treated carcinoma in situ without evidence of diseasecid129 History of leptomeningeal carcinomatosiscid129 History of active primary immunodeficiencycid129 History of allogenic an or tissue transplantationcid129 Clinical diagnosis of active tuberculosiscid129 Positive testing for hepatitis B virus surface antigen or hepatitis C virus RNA indicating acute or chronic infection or for human immunodeficiencyviruscid129 Current or prior use of immunosuppressive medication within days before the first dose of durvalumab The following are exceptions to thiscriterion Intranasal inhaled topical steroids or local steroid injections systemic corticosteroids at physiologic doses not to exceed mgday ofprednisone or its equivalent steroids as premedication for hypersensitivity reactionscid129 Receipt of live attenuated vaccine within days prior to the first dose of IPcid129 Female patients who are pregnant or breastfeeding or male or female patients of reproductive potential who are not willing to employ effectivebirth controlcid129 Known allergy or hypersensitivity to any of the IPs or any of the constituents of the productcid129 Any coexisting medical condition that in the investigator™s judgement will substantially increase the risk associated with the patient™s participationin the studycid129 Patient who has been incarcerated or involuntarily institutionalized by court order or by the authorities § Abs S Nr AMGcid129 Patients who are unable to consent because they do not understand the nature significance and implications of the clinical trial and thereforecannot form a rational intention in the light of the facts [§ Abs S Nr 3a AMG]safety leadin phase with stopandgo design will precedefull enrollment into the HYPOgroup Fig Toxicitywill be evaluated with a design that is based on thestatistical assumption that ‰¤ event in n patientsconforms to a nontoxicity scenario with œevent beingdefined as the occurrence of pneumonitis grade ‰¥ within weeks after end of TRT Consequently twoevents in the first six patients or two events in the first 0cBozmehr BMC Cancer Page of Table Schedule of assessmentsProcedure Point in timeInformed Consent eligibility criteria demographicsmedical and historyAllocation RandomizationPrior and Concomitant Medication ReviewDurvalumab administrationRadiotherapy CONa or HYPObAEsFull Physical ExaminationDirected Physical ExaminationVital Signs O2 Saturation and WeightPulmonary function tests12lead ECGECOG Performance StatusPregnancy Test CBC with Differential Serum ChemistryPanel Thyroid function testHBV HCVUrinanalysisTumor ImagingFACTL and G8 screening questionnaireTissueScreening Treatment Cycles Q4WScreening C1D1 C1D4 C2D1 C3D1 C4D1XC5D1C13D1EOT PostTreatmentEOT Safety FU FU Q12WXXXXXXXXXXXXXXXXXXXXXXXXXXXXXcXX cXXXXXXXXXXXXXtogether with staging XWhenever clinically indicatedXXXXXXXXXXXXXXXXWhenever clinically indicatedXdXdXeQ8WeXfXXXtogether with staging XXXgXhXXiOptional ReBiopsy at time of progressionXiXXBlood and stoolaCONgroup radiation scheme Patients receive conventional fractions of — Gy Gy within weeks of TRT to be started within h after start ofdurvalumab treatmentbHYPOgroup radiation scheme Patients receive hypofractionated thoracic radiotherapy consisting of — Gy Gy within weeks of TRT to be startedwithin h after start of durvalumab treatmentcTo be performed on C2D1 and C3D1 if in accordance with local standarddChest Xray to be performed on cycles and if in accordance with local standardeFirst onstudy CT imaging to be performed weeks after first durvalumab administration Further onstudy imaging to be performed Q8W days ± daysfOnly applicable if EOT not according to already detected disease progressiongIn patients with EOT not due to disease progression tumor imaging will be performed until the start of a new anticancer treatment disease progression deathwithdrawal of consent or the end of the studyhQuestionnaires will be collected until disease progression only and may be collected by telephone callsiBiomarker sample to be taken prior to first study drug medication either during screening or C1D1 visitX or two events in the first patients will result in termination of the HYPOgroup Fig During this safety stopandgo phase patients will beallocated to treatment arms asfollows While theHYPOarm is recruiting patients will exclusively enterthis treatment group During safety evaluation of the sixpatients of a HYPOcohort Stop Go decision patientswill be allocated to the CONgroup only If safety criteria in the HYPOcohort are met the HYPOarm willbe re ed to continue toxicity assessment and patients will solely be allocated to this arm Fig Inorder to avoid any bias patients will be allocated centrally by the study management during this phase If thenontoxicity criteria in the safety cohorts are met thestudy will proceed to theandremaining patients will be randomized into the twotreatment arms using an interactive web responseexpansion phasesystem integrated in the electronic case report formeCRF A possibly uneven distribution of patients between the treatment groups at this stage will be compensated by a randomization strategy based on the œbiasedcoin method [ ] In the randomization phase patients will be stratified according to tumor stage stageIIIA vs stage IIIBIIICIn total patients will be enrolled per group Aftern patients have been enrolled to the HYPO orCONtreatment arm respectively an interim efficacy analysis for the respective arm will be conducted based on theobjective response rate ORR at weeks after first durvalumab administration In case of insufficient efficacy ofone of the arms ie the number of patients who haveachieved a response is eight out of or lower this treatment arm will be terminated Recruitment will be halteduntil results of the interim efficacy analysis are available 0cBozmehr BMC Cancer Page of Tumor response will be assessed according to RECIST by CT and or MRI scans at baseline weeks afterdurvalumab initiation and then every weeks Safetymeasures willinclude physical examinations performance status ECOG clinical laboratory profiles and continuous assessments of AEs An overview of all studyprocedures is presented in Table An Independent Safety Monitoring Board ISMB willensure the continued safety of participants throughoutthe trial Data management and data quality assurancewill be conducted following the Standard OperationalProcedures of the Institute of Clinical Cancer ResearchIKF at Northwest Hospital GmbH Frankfurt GermanyAn eCRF will be carefully maintained for each participant for data collection also reporting serious and nonserious AEs following the common criteria for adverseevents CTCAE version throughout the entire trialAfter the end of the study participants will be proactively followed up regarding treatmentrelated AEsuntil resolved returned to baseline or considered irreversible until lost to followup or withdrawal of studyconsent All subjects will be followed for survival Patients who decline to return to the site for evaluationswill be offered a followup FU by phone every months as an alternative At the end of the study treatment the investigators are responsible for the furthertreatment of the patient and must ensure that he or shereceives appropriate standard of care or other appropriate therapiesSampling for translational researchIf patients participate in the translational research program blood samples will be collected prior to cycles and and at the time of disease progression or end oftreatment EOT along with stool samples Table Samples of untreated tumor lesions serving as baselinespecimens will be collected as paraffinembedded tissueIf rebiopsies are taken at the time of progression samples should also be also submitted for translationalresearchStudy endpointsThe primary endpoint of the study will be toxicity defined by the occurrence of treatmentrelated pneumonitis grade ‰¥ The ORR evaluated weeks after firstdurvalumab administration according to RECIST isset as the primary efficacy endpoint Secondary endpoints ofthe occurrence oftreatmentrelated AEs and serious AEs SAEsfrequency of prespecified abnormal laboratory parametersprogressionfree survival PFS and duration of clinicalbenefit metastasisfree survival overall survival OSand QoLcomprisethestudyPatient vulnerability and its association with survivaland outcome will be assessed as an exploratory endpoint To this end the G8screening questionnaire asimple and fast screening tool for identifying geriatricrisk profiles with a strong prognostic value for functionaldecline and OS in older populations with cancer will beused [] Furthermore biomaterials will be collectedduring the trial for correlation analyses on selected molecular parameters and clinical data in order to identifymolecular biomarkers predictive for response to therapyThisto obtainhypothesisgenerating data for future research due to theexplorative character of this trialapproach is deemed appropriateStatistical analysisSample size justificationSafety runin phase HYPOgroup With regard to thepneumonitis grade ‰¥ rate this phase is designed to distinguish between a toxicity scenario pTox and anontoxicity scenario pTox A sample size ofn will yield a probability of to correctly detectthe toxicity scenario while the nontoxicity scenario willcorrectly be detected with a probability of Theseprobabilities are based on the decision rule that if thenumber of patients with pneumonitis grade ‰¥ in thiscohort is ‰¥ recruitment to the HYPOgroup will beterminatedanalysisInterim efficacyregarding ORR andexpansion phase An interim efficacy analysis based onthe ORR will be conducted after n patients in eacharm have completed radiotherapy and the 18th patienthas undergone first radiographic assessment at weeksafter first durvalumab dosePreviously an ORR of after radiotherapy alonehas been reported in a Japanese population of elderly patients with unresectable stage III NSCLC [] Based onthis and the observation that Asian ethnicity is associated with a favorable prognosis we assume for theTRADEhypo trial that an ORR higher than in bothtreatment arms can be demonstrated ie the null hypotheses for arm HYPO and CON are defined as H0HYPO Ï HYPO ‰¤ and H0CON Ï CON ‰¤ where ÏHYPO and Ï CON denote the actual ORR in arm HYPOand CON respectively [ ] Under the alternativehypothesis it is assumed that both Ï HYPO and Ï CONamount to Using an optimal Simon™s twostage design with a onesided significance level of α and apower of 1β for each hypothesis test n patients per arm are required while the interim analysis isconducted after n patients per arm have been recruited to the trial After successfully passing the safetyanalysis in the HYPOgroup if among patients in the 0cBozmehr BMC Cancer Page of HYPO or CONarm the number of patients who haveachieved a response is eight or lower the respective armwill be closed Otherwise an additional number of patients will be enrolled into the respective arm Thenull hypothesis of the respective arm can be rejected ifat least of all patients per arm achieve a responseSample size calculation was done using the R packageœOneArmPhaseTwoStudy []To account for an estimated dropout rate of fourpatients will additionally be recruited to each treatmentarm Deviations from planned sample sizes will be handled as described by Englert Kieser allowing strictcontrol of the aspired type I error rate in each arm []Methods of statistical analysis The primary populationfor evaluating all efficacy endpoints and subject characteristics is defined as all patients enrolled according toinitial allocationrandomization intentiontotreat population IIT Secondary efficacy analyses will be carriedout on the perprotocol PP population The safetypopulation comprising all patients who received at leastone dose of the study medication will be used for allsafety endpoints and will be analyzed according to theactual treatment receivedResponse rates with confidence intervals CI and pvalues in both study arms will be estimated taking intoaccount the twostage nature of the design [ ] Secondary endpoints will be evaluated descriptively All toxicities will be summarized by relative and absolutefrequency and severity grade based on CTCAE V50 AEand SAE summary tables will provide the number andpercentage of patients with AEs and the ClopperPearson type CIs All analyses will be done using SASversion SAS Institute Cary NC or higherTrial statusAs of July 15th eight study sites are initiated Firstinitiation coincided with the beginning of the Covid19pandemic in Germany Therefore recruitment of patients was withheld On May 8th recruitment wasresumed after consultation with the ISMB The first patient was enrolled on July 13th renal carcinoma and NSCLCDiscussionIn recent years the concept of restoring the patients™ antitumor immunity by immune checkpoint inhibition hasrevolutionized cancer therapy especially in advancedmelanomaImmunecheckpoint molecules efficiently regulate T cell activation and thus enable tumor cells to evade the immunesystem for example by hijacking the PD1 PDL1 interaction to downregulate effector T cells [ ] To dateseveral human monoclonal antibodies pharmacologicallyblocking these interactions have been implemented incancer therapy such as the antiPD1 antibody pembrolizumab that has been approved in combination withchemotherapy for nonsquamous NSCLC irrespective ofPDL1 expression [ ]Several studies have shown that immune checkpointinhibition and radiotherapy in combination can act synergistically to further boost antineoplastic effects [“] Although in a large phase III trial no benefit ofblockade of cytotoxic T lymphocyteassociated antigen CTLA4 after radiotherapy was observed in metastaticprostate cancer [] clinical trials such as NICOLASNCT02434081 and DETERRED NCT02525757investigating concurrent PDL1directed immunotherapyand chemoradiot

Thyroid_Cancer

"Cardiac arrhythmias Atrial fibrillation Sudden cardiac death Long QT syndrome Torsade des pointes Ventricular tachycardia Ventricular fibrillation As the coronavirus COVID19 pandemic marches unrelentingly more patients with cardiac arrhyth mias are emerging due to the effects of the virus on the respiratory and cardiovascular CV systems and the systemic ‚ammation that it incurs and also as a result of the proarrhythmic effects of COVID19 pharmacotherapies and other drug interactions and the associated autonomic imbalance that enhance ar rhythmogenicity The most worrisome of all arrhythmogenic mechanisms is the QT prolonging effect of various antiCOVID pharmacotherapies that can lead to polymorphic ventricular tachycardia in the form of torsade des pointes and sudden cardiac death It is therefore imperative to monitor the QT interval dur ing treatment however conventional approaches to such monitoring increase the transmission risk for the staff and strain the health system Hence there is dire need for contactless monitoring and teleme try for inpatients especially those admitted to the intensive care unit as well as for outpatients needing continued management In this context recent technological advances have ushered in a new era in im plementing digital health monitoring tools that circumvent these obstacles All these issues are herein discussed and a large body of recent relevant data are reviewed Elsevier Inc All rights reserved Introduction The ongoing pandemic of coronavirus disease COVID19 has created a global tumult [] According to current data of patients with COVID19 infection are afflicted by acute my ocardial injury with an attendant higher mortality rate compared with those without cardiac injury commensurate with the degree of cardiac troponin cTn elevation [“] Furthermore of patients develop cardiac arrhythmias Table including malig nant ventricular arrhythmias VAs [ ] with a higher prevalence noted in patients admitted to the intensive care unit ICU [] Importantly clinically stable patients may have a low preva Abbreviations AAD antiarrhythmic drug AF atrial fibrillation APCs atrial pre mature complexes AZM azithromycin COVID19 coronavirus CQ chloro quine cTn cardiac troponin CV cardiovascular CYP cytochrome P450 ECG elec trocardiogram HCQ hydroxychloroquine ICU intensive care unit LQTS long QT syndrome NSVT nonsustained ventricular tachycardia OOHCA outofhospital cardiac arrest SCD sudden cardiac death TdP torsade des pointes VAs ventricular arrhythmias VF ventricular fibrillation VPCs ventricular premature complexes VT ventricular tachycardia ˆ—Corresponding author Email address asmotenetgr AS Manolis lence of arrhythmias [] however critically ill patients are at much higher risk for cardiac arrhythmias [] Cardiac arrhythmias including lifethreatening VAs may be the consequence of direct effects of COVID19 infection but also of the deleterious effects of systemic illness and the adverse reactions to drugs employed in the treatment of this pandemic Table Fig [ “ ] A recent study indicated that among patients with ± years men African Ameri COVID19 mean age can receiving care in the ICU there were cardiac arrests incident atrial fibrillation AF episodes bradyarrhythmias and nonsustained ventricular tachycardias NSVTs [] Arrhythmias occurring in patients admitted to the ICU included cardiac arrests all events of cardiac arrest occurred in this group AF odds ratioOR vs those not in the ICU and NSVT OR Car diac arrests were associated with acute inhospital mortality Among patients with confirmed COVID19 ex hibited myocardial injury as indicated by elevated cardiac troponin T cTnT levels [] During hospitalization patients de veloped ventricular tachycardia VTventricular fibrillation VF patients with elevated cTnT levels had more frequent VAs vs p compared with those with normal cTnT levels A recent singleday snapshot survey of stable patients with 101016jtcm202008002 Elsevier Inc All rights reserved Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Cardiac Arrhythmias Occurring in Patients with COVID19 Infection Sinus tachycardia Sinus bradycardia Conduction disturbances AVBBBB Atrial premature complexes Atrial fibrillation Supraventricular tachycardia Ventricular premature complexes Nonsustained ventricular tachycardia Polymorphic ventricular tachycardia Torsade des pointes Sustained ventricular tachycardia Ventricular fibrillation Pulseless electrical activity AVB atrioventricular block BBB bundle branch block Table Mechanisms of Arrhythmogenicity in Patients with COVID19 Infection Acute myocardial injury Myocarditis Hypoxia Systemic ‚ammation Autonomic imbalance SNS overactivity virusinduced vagal nerve injury Electrolyte abnormalities QT prolonging drugs antiCOVID pharmacotherapies AADs other agents Drugdrug interactions Cardiovascular comorbidities hypertension coronary artery disease cardiomyopathy AADs antiarrhythmic drugs SNS In this review we present current data about the whole spec trum of cardiac arrhythmias encountered in patients with COVID infection either attributable to the effect of the virus itself on the cardiovascular CV and the respiratory system andor to the effects of the treatments that these patients receive in combina tion with autonomic imbalance that is incurred by this unrelenting pandemic Acute myocardial injury and arrhythmias As mentioned in patients with evidence of acute myocardial injury the prevalence of cardiac arrhythmias is higher compared to patients without myocardial injury [] In a recent retrospec tive cohort study among patients with severe COVID19 had a cTnI level measured upon hospital admission of whom had positive results showing cardiac injury [] In patients with cardiac injury mortality was higher compared to patients without cardiac injury vs p Arrhythmias were found in of the patients with cardiac injury includ ing patients with VT or VF all of whom died [] A recent meta analysis of studies including COVID19 patients showed that patients with cardiac injury and newly occurring arrhythmias were at higher risk of developing severe disease or requiring ICU admission relative riskRR p [] sympathetic nervous system Sinus tachycardia Sinus tachycardia is the most common rhythm disturbance in patients with COVID19 infection due to multiple reasons such as fever respiratory insufficiencyhypoxemia hemodynamic compro mise fearanxiety pain and several other physical and emotional symptoms [] Bradycardiaconduction disturbances According to a retrospective series of patients transient si nus bradycardia lasting days is a possible manifestation of COVID19 hence another reason for close monitoring [] There may be many reasons for bradycardia but severe hypoxia ‚am matory injury of the sinus node by circulating cytokines and exag gerated response to medications are possible triggers Interestingly bradycardia has been suggested as a warning sign of the onset of a serious cytokine storm Fig The schema illustrates the various arrhythmias encountered in patients with COVID19 infection as a consequence of the virus infection affecting the heart and lung andor producing systemic ‚ammation the adverse proarrhythmic effects of COVID therapies and the drugdrug interactions that may occur see text for long QT discussion AF interval PEA pulseless electrical activity SB sud sinus tachycardia sympathetic nervous system STach den cardiac death SNS ventricular arrhythmias VF TdP ventricular fibril lation VT atrioventricular block LQT torsade des pointes VAs sinus bradycardia SCD atrial fibrillation AVB ventricular tachycardia COVID19 showed a incidence of arrhythmias limited to AF in and supraventricular tachycardia SVT in patients [] A Heart Rhythm Society HRS online survey of electrophysiology professionals n indicated that AF was the most commonly reported tachyarrhythmia whereas severe sinus bradycardia and complete heart block were the most common brad yarrhythmias in hospitalized patients with COVID19 [] Ven tricular tachycardiaVF arrest and pulseless electrical activity were reported by and of respondents respectively A meta analysis of retrospective cohort studies comprising pa tients with COVID19 showed that the pooled incidence was for cardiac arrhythmia for cardiac arrest [] p According to a retrospective cohort study of COVID19 pa tients multivariable logistic regression indicated that among other ECG abnormalities the presence of one or more atrial premature contractions APCs odds ratio OR a right bun dle branch block RBBB or intraventricular block IVB OR p increased the odds of death [] Another study analyzing the ECGs of COVID19 patients showed that abnormal PR interval behavior paradoxical prolonga tion or lack of shortening with increasing heart rate was associ and need ated with increased risk of death vs p for endotracheal intubation vs p compared to patients with PR interval shortening [] Atrial fibrillation According to a recent survey of electrophysiology professionals atrial fibrillation AF was the most commonly encountered car diac arrhythmia observed in patients with COVID19 infection [] Several mechanisms could be involved in the pathogenesis of AF in these patients virusinduced cardiac injury that could lead to perimyocarditis hypoxemia frequently occurring in these patients systemic infection common occurrence of the COVID19 infection Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx in older patients who are already susceptible to AF and sympa thetic nervous system overactivity could all account for such a high incidence of this arrhythmia in this particular population [ ] Guidance on acute management of AF In cases of AF associated hemodynamic compromise as done in all cases of hemodynamically unstable arrhythmias synchronized direct cur rent cardioversion should be used to restore sinus rhythm [] In all other cases one needs to initially proceed with a rate control strategy with use of a betablocker when there is no contraindi cation eg bronchospasm acute heart failure a calcium channel blocker in the absence of heart failure andor digoxin In cases of heart failure digoxin andor amiodarone may be used to achieve rate control For newonset AF within the last hours restoring sinus rhythm is the next target This can be achieved with use of class IA IC or III antiarrhythmic drugs AADs with the selection of the appropriate agent made as based on the presence where only amiodarone seems to be relatively safe or absence of under lying structural heart disease where all other options are available with the caveats being detailed in the discussion that follows be low also taking into account drug interactions with COVID phar macotherapies that are in use A major concern in using specific AADs relates to the baseline measurement of the QT interval and the coadministration of QTprolonging drugs see discussion be low Most importantly all AF patients should be receiving prophy lactic anticoagulation therapy with intravenous heparin Impact of national lockdown on newonset AF diagnosis An other aspect of the impact of national COVID19 lockdowns on the diagnosis of AF has been recently reported by a Danish study [] Using Danish registries the number of patients receiving a new onset AF diagnosis during the first months of and was compared A lower incidence of newonset AF during the weeks of lockdown was noted compared with the corresponding weeks in incidence rate ratios RRs for the weeks and There was a drop in total numbers vs [] Patients diagnosed during lockdown were younger and with a lower CHA2DS2VASc score while history of cancer heart fail ure and vascular disease were more prevalent During lockdown patients with newonset AF suffered an ischemic stroke and died compared with and pa tients during the corresponding period respectively The au thors concluded that following a national lockdown in Denmark a drop in registered newonset AF cases was observed indicat ing that the risk of undiagnosed AF patients developing complica tions could potentially translate into poorer outcomes in patients with AF during the COVID19 pandemic Ventricular arrhythmias In the setting of acute myocardial injury and acute myocarditis in patients with COVID19 infection various and serious ventricu lar arrhythmias VAs may occur [] Other important triggers in clude the severe respiratory insufficiency and the systemic ‚am mation incurred by COVID19 infection as well as the proarrhyth mic effects of COVID therapies and other drug interactions and also the autonomic imbalance superimposed in patients afflicted by the disease [ ] Furthermore hypoxemia which is common in these patients and electrolyte disturbances occurring for various reasons in this group of patients may aggravate arrhythmogenic ity Depending on preexisting or currently emerging CV disease various VAs may be encountered including ventricular premature complexes VPCs nonsustained VT NSVT and sustained VTVF Special attention is required for the development of polymorphic VT in the form of torsade des pointes TdP in the setting of QT prolongation either preexisting or acquired and induced by drugs especially when combination therapies are employed that are po tentially proarrhythmic [] Acute myocardial injury noted in of COVID19 patients can be the inciting factor for various VAs [ ] Among pa tients with confirmed COVID19 malignant VAs VTVF developed in patients during hospitalization patients with ele vated cTn levels had more frequent malignant arrhythmias vs [] A recent retrospective cohort study of patients with severe COVID19 indicated that among having a cTn level measured on admission with showing cardiac in jury arrhythmias developed in of the patients in cluding patients with VT or VF all of whom died [] Critically ill COVID19 patients often have comorbidities that can increase the risk for malignant VAs These include electrolyte abnormalities hypokalemia hypomagnesemia fever an ‚am matory state and most importantly COVID19 pharmacotherapies that are potentially proarrhythmic as they prolong the QT interval and may thus trigger TdP and sudden cardiac death SCD [] On the other hand the acute myocardial injury induced by the virus could also independently prolong the QT interval According to a recent report of a Kawasakilike syndrome temporally associated with COVID19 infection in children among whom myocardi tis was diagnosed in patients left ventricular ejection fractionLVEF range “ of these patients displayed im portant ECG changes that included QT interval prolongation and occasional VAs not attributable to any QTprolonging drug [] Inhospital cardiac arrest As mentioned among patients hospitalized with COVID19 infection all cardiac arrests occurred among patients admitted to the ICU [] In a retrospective cohort study inhospital VTVF occurred in of patients with cardiac injury all of whom died [] Outofhospital cardiac arrest OOHCA A recent Italian study compared all the consecutive outofhospital cardiac arrests OOHCA in the months following the first documented case of COVID19 in the region with those which occurred in the same time frame in [] The cumulative incidence of COVID19 from February to April in the study territory was COVID19100 inhabitants and the cumulative incidence of OOHCA was cases100 inhabitants with a increase as compared with OOHCAs in vs in p The authors concluded that the increase in OOHCAs in is significantly correlated to the COVID19 pandemic and is coupled with a reduction in shortterm outcome A French study comparing the OOHCAs of the pandemic period to the mean of the total over weeks in the non pandemic period indicated that the maximum weekly OOHCA in cidence increased from to per million inhabitants p before returning to normal in the final weeks of the pandemic period [] There was a higher rate of OOHCA at home less bystander cardiopulmonary resus vs p citation vs p and shockable rhythm vs and longer delays to intervention median p min vs min p The proportion of OOHCA patients ad mitted alive decreased from to p in the pan demic period After adjustment for confounders the pandemic pe riod remained significantly associated with lower survival rate at hospital admission odds ratio p COVID19 infection accounted for about one third of the increase in OOHCA incidence during the pandemic Druginduced prolongation of QTc interval and torsade des pointes Several agents employed for treating COVID19 infection may prolong the QT interval and lead to polymorphic VT in the form of TdP Table Chloroquinehydroxychloroquine and azithromycin which have been recently used for potential prophylaxis or treat ment for COVID19 infection are listed as definite causes of TdP Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table QTProlonging Drugs in COVID19 Infection Antibiotics Antiviral agents Anesthetics Antiemetics Antiarrhythmics Antipsychotics ChloroquineHydroxychloroquine Macrolides Azithromycin Quinolones LopinavirRitonavir Favipiravir Tocilizumab Fingolimod Propofol Domperidone Class IA Class III Haloperidol at crediblemeds [] According with the FDA azithromycin other macrolides and fluoroquinolones can cause lethal arrhyth mias as a potential consequence of QTinterval prolongation [] Chloroquine hydroxychloroquine Chloroquine CQ and hydroxychloroquine HCQ have been used for treatment and prophylaxis of malaria while they have also been employed for treatment of amebiasis that is occurring out side the gastrointestinal tract rheumatoid arthritis and lupus ery thematosus These agents were also found to have antiviral effects and have been proposed for the treatment of COVID19 infection [] However both these agents can be proarrhythmic by pro longing the QT interval and potentially initiating lifethreatening VAs including TdP they can also cause QRS widening Chloroquine interacts with multiple cardiac ion channels including the human etheragogorelated gene hERG potassium channel a reduction in hERG channel potassium current is the main cause of acquired druginduced long QT syndrome Recent experimental data indi cated that HCQ markedly increases the action potential dispersion and results in the development of repolarization alternans and ini tiates polymorphic VT [] Preliminary findings from a recent study suggested that the QTc prolonging effect of CQ is dosedependent [] Among pa tients enrolled with COVID19 infection were allocated to highdosage group ie mg CQ bid for days and to lowdosage group ie mg bid on day and qd for days Lethality until day was in the highdosage group of and in the lowdosage group of The highdosage group presented more instance of QTc interval ms compared with the lowdosage group Respiratory secre tion at day was negative in only of patients The authors suggested that the higher CQ dosage should not be rec ommended for critically ill patients with COVID19 because of its potential safety hazards especially when taken concurrently with azithromycin and oseltamivir A recent disproportionality analysis of HCQassociated CV ad verse reactions using the FDA adverse event reporting system FAERS database of datasets and patient records indicated that HCQ was associated with higher reporting odds ratios ROR of TdP ROR CI to complete atrioventricular AV block ROR CI to and QT prolongation ROR CI to [] QT prolongation and TdP are more frequent with high doses for a comparatively short period and represent the most common HCQassociated side effects A systematic review of data on COVID19 patients showed that of COVID19 patients treated with CQHCQ developed QT prolongation [] Ventricular arrhythmias developed in COVID patients from a group of treated with highdose CQ The authors suggest daily ECG monitoring and other risk mitigation strategies to be adopted in order to prevent possible arrhythmic sideeffects Macrolide antibiotics Azithromycin AZM also can cause modest QT interval pro longation but not through potent hERG channel blockade rather when used chronically through an increase in peak and late cardiac sodium current to cause potential loading of cardiomyocytes with sodium and calcium to produce calcium overload Advanced age and female gender are considered risk factors [] Azithromycin can also provoke nonpause“dependent polymorphic VT in the ab sence of QT prolongation [ ] After reviewing the data of AZM regarding risk of QT prolonga tion and associated TdP the FDA revised AZM product labels ad vising against its use in patients with known risk factors such as QTinterval prolongation hypokalemia hypomagnesemia bradycar dia or use of certain QTprolonging antiarrhythmic agents includ ing class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents [] Antiviral agents The combined antiviral regimen of ritonavirlopinavir approved for human immunodeficiency virus HIV infection was also con sidered to be able to suppress SARSCoV2 replication [ ] Lopinavir is metabolized by the hepatic cytochrome P450 sys tem CYP3A [] it also inhibits drug transporters such as Pglycoprotein Pgp [] Thus ritonavirlopinavir may increase plasma concentrations of drugs primarily metabolized by CYP3A or substrates of these drug transporters Ritonavirlopinavir may require dose reductions or avoidance of CYP3Amediated drugs such as rivaroxaban and apixaban Ritonavirlopinavir has also been shown to cause QT and PR interval prolongation or occasionally second or thirddegree AV block particularly in patients with un derlying structural heart disease and preexisting conduction sys tem abnormalities [] Due to its competitive inhibition of the RNAdependent RNA polymerase favipiravir is being evaluated in treating patients with COVID19 alone or in combination therapies the risk for QT inter val prolongation by favipiravir is considered to be low [] Other agents Fingolimod is an immunomodulator and immuno suppressant which reduces lymphocyte migration and is used in the treatment of multiple sclerosis [] it has been proposed as a potential adjuvant therapeutic agent against COVID19 [] Fin golimod has Ltype calcium channel blockade effect causing pro longation of PR RR and QT interval It also activates acetylcholine dependent potassium channels IKach in sinoatrial node causing dosedependent bradycardia [] Thus fingolimod increases the risk of bradycardia and heart block through Ltype calcium channel and IKach blockade [] Combined therapies Treatments employed for COVID19 may increase arrhythmia risk particularly the risk for VAs through drug interactions Drug combinations can lead to greater prolongation of cellular action potential duration analogous to QT prolongation compared with single drug therapies [] The combination effect can result from both pharmacokinetic and pharmacodynamic drug interactions Importantly females with preexisting CV disease seem to be more susceptible to druginduced arrhythmias compared to males with CV disease or healthy persons of either gender Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Measures to Prevent Arrhythmias in Patients with COVID19 Infection ¢ Withhold QT prolonging drugs in patients with baseline QTc ¢ Withdraw QTprolonging drugs when QTc increases to ¢ Do not use chloroquinehydroxychloroquine azithromycin other macrolides fluoroquinolones lopinavirritonavir or favipiravir in patients with known risk factors such as prolonged QTc hypokalemia hypomagnesemia bradycardia or concomitant use of certain QTprolonging antiarrhythmic drugs including class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents ¢ Maintain K ¢ Monitor QTc via ECG or preferably via telemetry monitor or smart phone measurements ms compared to baseline measurement ms or if QTc is prolonged by ms or with known LQTS mEqL and Mg level to level to mgdL An online survey of electrophysiology professionals revealed that of respondents reported having to discontinue therapy with HCQ AZM due to significant QTc prolongation and reported cases of TdP in patients on HCQCQ and AZM [] Amiodarone was the most common antiarrhythmic drug used for VA management Among COVID19 positive suspected patients stud ± years male received AZM HCQ ied age ± and received both drugs [] Baseline mean QTc was ± ms p with medications Sig ms and increased to ± ms vs nificant prolongation was observed only in men ± ms in women p of patients reached critical ms or QTc QTc prolongation maximum QTc ‰¥ ms Changes in ‰¥ ms if QRS QTc were highest with the combination compared to either drug ± with much greater prolongation with combination vs AZM vs ‰¥ ms or QTc increase of No patients manifested TdP ‰¥ ms if QRS ± ms p ± ms p ± ms vs Another recent cohort study of patients treated for COVID with CQHCQ reported that patients received CQ received HCQ and also received AZM [] Although the maximum QTc during treatment was signifi cantly longer in the combination group vs the monotherapy group TdP was not ob served in the entire population and there were no arrhythmogenic deaths reported A study of COVID patients receiving combined HCQAZM therapy indicated longer QTcinterval than before ther ‰¥ ms had apy vs ms p higher values of transaminases p compared with those with ms [] At h Holter ECG monitoring COVID19 QTc patient and no control had No patients showed R on T VPCs Analysis of h QTc dynamics revealed that COVID19 patients had higher QTc values than controls with no significant hourly variability Therapy with HCQ and AZM pro longs QTc interval in patients with COVID19 particularly in those with high levels of transaminases ‰¥ run of NSVT p patients with a QTc Interestingly in nonCOVID patients a retrospective cohort study identified only two SCDVA events among com bination users CQHCQ plus AZM [] However the doses were lower in this study compared to doses used in COVIDpatients drugs were not used acutely in a hospital setting as currently done for COVID patients fewer cardiac patients received the drugs all suggesting an attenuated risk for cardiac arrhythmias in this par ticular cohort Nevertheless when all measures and precautions are taken Table the incidence of QT prolongation and the TdPevent rate may remain low In a recent study of patients with COVID ± years male HCQAZM was infection mean age ‰¤ 480ms and potassium level initiated only if baseline QTc was mmolL [] Two patients were not eligible for drug ± ms initiation QTc ± ms after h of combined therapy The and increased to treatment had to be stopped because of significant QTc prolonga tion in patients No druginduced TdP nor death was ob served In this specific population HCQAZM could not be initiated or had to be interrupted in ‰¥ ms Baseline average QTc was of the cases QTc monitoring Congenital long QT syndrome LQTS with a prevalence of in the general population may often be asymptomatic and if an ECG has not been recorded it will remain unknown to the affected person and the first manifestation may be SCD usually triggered by a drug [] Furthermore silent genetic variants or œforme fruste  of congenital LQTS encountered in of people may render a person vulnerable to QT prolongation TdP and SCD [] Therefore a large number of healthy individuals will be at an increased risk of a druginduced LQTS Data suggest that in African Americans may be at a higher risk of druginduced TdP during the COVID19 pandemic due to clustering of intrinsic genetic susceptibility ie exclusive oc currence of the proarrhythmic ion channel variant pSer1103Tyr SCN5A acquired risk factors eg electrolyte disturbances and QTcprolonging drug use and COVID19“specific risk factors eg profound hypoxemia and cytokine storm [] A heart ratecorrected QT QTc interval is measured with use ˆšof various formulas among which the Bazett™s correction formula is most commonly used QTc QT RRsec QTc is defined as pro longed when it exceeds ms in males and ms in females as measured preferably in lead II or V on a standard 12lead ECG [] A prolonged QTc predisposes to polymorphic VT in the form of TdP that may degenerate into VF and SCD For the wideQRS adjusted QTc methods that have been suggested include the JT ad justment obtained as QTc“QRS [] or subtracting of the QRS duration from the measured QT [] For patients receiving QTprolonging drugs it is imperative to monitor the QTc interval during treatment Table Traditionally this can be accomplished by obtaining a 12lead ECG however in the era of the COVID19 pandemic this poses a certain risk and puts considerable strain on medical personnel and the health sys tem [] Many telemetry systems are equipped with features of real time QTc monitoring and could be used in hospitalized pa tients and those managed in the ICU setting In addition smart phone heart monitors are also capable of providing remote accu rate QTc measurements [] In this context AliveCor has recently received clearance from the FDA to market the KardiaMobile6L device a previously FDAapproved device for AF detection for QTc monitoring of COVID19 patients treated with QT prolonging drugs such as CQHCQ [] Similarly the Apple Watch ECG an F

Thyroid_Cancer

emergence of promising targeted therapies for the treatment of hepatocellular carcinoma HCCSorafenib has been the mainstay of treatment for a decade and newer modalities were ineffective and did not confer any increasedtherapeutic benefit until the introduction of lenvatinib which was approved based on its noninferiority to sorafenib Thesubsequent success of regorafenib in HCC patients who progress on sorafenib treatment heralded a new era of secondlinetreatment and was quickly followed by ramucirumab cabozantinib and the most ‚uential immune checkpoint inhibitors ICIsOver the same period combination therapies including antiangiogenesis agents with ICIs dual ICIs and targeted agents inconjunction with surgery or other locoregional therapies have been extensively investigated and have shown promise andprovided the basis for exciting clinical trials Work continues to develop additional novel therapeutic agents which could potentiallyaugment the presently available options and understand the underlying mechanisms responsible for drug resistance with the goalof improving the survival of patients with HCCSignal Transduction and Targeted Therapy 101038s4139202000264xINTRODUCTIONPrimary liver cancer remains a major problem for all health caresystems worldwide and is associated with a significant clinicaleconomic and psychological burden Hepatocellular carcinomaHCC accounts for of cases of nonmetastatic tumors of theliver1 During the past decades research has shed light on theepidemiology risk factors and molecular and genetic profiles ofHCCš contributing to the evolution of strategies for preventionsurveillance early diagnosis and treatment23 Liver resectionablation and liver transplantation are potentially curative butrequire diagnosis at a sufficiently early stage Unfortunately asignificant proportion of HCC patients present with intermediateand advanced stage disease often despite diligent surveillanceand curative treatments are frequently not possible4 In thesepatients systemic therapy remains essential and its pivotal roleand potential have stimulated considerable research over the pastdecade In this review we examine recent advances in targetedtherapy and discuss the impact this has had on the managementof HCC We also provide an overview of the most important areasof HCC research including novel clinical trials and technicalplatforms which promise to facilitate substantial progress withinthe next decadeAPPROVED FIRSTLINE AGENTS FOR HCCSorafenibThe success of SHARP and AsiaPacific trial promoted the approvalof sorafenib as firstline targeted therapy for advanced HCC5“ushering in the era of systemic treatment Subsequently virtuallyall trials were centered around sorafenib and it was used as acontrol with which novel firstline agents were compared andevaluated in an attempt to improve the prognosis of patients withHCC Unfortunately despite a number of trials which comparedthese novel agents including sunitinib10 brivanib11 cediranib12linifanib13 dovotinib14 and immunecheckpoint inhibitors ICIs tosorafenib none achieved the predefined primary end points Fig In addition during the decade when these agents were investigated the median overall survival OS of sorafenib monotherapy asfirstline treatment for advanced HCC increased from monthsSHARP to months CheckMate459 further consolidating itsposition Meanwhile the antitumor activity and safety of sorafenibhave been validated in realworld setting Subanalyses of the SHARPand AsiaPacific trials found sorafenib was effective and safeirrespective of disease etiology disease burden ECOG EasternCooperative Oncology Group performance status status etc15“The safety of sorafenib was consistent across ChildPugh A and Bpatients in clinical practice18 and the occurrence of side effects suchas handfoot syndrome and diarrhea were associated with animproved OS19 Baseline hepatic function clinicopathologicalfactors and etiology also affect the prognosis in HCC patientstreated with sorafenib20 In addition sorafenib exerts antitumoreffects with recurrenttransplantationconferring a survival advantage when compared with bestsupportive care BSC21“ Noticeably the application of sorafenibin clinical practice displays significantregional variations andincompliance with guidelines besides its usage as firstline therapyIt is common that initially unresectable HCCs got downstaged aftersorafenib treatment and underwent curativeintent surgery24“ andlocoregional therapies before sorafenib were commonly encountered in realworld settings2930tumors following liver1Department of Liver Surgery and Transplantation Liver Cancer Institute Zhongshan Hospital Fudan University Shanghai China 2Key Laboratory of Carcinogenesis and CancerInvasion Fudan University Ministry of Education Shanghai China 3Shanghai Key Laboratory of an Transplantation Zhongshan Hospital Fudan University Shanghai China4Department of Hepatobiliary and Pancreatic Surgery University Hospitals of Leicester NHS Trust Leicester UK 5Institute of Biomedical Sciences Fudan University ShanghaiChina and 6State Key Laboratory of Genetic Engineering Fudan University Shanghai ChinaCorrespondence Jian Zhou zhoujianzshospitalshcnThese authors contributed equally Ao Huang XinRong YangReceived April Revised July Accepted July The Authors 0cTargeted therapy for hepatocellular carcinomaHuang et alFirstlineSorafenibSharpAsiaPacificCediranibNCT00427973SunitinibNCT0069937BrivanibBRISKFLLinifanibNCT01009593NintedanibNCT01004003DovitinibNCT01232296ATEZOBEVGO30140IMbrave150LenvatinibREFLECTPEMLENKEYNOTE524NivolumabCheckMate459DonofenibChina SecondlineBrivanibBRISKPSEverolimusEVOLVE1AxitinibNCT01210495RamucirumabREACHRegorafenibRESORCENivolumabCheckMate040PEMKEYNOTE224TivantinibMETIVHCCS1SCUBEPEMKEYNOTE240CabozantinibCELESTIALRamucirumabREACH2NIVIPICheckMate040CAMChinaApatinibChinaFig Overview of the targeted agents approved for HCC ATEZO atezolizumab BEV bevacizumab CAM camrelizumab LEN lenvatinib PEMpembrolizumab NIV nivolumab IPI ipilimumabThe clinical benefit of sorafenib however remains modest andthe complex molecular pathogenesis of HCC stimulated theinvestigation of combinations of sorafenib with other moleculartargeting drugs Sorafenib has been combined with antiangiogenic agents MEKERK pathway inhibitors mTOR pathwayinhibitors histone deacetylase inhibitors EGFEGFR pathwayinhibitors and HGFcMet pathway inhibitors31 Other agentssuch as interferon32 selumetinib33 capecitabine34 tegafururacil35 gemcitabine and oxaliplatin GEMOX3637 and gemcitabinealone38 have also been evaluated but to date no treatmentsinvolving combinations containing sorafenib have succeeded inphase III trialsSince sorafenib and TACE are both recommended therapies foradvanced HCC it is reasonable to expect that their combined usewould confer benefits when compared with monotherapy Resultshowever highlighted regional differences and the heterogeneityof the trial protocolsIn TACE the multicenter randomizedplacebocontrolled phase European trial when compared withTACE alone the addition of concurrent sorafenib unlike the SPACEtrial39 did not improve progression free survival PFS40 It was alsoshown that the addition of sorafenib did not confer any survivalbenefit in patients with unresectable HCCs who had alreadyresponded to TACE41 Contrasting with these findings retrospective studies from China have shown that combination therapywith sorafenib and TACE increased OS by more than compared with TACE alone42“ which was supported by thefindings from a number of other groups5455 RecentlytheTACTICS trial a randomized multicenter prospective trial fromJapan reported an improved PFS for TACE plus sorafenibcompared with TACE alone vs months p although this trial used a redefined PFS not conventional PFS buttime until œunTACEable progression The TACTICS trial also usedtime to any cause of death plus OS as primary endpoints resultsnot reported and compared with sorafenib monotherapy TACEplus sorafenib was only superior in controlling tumor progressionand did not prolong OS5758The acceptance of sorafenib as the standard to which othernewer agents and nonsurgical interventions are compared hasresulted in studies comparing its use as monotherapy with TACEplus external beam radiotherapy59 and TACE plus intensitymodulated radiotherapy combined with sorafenib60 In the SARAHstudy selective internal radiotherapy with yttrium90 resin microspheres did not produce any survival benefit compared withsorafenib in locally advanced and inoperable HCC median OS vs p and did not meet the primary endpoint of OS61Similarly the addition of selective internal radiation therapy tosorafenib did not result in a significant improvement in OScompared with sorafenib alone62 Bettinger et al63 however diddemonstrate that stereotactic body external beam radiotherapyemployed as monotherapy SBRT was able to improve OScompared with sorafenib and SBRT with TACE also providedimproved OS and PFS when compared with sorafenib and TACE incombination64 In a recentinfusionchemotherapy HAIC NCT02774187 He et al65 reported thatsorafenib plus HAIC with FOLFOX improved OS compared withsorafenib alone in advanced HCC when portal vein invasion waspresent which was supported by other studies6667 Although theSCOOP2 trial found sequential HAIC with cisplatin and sorafenibdid not improve the survival benefit compared with sorafenibalone this is likely to have resulted from the study beingunderpowered for the primary and secondary endpoints68trial of hepatic arterialDue to the high recurrence rates following hepatectomy fortherapy has been extensivelyHCC approaches to adjuvantinvestigated although previous attemptsincluding the use ofantiviral agents have been largely unsuccessful Based on thepalliative use and success of sorafenib its potential in the adjuvantsetting was investigated and improved survivals following surgeryanticipated Unfortunately this has not been demonstrated and itfailed to reduce postoperative tumor recurrence in the STORMtrial69 and other western studies70 Explanations for the negativeoutcome in the STORM trial include highdose modification ratesshort treatment durations and the enrollment of patients whowere not at high risk of tumor recurrence with no evidenceof tumor satellites with one lesion and with nomicroscopic vascular invasion71 Consistent with this viewpointWang et al72 reported no case of recurrence during the sorafenibdosing period whereas patients suffered recurrence of theirtumor within months of discontinuation of sorafenib72 andpersistent sorafenib intake following postoperative recurrenceimproved OS73 Considering that patients who respond tosorafenib may belong to limited clinical or biological subsetsthe effectiveness of sorafenib in an unselected population cohortsupports its use in the adjuvant setting A number of studies fromthe Far East including China Japan and Korea include patientswith HCCs who are treated with hepatectomy despite their tumorsbeing outside Barcelona Clinic Liver Cancer Classification BCLCguidelines and although the results are difficult to compare dueto heterogeneity ofthe protocols the results are positiveSorafenib significantly reduces tumor recurrence in BCLC stage Cpatients7475 and increases diseasefree survivalDFS76 andSignal Transduction and Targeted Therapy 0cZhuang et al77 demonstrated that adjuvant therapy increaseddisease free survival DFS and OS Sorafenib treatment followinghepatectomy significantly prolonged the OS of advanced HCCthan intermediate HCC78 In addition to BCLC stageratherpatients who underwent hepatectomy and were pathologicallydiagnosed with microvascular invasion MVI also benefited fromadjuvant sorafenib treatment79 In line with these results a largerecent study with propensity score matching analysis alsodemonstrated that sorafenib significantly improved overall andrecurrencefree survival following resection80 The results fromthese studies which include all eligible patients suggest that moreprecise stratification would enable the identification of thosepatients who will benefit most from the use of adjuvant sorafeniband those in where additional treatment is not appropriateOngoing trials are attempting to evaluate the role of sorafenib inpatients with MVI following radical resection NCT02867280 andNCT02537158LenvatinibFollowing the approval of sorafenib for use in the treatment ofHCC it takes more than a decade before the second firstlinetargeted agent for HCC emerged Lenvatinib was approved for thefirstline therapy in advanced HCC following the results of theREFLECT trial a randomized phase III noninferiority trial publishedby Kudo et al81 Although not approved for long further multicenter data from œrealworld conditions confirmed the efficacy oflenvatinib regardless of previous tyrosine kinase inhibitor TKItherapies8283 and lenvatinib monotherapy demonstrated antitumor activity for more than years in unresectable HCC whenportal vein invasion was present84 In intermediatestage HCCpatients with tumors exceeding the uptoseven criteria for whomTACE is not helpful lenvatinib could provide significant longer OS vs months and PFS vs months85 Lenvatinibpharmacokinetics in HCC is affected by body weight8687 and asufficient dose relative dose intensity RDI is required to achieve agood therapeutic effect and consequently improved outcomesand prognosis are associated with the preservation ofliverfunction which reduces the number of patients who need todiscontinue their treatment88“ With lenvatinib unlike other TKIsthere are issues with thyroid toxicity and surveillance for thyroidabnormalities during treatment is important92 Hypothyroidism isnot unusual and there are also fewer common reports ofthyrotoxicosis and destructive thyroiditis93 From a healtheconomics standpoint however lenvatinib is more cost effectivethan sorafenib9495Secondline targeted agents for HCCStill sorafenib displays limited antitumor activity and someinitially sorafenibsensitive would eventually succumb to thedisease indicating the acquired resistance to sorafenib reducesits beneficial effects and an urgent need for secondline therapyRegorafenibInitial attempts to discover effective secondline agents wereunsuccessful and mirrored attempts to develop firstline agentswhich were superior to sorafenib96 The RESORCE trial was arandomized double blind placebocontrolled and phase III trialdemonstrating the effectiveness of regorafenib in patients whohad progressed on sorafenib treatment Thisstudy finallyconfirmed the potential of secondline agents and ushered inthe era of secondline and sequential therapy97 Regorafenibprovided survival benefitthe rate of diseaseprogression during prior sorafenib treatment or since the lastsorafenib dose98 This was confirmed by Yoo et al99 in aretrospective study of safety and efficacy in Korean patientswhere data were consistent with those from the RESORCE trialRegorafenib was even shown to be effective in patients with HCCrecurrence following liver transplantation with a median OS ofregardless ofSignal Transduction and Targeted Therapy Targeted therapy for hepatocellular carcinomaHuang et al months following regorafenib initiation and monthsfollowing sorafenib initiation CI “ for the sorafenibfollowed by regorafenib sequential therapy100However not all patients who progress on sorafenib arecandidates for secondline therapy101 In clinical practice only of patients are eligible forsecondline regorafenibtreatment102 Good liver functional reserve and ECOG performance status during sorafenib treatment contributed to theefficacy and better outcomes of subsequent treatment103104including lenvatinib105 This may in part be due to the RDIrequired to achieve a clinically significantinprognosis106 This is supported by the demonstration that thenew liver reserve function biomarker albuminbilirubin gradeALBI107 successfully identified regorafenib candidates and thatin the selected cohort a median OS of months was achievedcompared with months for noncandidates108 Even in patientsnot eligible for regorafenib the ones with an ECOGPS score of the absence of MVI and TTP time to progression ‰¥ monthscould still have acceptable postprogression survival109 Longtermtreatment with regorafenib has also been shown to reduceangiogenesis and improve portal hypertension PHT and acuteadministration ameliorates portal haemodynamics suggestingthat it may be especially suitable for patients with PHT andpreserved liver function110improvementCabozantinibCabozantinib is another small molecule inhibitor of the tyrosinekinases which are implicated in the progression of HCC and theacquired resistance to sorafenib Cabozantinib blocks the receptors involved in oncogenesis and angiogenesis including VEGFR hepatocyte growth factor receptor MET AXL and theangiopoietin receptors TIE2 RET cKit and FLT3 in vitro andin vivoIn CELESTIAL trial cabozantinib achieved the primaryendpoint with median OS of months compared with months for the placebo group111 and was consequentlyapproved in the EU and USA There remains a paucity of datahowever from realworld clinical practice examining the sequentialtreatment utilizing cabozantinib as the secondline agent it is acostly option associated with frequent highgrade adverse eventsConsequently several studies have addressed the costeffectivenessof cabozantinib using the cost and utility data extracted from theCELESTIAL trial The conclusion from these studies is consistent andconfirms that at its current cost point the gain of qualityadjustedlifeyears for cabozantinib QALYs “ and the incrementalcosteffectiveness ratio ICER “ mean that it isnot a costeffective treatment option for patients with sorafenibrefractory HCC112“ compared with regorafenib QALY “and ICER “RamucirumabRamucirumab is a fully human recombinant IgG1 monoclonalantibody targeting the VEGF2 receptor Although ramucirumabfailed to meet its primary endpoint as secondline treatment in theREACH trial117 subgroup analysis found survival benefit in patientswith AFP of ngml or higher118“ This was later confirmed inthe REACH2 trial122 which led to the approval of ramucirumab assecondline treatment for advanced HCC REACH2 is the firstpositive phase trialin patients with HCC performed in abiomarkerselected patient cohort and more recent findingsdemonstrated that AFPenriched HCCs displayed significantactivation of VEGF which suggests the underlying mechanism ofaction and confirms the potential value of biomarkerdrivenclinical trials123Immune checkpoint therapy and TKI inhibitorsICIs stand as the mainstream of immunotherapy The CheckMate and KEYNOTE224125 studies evaluated the safety andefficacy of nivolumab and pembrolizumab in patients with 0cTargeted therapy for hepatocellular carcinomaHuang et alphaseIIrandomizedparallelgrouptislelizumab sintilimabrealworld cohort studyadvanced HCC refractory to previous sorafenib treatment whichestablished the basis for accelerated approval by the FDA assecondline treatment Subanalysis of CheckMate040 data validated the safety and efficacy of nivolumab in Asian cohort126 Inan internationalICIs have showedpromising efficacy and safety in advanced HCCs as systemicfirstsecondthirdfourthline treatment with median OS andPFS of and months respectively127 and an excellentresponse to antiPD1 therapy has also been described in casereport128 Although the subsequent phase III KEYNOTE240 trialdid not meet its prespecified statistical significance in respect ofimproved PFS and OS the results were consistent with previousKEYNOTE224129 The KEYNOTE394 presently underway in Asianpatients may clarify the role of pembrolizumab in cases ofadvanced HCC with a viral background NCT03062358 RecentlyCheckMate459 the multicenter phase III randomized sorafenibcontrolled trial evaluating nivolumab as firstline treatment foradvanced HCC failed to achieve its endpoints ESMO butnivolumab did prolong OS vs months and achievelongtime disease control less adverse events AEs and survivalbenefit regardless of the level of PDL1 expression Furthermorenivolumab improved the survival of HCC patients whose etiologywas HBVHCV and did not reactivate hepatitis CamrelizumabSHR1210 Hengrui Pharmaceutical is an antiPD1 inhibitor fromChina investigated for the treatment of Hodgkin lymphoma andHCC It has been shown to have antitumor activity in previouslytreated Chinese patients with advanced HCC in a multicenteropenlabeltrialNCT02989922130 providing evidence for the effectiveness ofPD1 therapy for HBV related HCC in Chinese patients The resultsICIs including durvalumabfrom other trials investigating novelavelumabtremelimumabipilimumabspartalizumab and toripalimab will hopefully yield positive resultsand provide further options for the treatment of patients withHCC particularly those who have relapsed on firstline treatmentsFurther efforts to enhance the treatment effect of ICIs includedual ICIs treatment and combination therapy of ICIs with otherkinds of targeted agents For dual ICIs treatment the initial resultsfrom CheckMate 9DW were astonishing the objective responserate was higher than monotherapy of any ICIs alone FDA hasapproved nivolumab in combination with ipilimumab for patientswith HCC previously treated with sorafenib Treatment modalitiessuch as radiotherapy and antiangiogenesis agents which affectantigen release or modulate the tumor microenvironments havethe potential to increase the efficacy of immunotherapy and thecombination oftargeted agents with ICIs are attracting theattention of a number of research groups and in vitro studies andearlyphase clinical trials assessing combination treatments haveshown promising antitumor effects in patients with advancedIn vitro evidence by Qui et al131 demonstrated thatHCClenvatinib and regorafenib could affect the expression of PDL1and realtime PCR results suggested that the mRNA expression ofPDL1 in the lenvatinib group was significantly higher than that inthe control group while its expression in the regorafenib groupwas significantly lower When combined with antiPD1 lenvatinibcan modulate cancer immunity in the tumor microenvironmentand enhance antitumor activity132133 In July the FDAannounced its approval of the first combination therapy employing the TKI lenvatinib with the ICI pembrolizumab based on theresults from the KEYNOTE524Study NCT03006926 for thetreatment of HCC Recently results from Study Phase IbNCT03418922 showed marginally better results for lenvatinibwith nivolumab than lenvatinib with pembrolizumab METmediated phosphorylation leads to a decreased expression ofPDL1 using the combination of MET inhibitors tivantinib andcapmatinib antiPD1 and antiPDL1 produced an additive effectwhich slows the growth of HCCs in mice134 Clinically based onthe results from the experimental arm A of the GO studyNCT02715531 the FDA approved atezolizumab plus bevacizumab as breakthrough therapy for untreated advanced ormetastatic HCC135 Individual case studies also reported promisingresults for the use of combined TKI and antiPD1PDL1 agents foradvanced HCC136“ Such results were confirmed in the phase IIItrialIMbrave study NCT03434379 which reported thatatezolizumab combined with bevacizumab resulted in better OSand PFS than sorafenib in patients with unresectable HCC139Other combination therapies include Galunisertib with nivolumabNCT02423343 spartalizumab with and without capmatinibNCT02795429 FGF401 with spartalizumab NCT02325739regorafenib with pembrolizumab NCT03347292 cabozantinibwithaxitinibNCT03289533 ramucirumab with durvalumab NCT02572687and XL888 with pembrolizumab NCT03095781 Table avelumab withNCT03299946nivolumabImmunerelated adverse events IRAEs occur frequently duringtreatment with ICIs and the clinical consequences can besignificant140 Activation of the immune system leads to damageof normal healthy tissues and IRAEs can have myriad effects andinvolve a number of different ans and have been reported toproduce colitis hepatitis pneumonitis dermatitis myocarditisendocrine glands ‚ammation and rheumatic and musculoskeletal phenotypesincluding ‚ammatory arthritis arthralgiamyositis and sicca syndrome141 Although the precise pathophysiology underlying the IRAEs side effects during treatment withICIs remains unknown discontinuing administration and the useof steroids is generally effectiveIn severe cases howeveradditional immunosuppressants may be required but based oncurrent available evidence immunosuppression for IRAEs does notappearresponse to the ICItreatment142143to compromise the antitumorPromising agents and treatment regimensDespite abovementioned targeted drugs novel agents have beencontinuously under development Table Of note apatinib anovel inhibitor of VEGFR2 tyrosine kinase has attracted considerable attention and there is now a significant body of workdescribing clinical experience with its use Although less effectivethan sorafenib as a firstline treatment in a retrospective study144apatinib still displayed promising antitumor effects in sorafenibresistant HCC145“ where portal vein invasion was present148when metastases have occured149150 and for unresectable andrelapsed HCCs151152 Combination therapy in studies utilisingapatinib with TACE have achieved better clinical effectivenessthan TACE alone with tolerable AEs153“ Recentlythecombination of apatinib with the antiPD1 monoclonal antibodycamrelizumab achieved partial response rates of Theresults of other ongoing trials including the phase IIItrialcomparing TACE and apatinib with sorafenib as firstline treatmentfor locally advanced or metastatic and unresectable HCC NCT and the adjuvant apatinib after hepatectomy for theprevention of tumor recurrence NCT03722875 and NCT03261791will hopefully prove effective and add to the presently availabletherapeutic optionsThese promising results have stimulated the investigation ofother new agents the combinations of agents and regimenswhich have been thoroughly discussed in a recent review fromZhu and Sun154 The combination of bevacizumab and erlotinibhas been extensively evaluated as first155 or secondline inadvanced HCCs156“ but unfortunately the heterogeneousnature of the results precludes firm conclusions and recommendations Recently a singlearm metaanalysis of prospectivestudies found that combination therapy with bevacizumab anderlotinib used as secondline treatment was associated with afavorable PFS weeks P and OS months P suggesting that future welldesigned and sufficiently poweredlargescale RCTsshould be able to identify the potentialcontribution of these agents163Signal Transduction and Targeted Therapy 0cTable Trials investigating the combination therapy of ICIs and TKIs in HCCTrial nameidentifierPatient No Study type LineInterventionsPrimaryendpointStudy statusTargeted therapy for hepatocellular carcinomaHuang et alLEAP002NCT03713593Phase IIIFirstLenvatinib pembrolizumab vs lenvatinib PFSOSPhase IIIFirstPhase IIIPhase IIIPhase IIIFirstFirstFirstPhase IIIFirstFirstPhase IIFirstPhase IIPhase Ib II FirstFirstPhase IIPhase IbFirstNivolumab ipilimumab vs lenvatinib orsorafenibCabozantinib atezolizumab vs sorafenib PFSOSSintilimab IBI305 vs sorafenibOS ORRCamrelizumab apatinib vs sorafenibOSPFSOSCamrelizumab apatinib vs FOLFOX orsorafenibNivolumab lenvatinibNivolumab sorafenibSorafenib pembrolizumabAnlotinib sintilimabAvelumab axitinibOSORR AEMTD ORRORRORR AEAEActive notrecruitingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingCompletedCheckMate 9DWNCT04039607COSMIC312NCT03755791ORIENT32NCT03794440SHR1210III310NCT03764293SHR1210III305NCT03605706IMMUNIBNCT03841201NCT03439891NCT03211416KEEPG 04NCT04052152VEGF Liver NCT03289533KN743NCT03347292GOINGNCT04170556REGOMUNENCT03475953NCT02423343aaRegorafenib pembrolizumabFirstPhase ISecond Regorafenib nivolumabPhase IISecond Regorafenib avelumabPhase IIIPhase Ib II Second Galunisertib nivolumabAEAECR PRPhase Ib MTDOngoingOngoingOngoingOngoingPFS progressionfree survival OS overall survival ORR objective response rate AE adverse events MTD maximum tolerated dose CR complete response PRpartial responseaTrials enroll not only HCC patientsTable Trials investigating targeted therapy in advanced HCCTrial nameidentifierPatient noStudy typeLineInterventionsPrimary endpointStudy statusIMbrave150 NCT03434379ZGDH3NCT02645981HIMALYYA NCT03298451RATIONALE301 NCT03412773PHOCUSNCT04344158ALTER0802 NCT02809534AHELPNCT02329860KEYNOTE394 NCT03062358NCT04080154Phase IIIPhase IIIIIPhase IIIPhase IIIPhase IIIPhase IIIPhase IIPhase IIIPhase IIIPhase IIFirstFirstFirstFirstFirstFirstFirstSecond Apatinib vs placeboSecond Pembrolizumab BSC vs placebo BSCSecond AnlotinibAtezolizumab bevacizumab vs Sorafenib OS PFSDonafenib vs sorafenibOSDurvalumab tremelimumab vs sorafenib OSOSTislelizumab vs sorafenibPexaVec sorafenib vs sorafenibOSAK105 anlotinib vs sorafenibOSPFS 12WAnlotinibOSOSPFSOS overall survival PFS progressionfree survival BSC best supportive careCompletedCompletedOngoingOngoingOngoingOngoingOngoingCompletedOngoingOngoingundergoingevaluationandPreclinical evidence for cyclindependent kinase CDK targetingtherapies in HCC has showed promise and supports theirinvestigation164“ especially with the potential ability toabrogate the emergence of sorafenib resistance167 and sensitizeHCC to regorafenib treatment168 A number of CKD inhibitors arepalbociclibpresentlyNCT01356628 milciclibribociclibNCT02524119 The antiMET monoclonal antibody emibetuzumab exhibited the greatest antitumor activity in HCC whencombined with ramucirumab and had an excellentsafetyprofile169 and for HCC with high MET expression there was analmost 3fold increase in PFS vs months relative to thosewith low MET expression suggesting the potential for furtherbiomarkerdriven clinical trials Rigosertib is a synthetic benzylstyryl sulfone small molecule inhibitor which has been used in theNCT03109886includingtreatment of monomyelocytic leukemia and due to its activity as aRAS and PLK1signaling inhibitorit was investigated in HCCpatients who demonstrate upregulation of PLK1 during tumordevelopment and HRAS expression in advanced HCC Highexpression levels of PLK1 are also significantly correlated withpoor patient survival and the multiple effects of rigosertib couldbe beneficially employed to produce a therapeutic œdualhitapproach in selected patients170 Donafenib is a novel multikinaseinhibitor which is similar to sorafenib displaying comparable orbetter safety and efficacy when treating advanced HCC in phase1b trial and phase studies using sorafenib as the controlNCT02645981171 There are ongoing trials evaluating novelagents such as anlotinib another multikinase inhibitor which isorally administered and targets VEGFR fibroblast growth factorreceptor FGFR plateletderived growth factor receptors PDGFRSignal Transduction and Targeted Therapy 0cTargeted therapy for hepatocellular carcinomaHuang et alTable Molecular classification of HCCResearcherBoyault et alHoshida et alSchulze et alSia et alKurebayashi et alShinata et alJiang et alYearClassificationG1“G6S1“S3Msig “iC1“iC3 HCCs with adaptive or exhausted immune responsesImmunehigh mid and “lowMS1 ˆ’ ˆ’SI SII and SIIITypeTranscriptomeTranscriptomeExome sequencingMultiomocisImmune cell profilingImmunomicroenvironmentTranscriptome and gonomeProteomicsCase no and ckit NCT02809534 Tivozanib is another oralinhibitor ofVEGFR123 with promising activity against HCC in vivoNCT01835223 and TRC105 which despite demonstrating clinicaltolerated in HCC patients followingactivity and being wellsorafenib has notto date met prespecified criteria and itsdevelopment in HCC continues as combination therapy withsorafenib NCT02560779Biomarkerdriven targeted therapyDespite extensive research investigating potential biomarkers to aidthe development of protocols for the treatment of HCC none haveso far been identified to be able to predict the effect of or responseto treatment with sorafenib172“ Although the molecularclassification of HCC has been widely reported Table to date itremains unclear whether this basic genomic and proteomic datawill prove valuable in guiding targeted therapies183“The continued belief that the future lies with personalizedtreatment which will be made possible through the rapiddevelopments in next generation sequencing and the precisionmedicine that it underpins have encouraged the development ofnovel trial designs191 These novel trials designs offer new hopethat biomarkerdriven targeted therapies can be modul

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

Deacetylases inSkeletal Muscle Physiology andSystemic Energy HomeostasisImplications for Metabolic Diseasesand TherapyHaili Tian1  Sujuan Liu2  Jun Ren3 Jason Kai Wei Lee456 Ru Wang1 and Peijie Chen1 School of Kinesiology Shanghai University of Sport Shanghai China Department of Anatomy and Histology Schoolof Basic Medical Sciences Tianjin Medical University Tianjin China Department of Cardiology Shanghai Instituteof Cardiovascular Diseases Zhongshan Hospital Fudan University Shanghai China Department of Physiology Yong LooLin School of Medicine National University of Singapore Singapore Singapore Global Asia Institute National Universityof Singapore Singapore Singapore N1 Institute for Health National University of Singapore Singapore SingaporeSkeletal muscle is the largest metabolic an in the human body and is able torapidly adapt to drastic changes during exercise Histone acetyltransferases HATs andhistone deacetylases HDACs which target histone and nonhistone proteins are twomajor enzyme families that control the biological process of histone acetylation anddeacetylation Balance between these two enzymes serves as an essential elementfor gene expression and metabolic and physiological function Genetic KOTG murinemodels reveal that HDACs possess pivotal roles in maintaining skeletal muscles™metabolic homeostasis regulating skeletal muscles motor adaptation and exercisecapacity HDACs may be involved in mitochondrial remodelinginsulin sensitivityregulationturn onoff of metabolic fuel switching and orchestrating physiologicalhomeostasis of skeletal muscles from the process of myogenesis Moreover manymyogenic factors and metabolic factors are modulated by HDACs HDACs areconsidered as therapeutic targets in clinicaltreatment of cancer‚ammation and neurological and metabolicrelated diseases This review will focuson physiological function of HDACs in skeletal muscles and provide new ideas for thetreatment of metabolic diseasesresearch forKeywords histone deacetylases exercise capacity skeletal muscle metabolism muscle physiologyINTRODUCTIONSkeletal muscle is the largest metabolic an in the human body consuming about of theentire body daily expenditure of energy Durnin It produces various secrete factors andparticipates in the interplay among multiple tissues and ans Pedersen and Febbraio Mizgier Muscular contraction is one of the main physiological functions of skeletalmuscles and plays a role in maintaining an and systemic metabolic homeostasis Guo Proper exercises help build up body defense to combat various diseases including obesitytype diabetes Alzheimer disease osteoarthritis and so on Luan These importantEdited byBrian James MorrisThe University of Sydney AustraliaReviewed bySean L McGeeDeakin University AustraliaViviana MoresiSapienza University of Rome ItalyCorrespondenceRu WangwangrutysuseducnPeijie Chenchenpeijiesuseducn These authors have contributedequally to this workSpecialty sectionThis was submitted toIntegrative Physiologya section of the journalFrontiers in PhysiologyReceived May Accepted July Published August CitationTian H Liu S Ren J Lee JKWWang R and Chen P Roleof Histone Deacetylases in SkeletalMuscle Physiology and SystemicEnergy Homeostasis Implicationsfor Metabolic Diseases and TherapyFront Physiol 103389fphys202000949Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise Capacityfunctions require delicate regulations in muscle from thelevel of genome to signal transduction establishing muscleplasticity and responses to environmental stress AcetylCoAas a central metabolite of amino acidfatty acidglucose notonly serves as fuel for energy expenditure but also bridgesthe gap between environmental stress and anismal responseLempradl Liu This control occursthrough posttranslational modification on nucleosomes histonelysine residues and other signal transducing proteins whichin turn controls accessibility of DNA to regulatory factorsand protein activity Acetylation one of the most prevalentmodifications of proteinis believed to function as a keymodulator of chromatin structure and signal transduction andprovides an avenue to couple extracellular stimuli with genomeduring muscle metabolism process by regulating acetylation anddeacetylation Haberland histone deacetylasesHDACs as a family of proteindeacetylases have been demonstrated to moderate physiologicalhomeostasis and development by deacetylation Haberland Table In humans there are types ofHDACs which are classified into four categories based onhomologous proteins in yeast namely Class I Rpd3like proteinHDAC1 Class II Hda1like proteins are classifiedas Class IIa HDAC4 and Class IIb HDAC6 Class III Sir2plike nicotinamide adenine dinucleotideNADdependent SIRT1 and ClassIV HDAC11 containing homologous domain with bothRpd3 and Hda1 Seto and Yoshida HDACs modulategene expression and protein activity through deacetylatingproteins When histone lysine εamino acid in the nucleosomeis acetylated it can neutralize positive charge before looseningchromatin structuretranscriptionfactors to DNA and expression of downstream target genesBy contrast histone deacetylation compresseschromatinstructure thereby inhibiting transcriptional gene expressionShahbazian and Grunstein to promote binding ofBiochemical properties of HDACs have been comprehensivelyreviewed Shahbazian and Grunstein Howeverthephysiological functions of HDACs have yet to be well examined inskeletal muscles A series of researches shed light on the potentialof drugs targeting HDACs to improve muscle fitness and cardiacmuscle disease which needs further clarification of HDACsphysiological function to understand the mechanisms Bai Galmozzi Xie Zhang and Ren Gaur Ample work has revealed the role of HDACin muscle physiology such as skeletal muscles me olism and thusexercise capacity McGee and Hargreaves Table Class IHDACs can interact with myocyte enhancer factor MEF2MyoD regulating myogenesis and exercise capacity Mal Puri Ohkawa Gregoire HDAC3 participates in myocytes diï¬erentiation and is linked toskeletal muscles metabolic fuel switching Gregoire Hong Class II HDACs can be phosphorylated byHDAC kinases and are transduced from nucleus to cytoplasmin response to cellular stress mediating muscle fiber switch andaï¬ecting the pathway of insulin sensitivity such as Glut4 andAKT Haberland McGee and Hargreaves Class III HDACs target a crucial mitochondrial biogenesis factorperoxisome proliferatoractivated receptor gamma coactivator alpha PGC1α in skeletal muscles liver and fat tissue Whiteand Schenk Therefore we will further discuss howthese HDACs ‚uence respective downstream targets exercisecapacity and therapeutic eï¬ects in human diseasesCLASS I HDAC HDAC123Regulation of Myogenesis by Class IHDACsIn previous studies HDAC123 was shown to be associated withthe process of myogenesis or myocyte diï¬erentiation Mal Puri Ohkawa Gregoire HDAC1 tightly binds to MyoD and deacetylates specific sites toinhibit the expression of musclespecific genes such as MHC andMCK in myoblasts Mimicking muscle diï¬erentiation conditionby serum deprivation HDAC1 protein gradually decreasesduring myocyte diï¬erentiation and transfers to bind to the tumorsuppressor pRb accompanied by isolation of HDAC1MyoD andtranscriptional activation of musclespecific genes Puri A HDAC1 H141A mutant incapable of binding withMyoD loses its inhibitory property on musclespecific genes inmyoblast state Mal In the late stage of myocytediï¬erentiation activating factor gradually switches from MyoDto myogenin occurring with a decrease in the MEF2D inhibitoryregulator HDAC2 Simultaneous overexpression of myogeninand MEF2D can enhance the expression of the musclespecificgene MHC in the absence of MyoD Ohkawa MEF2D a key factor that controls myocyte diï¬erentiationcan only be eï¬ectively deacetylated by HDAC3 rather thanHDAC128 Gregoire In addition HDAC3 caninhibit autoacetylation of acetyltransferases p300 and p300CBPassociated factor PCAF Thus HDAC3 impedes MyoDMEF2PCAF to form a multicomplex disturbing MEF2dependentmyogenic transcription Gregoire Figure Redundant Roles of HDAC12 inMaintaining Sarcomere Homeostasis inMuscleLoss of function of HDAC12 inhibits autophagy flux in skeletalmuscles tissue accompanied by decreased LC3 III ratio andp62 accumulation under fasting condition Moresi Toxic autophagy intermediates accumulate in muscle fibersin which class I HDACs deficiency led to impaired exercisecapacity Moresi This oï¬ers convincing evidenceon the role of HDAC12 in stabilizing basic structure andfacilitating development in muscles Genetic models suggestthat Class I HDACs control the physiological homeostasis ofskeletal muscles A germline deletion shows that wholebodyknockout of either HDAC1 or HDAC2 leads to mortalityin mice prior to the perinatal period Montgomery However a tissuespecific single deletion of HDAC1or HDAC2 in myocardium does not cause any phenotypeMontgomery Double knockout HDAC12 in theFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityTABLE The targets of HADCs in skeletal muscles and their physiological functionsClassMembersDown targetsGenetic KOTG phenotypeReferencesIHDAC1MyoD†“ PTEN†“ FoxOHDAC2HDAC3HDAC8HDAC4HDAC5HDAC9HDAC7HDAC6MyoD†“ MEF2D†“ NFκBp65†“MEF2D PCAF†“ Ampd3†“RCAN1 MEF2ACpt1b†“MEF2†“ Glut4†“ Myosin HeavyChain PGC1α and Hsc70“Pax7†“MEF2†“ Glut4†“ Baf60c†“Atg7 Beclin1 LC3†“Dach2Myog Gdf5†“MEF2†“Pax7 MFN1†“ Fam65bdysferlin and MAFbxHDAC10SIRT1“PGC1α STAT3†“IIAIIBIII“MnSOD Hexokinase II PDHsubunit E1αMalonyl CoA decarboxylase†““NFκBMstnSIRT2SIRT3SIRT4SIRT5SIRT6SIRT7Functionally redundant HDAC12 DKO mice causesmitochondrial abnormalities and sarcomeredegenerationPuri Ohkawa Beharry Cho Zhu Bin mKO mice Enhanced amino acid\\lipid metabolism andendurance exercise Causes IR under basal conditionGregoire Han Yuan Hong “Functionally redundant HDAC45 DKO HDAC59 DKOHDAC459 TKO increases type I fiber percentage andMcKinsey 2000a Choi Niu Luo oxidation metabolismYuan Meng Niu Macpherson Zhang Dressel “KO mice Causes mitochondrial oxidative damagemitofusion defect Protect against muscle wastingBalasubramanian Lee Ratti “mKO mice Mediates CR induced insulin sensitivity hasno effect on glucose homeostasis or exercise capacityTG mice no effect on glucose homeostasis or exercisecapacityKO mice Exacerbates obesity and IR in HFDKO mice Exacerbates obesity and IR in HFDKO mice Increased exercise tolerance and protectagainst HFDinduced obesity“Rodgers Schenk Boyle Jing Lantier Laurent KO mice Abnormal hypoglycemia TG mice Protectagainst HFD mKO mice Impaired insulin sensitivity lossKanfi Xiao Cui Samant of muscle mass““HDAC11IVActivation inhibition†“ unknown KO wholebody deletion TG wholebody overexpression DKO double knockout TKO triple knockout mKO musclespecificknockout CR caloric restriction HFD high fat diet IR insulin resistance““““heart leads to severe cardiomyopathy in mice indicating theobligatory role for HDAC12 in specific tissues Montgomery In skeletal muscle double knockout of HDAC12by myogeninCre causes mitochondrial abnormalities andsarcomere degeneration disrupting fundamental structural unitsof myofibers Moresi Therefore these findingsdemonstrate that HDAC12 redundantly maintains sarcomerehomeostasis in skeletal muscleHDAC3 Functions as a Fuel Switch inMuscle Energy Metabolismthe enzymatic core of nuclearHistone deacetylases isreceptor corepressorNCoR and silencing mediator ofretinoic acid and thyroid hormone receptors SMRT corepressor correspondingly NCoR and SMRT corepressoractivate HDAC3 through its SANT domain enzyme activityKaragianni and Wong Mice suï¬er from insulin resistanceafter specifically knocking out HDAC3 in skeletal musclesmKO while their endurance exercise abilities are enhancedHong It seems a selfcontradictory phenomenonbecause former studies pointed out that increased enduranceexercise capacity enhances insulin sensitivity The study showsthat insulin signaling cascades including pAKTAKT pIRS1S1101 and pGSKGSK have no change in HDAC3 mKO musclewhile glucose uptake and insulin sensitivity are impaired inglucose tolerance test GTT and insulin tolerance test ITTHong which is called the œdissociation eï¬ect byresearchers Lipid tends to be used as energy fuel in HDAC mKOmice which inhibits glucose absorption but does not ‚uenceinsulin signaling sensitivity Hong Further workfound that the HDAC3 knockout upregulates the expressionof AMP deaminase AMPD3 the first ratelimiting enzymein purine metabolism in skeletal muscles Fortuin Hong AMPD3 can deaminate AMP to form IMPfacilitating aspartic acid to transmit into fumarate and malate theintermediate metabolites of tricarboxylic acid cycle TCA cycleHong Research studies found that exercise inducedglucose labeled 13C6 expressed a lower glycolysis flux rate inmuscles of HDAC3 mKO but a higher expression in the TCAFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Controls of myogenesis by Class I HDACs The inhibitory effects of HDACs on myogenic factors MEF2 and MyoD maintain a primary myoblast stateDuring myocyte differentiation and myogenic process the inhibition is lifted by other factors facilitating MEF2MyoD complex formation to promote myogenesis“Ac deacetylationcycle intermediates Hong Gong Thus ageneral increase in TCA cycle metabolites activates the oxidationin mitochondria In vitro radioactive aspartic acid isotope tracertest showed that inhibiting HDAC3 or overexpressing AMPD3can increase amino acid metabolism rate and enhance fatty acidmetabolism thereby downregulating glucose metabolism Hong Figure In liver knockout HDAC3 causes severeliver steatosis and can be rescued by wildtype or catalyticallyinactive mutants of HDAC3 indicating an enzymatic activityindependent function Sun However the musclefuel switching in HDAC3 mKO mice cannot be rescued by anenzymatic inactivity mutant HDAC3 Song Thisfinding demonstrates that HDAC3 controls the fuel utilizationin skeletal muscles and is dependent on its enzymatic activityThe relationship between exercise and glucose uptake is far morecomplex than one could expectCLASS II HDACs HDAC45796Phosphorylation and NucleusCytoplasmShuttle of Class II HDACsThe MEF2 family is a class of transcription factors that areclosely associated with myogenic basic helixloophelix domainsincluding MyoD Taylor and Hughes MEF2 may interactwith Class IIa histone deacetylase HDAC45 to inhibit thetranscription of MEF2dependent genes thereby impeding thediï¬erentiation from myoblast to myotube McKinsey et al2000a During exercise Ca2 flows out from sarcoplasmicreticulum and activate calciumcalmodulindependent proteinkinase CaMK which phosphorylates HDAC45 and mediatesHDAC45 shuttle from the nucleus to the cytoplasm thusreleasing the repressive eï¬ect of HDAC45 on MEF2dependentgenes McKinsey 2000b Yuan Further researchdemonstrates that the nucleoplasmic shuttle of HDAC4 relies on binding while the shuttle of HDAC5 depends on CaMKphosphorylation at serine and sites first and thenexporting from the nucleus by binding with McKinsey 2000b As expected HDAC7 in Class IIa also possessesthe ability to be exported from nucleus to cytoplasm Gao suggesting that there may be redundant functions of ClassIIa HDACs Except for Class IIa knocking out Class IIb HDAC6in embryonic stem cells ESCs can promote ESCs to diï¬erentiateinto myoblast cells and transplantation of ESCs with HDAC6knockdown can also help the regeneration of wound skeletalmuscles Lee type I fiberthe proportion ofRegulation of Class II HDACs onFiberType Switch MitochondriaRemodeling and Energy Stress inSkeletal MuscleEvidence showsinthatskeletal muscles has no change in animal knocked out aloneany member of Class IIa Potthoï¬ Howeverthe proportion of type I fiber is significantly increased inHDAC45 DKO HDAC59 DKO HDAC459 TKO miceas well as exercise capacity of skeletal muscles Potthoï¬ Altogether the finding indicated a functionalredundant mechanism in Class II HDACs family Previousstudy found that Class IIa HDACs are closely associated withthe MEF2 family and can repress their downstream targetgenes McKinsey 2000a Therefore similar to Class IIaHDACs DKO the proportion of type I fiber is decreased in theMEF2C and MEF2D knockout mouse models overexpressingMEF2C results in increasing type I fiber and exercise capacityHDAC45 not only regulates exercise capacity but also governsmotor adaptation Several studies had shown that exerciseFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class I HDACs regulates sarcomere structure and exercise capacity in mature muscle A schematic diagram shows Class I HDACs regulate exercisecapacity on different levels HDAC12 have redundant roles in modulating autophagy flux in muscle eliminating toxic factors in muscle HDAC3 serves as a fuelswitch and promotes muscle to use fatty acid to adapt endurance exercisepromotes phosphorylation of HDAC45 mediated by CaMK andadenosine monophosphateactivated protein kinase AMPKthen increasing a MEF2dependent transcription of Glut4 andmusclespecific genes which participates in the adaptationand plasticity of skeletal muscles McGee McGee and Hargreaves Besides exercise glucose can alsoactivate KATP channeldependent calcium signaling and CaMKwhich induce phosphorylationdependent nuclearcytoplasmtransportation of HDAC5 Meng The leavingHDAC5 releases its repressive eï¬ect on Baf60c and enhancinginsulinindependent AKT activation Meng Usingspecific inhibitor of class IIa HDAC activity myosin heavy chainPGC1α and heatshock cognate HSC70 are confirmed to beone of the substrates of HDAC4 in denervationinduced atrophymuscle in comparison with acetylated protein enrichment withuntreated group Luo Taken together Class IIaHDACs play critical roles in exercise capacity remodeling andnutrient sensing of skeletal muscles Figure For Class IIbit was revealed that glucose deprivationinduced mitochondrialfusion mediated by mitofusion1MFN1 is significantly disrupted in HDAC6 KO mice causingmitochondria degeneration which isreversed followingapplication of MFN1 acetylationresistant mutant Lee This study suggested that MFN1 deacetylation by HDAC6plays an important role in mitochondrial adaptive energyproduction and remodeling of skeletal musclesCLASS III HDACsSir2HOMOLOGSIRT1634NuclearLocated SIRT16Numerous studies have found that caloric restriction CRextends lifespan of mammals Caenorhabditis elegans and fruitflies while such eï¬ect is lost in SIR2 or NPT1 mutant C elegansstrains Imai Lin SIR2 encodes thesilencing protein Sir2p and NPT1 participates in the synthesis ofNAD NAD is an essential cofactor for Sir2pClass III HDACsto exert enzyme activity rather than Class I II and IV relayingon the zinc Imai Lin In mammalsSir2 has homologous proteins and these proteins are essentialto mitochondrial energy homeostasis antioxidant defense cellproliferation and DNA repair VargasOrtiz SIRT1 Mediates Energy Stress Adaptation in SkeletalMuscles reported that SIRT1 promotes theRodgers et altranscriptional activity of PGC1α by deacetylating the PGC1αat K13 site and mediates CRinduced gluconeogenesisrelatedgenes G6P PEPCK expression in liver Further research showedthat the NADNADH ratio and the enzymatic activity of Sir2 asensor of redox state were decreased in diï¬erentiating myocyteswhich relieved the inhibitory eï¬ect on MyoD and then promotedcell diï¬erentiation Resveratrol RSV a compound that maytarget SIRT1PGC1α can improve mitochondrial function andmetabolic homeostasis owning a potential to prolong lifespanRSV loses its activating eï¬ect on PGC1α in SIRT1 ˆ’ˆ’ MEFsLagouge Moreover RSVtreated mice at a dose of gkg increased their exercise capacity oxygen consumptionand improved insulin sensitivity against highfat induced obesityLagouge In addition RSV could enhance thedeacetylation of PGC1α in brown fat tissue BAT and skeletalmuscles which promotes mitochondrial production oxygenconsumption and thus improves metabolic syndrome Lagouge The function of SIRT1 in regulating skeletal musclesmetabolic homeostasis and exercise capacity has attracted greatattention from researchers Researchers further generated skeletalmusclespecific SIRT1mKO mice and found that mKO miceFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Control of muscle exercise adaption and mitochondrial function by Class II HDACs A Schematic of the Class IIa HDACs nucleoplasm shuttle Understress condition CaMK and AMPK could be activated and phosphorylate Class IIa HDACs Phosphorylated HDACs then bind with and shuttle to cytoplasmrelieving inhibitory effects on MEF2 and Baf60c B Class IIb HDAC6 can deacetylate MFN1 and facilitates its mitofusion function P phosphorylationlost the eï¬ect of CRinduced increasing insulin sensitivity andwere unable to deacetylate and inactivate STAT3 resulting inupregulating the phosphatidylinositol3kinase PI3K inhibitoryregulator p55αp50α expression Schenk Althoughknockout or overexpression of SIRT1 in skeletal muscles hasno eï¬ect on endurance capacity or glucose homeostasis in miceGurd Philp White Svensson some studies have shown that AMPK regulatesenergy metabolism of skeletal muscles partially mediated bySIRT1 and SIRT1 is significantly increased after enduranceexercise Suwa Canto Overexpressionof Sirt1 in skeletal muscles by adenoassociated virus AAV1promotes the expression of oxidationrelated genes includingPpargc1a Tfam Cpt1b and Pdk4 while it has no eï¬ect on insulinsensitivity of body Vila But overexpressing Sirt1in liver by AAV8 can protect from fatty liver induced by highcarbohydrate food HCD Vila Taken togetherthe aforementioned studies suggest that SIRT1 possesses limitedregulation capacity of skeletal muscles motility and SIRT1may modulate mitochondrial homeostasis and mediate skeletalmuscles adaptation under certain physiological conditions suchas CR aging and regeneration Gomes Ryall Figure In addition there may be more beneficial eï¬ectsof SIRT1 on metabolism in the liver or fat tissue Lagouge Vila Stefanowicz SIRT6 Improves Muscle Fitness andExercise CapacitySIRT6 is another Sir2like deacetylase located in the nucleusWhole body deletion of SIRT6 causes mice death around weeks owning to hypoglycemia Xiao Neverthelessthe skeletal musclespecific knockout of SIRT6 causes insulinresistance and impairs glucose homeostasis of mKO mice Cui Because the activity of AMPK is reduced inSIRT6mKO mice the glycolipids absorption and utilizationof skeletal muscles are impaired and the exercise capacity ofthe mice was also attenuated Cui Furthermoreresearchers constructed the overexpressing SIRT6 Sirt6BACmice and found that their body weight and fat content werenormal but the Sirt6BAC mice could protect from HCDinducedhyperglycemia via increasing pAKTAKT induced by insulinstimulation and glucose absorption Anderson Vitroexperiments further demonstrated that the ability of glucoseuptake was enhanced mainly in skeletal muscles not in brainiWAT eWAT tissues of Sirt6BAC mice Anderson These results shows the potential eï¬ects of SIRT6 targeted inskeletal muscles on improving exercise performance and treatingmetabolic diseases Figure MitochondriaLocated SIRT34SIRT34 Maintains Mitochondria Function in SkeletalMusclesSIRT34 are mitochondrialocalized deacetylases and responsiblefor regulating the deacetylation of proteins in mitochondria andmaintaining mitochondrial homeostasis After feeding highfatdiet HFD accelerated obesity attenuated insulin sensitivityworsened fatty liver and increased acetylation of proteins inmitochondria were observed in SIRT3 KO mice Hirschey Multitissue proteomics and physiological examinationreveals that SIRT3 is responsible for mitochondrial acetylatedFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class III HDACs promote a stressinduced adaptation in muscle and regulate mitochondrial function Class III HDACs are NADdependentdeacetylases and may function as a redox sensor in muscle cell SIRT1 could activate PGC1alpha by directly deacetylasing K13 site and regulate insulin sensitivityby targeting STAT3 Mitochondrialocated Class III HDACs control mitochondrial function by deacetylating certain mitochondrial proteins modulating muscle fuelutilization and exercise capacityproteome regulation and metabolic fuel switching in brainheart kidneyliver and skeletal muscles DittenhaferReed Protein enrichment analysis discovers that itsregulatory proteins were mainly concentrated in lipid metabolismDittenhaferReed Knocking out SIRT3 leadsto reduction in the levels of deacetylation of manganesesuperoxide dismutase MnSOD mitochondrial complex II andpyruvate dehydrogenase PDH subunit E1α a downregulationof hexokinase II HK II binding with mitochondria resultingin impaired glucose and lipid metabolism of skeletal musclesLantier Interestingly SIRT3 liverspecific knockouthepˆ’ˆ’ and skeletal musclespecific knockoutskmˆ’ˆ’mice did not aï¬ect glucose homeostasis under chow or HFDconditions FernandezMarcos The above resultsindicate that SIRT3 has an important role in metabolic regulationbut in specific physiological processes such as redox stateexercise and aging and its regulatory mechanism is yet definedin skeletal muscles Kong Robin Williams A recent study found that SIRT4 knockoutcan resist HFDinduced obesity and increase endurance exercisein mice by repressing malonyl CoA decarboxylase which wasa key enzyme controlling fatty acid betaoxidation and wasreported to regulate muscle fuel switching between carbohydratesand fatty acids Koves Laurent Moreover knockdown of SIRT4 by adenoviral shRNA canincrease the mRNA and protein content of SIRT1 therebyenhancing the expression of fatty acid oxidation genes andmitochondrial oxidation capacity in hepatocytes and myotubesNasrin Table THERAPEUTIC TARGETS OF HDACsAND THEIR POTENTIAL FORMETABOLIC DISEASESA comparative analysis used HDAC paninhibitor SAHA aclass I HDAC selective inhibitor MS275 and a class IIHDAC selective inhibitor MC1568 to treat C2C12 separatelyIt was found that only MS275 could significantly stimulatemitochondria biogenesis and oxygen consumption Galmozzi In obese diabetic mice it was found that specificallyinhibiting Class I rather than Class II HDACs improvedGTT and insulin sensitivityincreased oxidative metabolismof skeletal muscles and adipose tissue and reduced bodyweight Galmozzi Similar to the eï¬ect of MS275knockdown HDAC3 in C2C12 could also increase Pgc1αGlut4 Tfam Idh3α transcription suggesting that HDAC3 andits target genes played an important role in the above eventsGalmozzi For Class II HDACs studies have found that scriptaid a ClassIIa HDAC inhibitor has similar eï¬ects to exercise Six weeksof scriptaid administration significantly improved enduranceperformance and significantly increased the wholebody energyexpenditure and the expression of lipid oxidation related genesPdk4 Cpt1b Pgc1α Pparδ etc of C57BL6 mice Gaur One possible explanation of above observation is thepotential target of scriptaid inhibition of titin a structure proteinof sarcomere deacetylation and downstream genes of Class IIaHDACs Gaur Huang Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityIn mice treated with SPT1720 another activator of SIRT1overtly enhanced endurance exercise ability was noted Moreoverthese mice were protected from HFDinduced obesity and insulinresistance owing to an upregulation of the oxidative metabolismin skeletal muscles liver and BAT tissues Pacholec In order to further explore the principle of SIRT1 activation theresearchers purified SIRT1 in vitro and added SRT1720 and itsanalogs SRT2183 SRT1460 and RSV and they found that theenzymatic activity of SIRT1 was not enhanced suggesting thatthese drugs may indirectly activate SIRT1 Pacholec The enzymatic activity of SIRT1 largely depends on the contentof cofactor NAD This implied that the enhancement of SIRT1activity may be achieved through indirect upregulation of NADStudy has discovered that knocking out poly ADPribosepolymerase1 PARP1 which is a NAD consuming enzymecould increase NAD content and enhance the activity of SIRT1in BAT and skeletal muscles Bai PARP1 inhibitorscan upregulate the proteins of mitochondrial respiratory chaincomplexes in mice enhance the aerobic oxidation capacity ofmitochondria and improve mitochondrial defects in the primarymyotubes of obese humans Bai Pirinen et alResveratrol was initially reported as an SIRT1 activatorthat improves mitochondrial function and exercise capacity inmice and resists HFDinduced obesity Lagouge However subsequent studies have discovered that administeringthe same dose of RSV to rats or mice does not increasemitochondrial protein content Higashida Bycontrast overexpression of SIRT1 in the triceps muscle of ratsdecreases the mitochondrial protein content Higashida In a doubleblind human trial healthy and obesemen were supplied for 30day RSV in which the data showedthat RSV can improve systolic blood pressure and homeostasismodel assessment HOMA indexindicating glucose metabolismability simulating the eï¬ect of CR Timmers However some researchers found that the overexpression ofSIRT1 alone cannot mimic the CR eï¬ect in transgenic mice andthe transcriptomic changes in various tissues were quite diï¬erentor even opposite Boutant Such an opposite situationmay explain that the genetic model and compound stimulationare not completely consistent RSV as a potential metabolicsyndrome treatment drug still needs largescale populationsample verificationCONCLUSIONThe very first mammalian histone deacetylase HDAC1 wascloned and isolated by Taunton and sabout HDACs have been published in the last years CurrentlyREFERENCESAnderson J G Ramadori G Ioris R M Galie M Berglund E D CoateK C Enhanced insulin sensitivity in skeletal muscle and liver byphysiological overexpression of SIRT6 Mol Metab “ 101016jmolmet201509003we know at least HDAC proteins They are responsible foreradicating epigenetic modifications establishing an epigeneticoï¬ chromatin state and regulating heritable gene expressionYang and Seto Haberland In these processeseach HDACs may play a diï¬erent role Table What kind ofgeneprotein is the specific downstream target of these HDACsIs its enzyme activity related to intracellular localization andthe formation of multiprotein complex It is still one of thekey and difficult issues in this field Based on previous researchexperience some techniques may be used to further experimentsas follows HDACs interacting proteinscomplex coIP Proteinacetylation western Histone acetylation target geneChIP High through put proteomicsacetylome with specificHDACs inhibitor etctherapeuticare potentialHistone deacetylasestargetsand clinical drugs such as SAHA Vorinostat and FK228romidepsin have been used for antitumor treatment Bolden However they have two disadvantages greattoxic and side eï¬ects and difficulty in specific inhibition ofHDACs activity With the development of computer simulationtechnology and structural biology it is believed that more specificHDACs inhibitorsactivators can be constructed And researchesshould attach importance to HDACs regulatory factors likePARP1 when direct targeting on HDACs fails to show its eï¬ectsFurther development of their inhibitors with more specificityand trials for the treatment of metabolic diseases may have greatpotential as well

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

Microbial colonisation of the gastrointestinal tract of newly hatched chicks starts at hatch seeded from the immediate hatching environment and quickly results in dense colonisation The role of ecological factors in gut colonisation has been extensively investigated as well as the role of micro and macronutrients in supporting and selecting for bacterial species highly adapted for utilising those nutrients However the microbial community contained in poultry feed and its influence on colonisation and maturation of gut microbiota has not been directly addressed In this study we compared the microbiota found in poultry feed with the microbiota of ileum cecum and excreta to identify substantial overlap in core microbiotas of the compared groups We then investigated the microbiota present in raw feedstuffs meat and bone meal wheat corn canola barley soybean millrun shum poultry oil oats limestone and bloodmeal from four geographically distinct feedstuff suppliers Each of the feedstuffs had diverse microbial communities The meat and bone meal and bloodmeal samples had the most complex and distinct microbial populations There was substantial overlap in the phylogenetic composition found in the grain and seed samples barley canola corn millrun oats shum soybean meal and wheat Issues related to methodology viability of microbial communities in the gut and feed and the implications for biosecurity are discussedKeywords Feed Microbiota Colonisation ChickenKey points¢ Rapid microbiota colonisation starts from birth or hatch in poultry¢ Feed carries rich microbial community within and seeds the host during colonisation¢ In poultry feed ingredients grains have similar microbiota¢ The meat and bone meal and bloodmeal had the most complex and distinct microbial populationsCorrespondence DStanleycqueduau Sarah Haberechta and Yadav S Bajagai contributed equally to this work Institute for Future Farming Systems Central Queensland University Rockhampton QLD AustraliaFull list of author information is available at the end of the IntroductionUntil recent advances in technology allowed us to sequence total DNA from any environmental sample and to identify almost all bacteria including uncultured our knowledge was limited to a small proportion of bacteria we could grow and investigate using classic microbiology growth methods Nowadays instead of taking a fecal sample and pulling out only targeted bacteria on specially selected microbiological plates we could see thousands of species in a sample and investigate their role in the gut This revolutionised our knowledge of the intestinal microbiota and its role in health and digestion We now know that the number of gut bacteria outnumber our own cells up to ten times and contribute around of unique genetic material to our genetic pool Cebra Fujimura et a0al Joyce and Gahan 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 0cHaberecht a0et a0al AMB Expr Page of The poultry intestinal microbiota has evolved into its present form incorporating many different communities from the environment and the animals and humans they contact This means that the phylogenetic composition of chicken gut microbiota strongly but not entirely overlaps with the microbiota of humans and other farmed animals However recent research into chicken gut microbiota has suggested that industrialization of chicken production has transformed the chicken gut microbiota to such an extent that modern commercial chicken microbiota is probably very different in composition to that which would be found in native jungle fowl the wild precursors of the highly selected modern chicken due to the unnatural hatching practices with separation of chicks from hens and natural nest environments In hatcheries chicks are immediately exposed to bacteria different from bacterial communities to those that were selected in chicken guts and historically adapted to chicken as host Stanley et a0al The practices of commercial poultry production expose newly hatched chicks to microbes from the hatchery environment from human handlers transport boxes and transport vehicles prior to arrival at the farm Stanley et a0 al This process is typically carried out in the first days of life during the period when there is a rapid increase in bacterial diversity and load in the gut These environmental sources of bacteria appear to have a significant influence on the establishment of intestinal microbiota given that most significant colonisation in chickens occurs within the first few days posthatch Lu et a0al In the absence of the natural chicken feeding brooding and nesting habits CrisolMartinez et a0al the chicken gut bacterial community becomes susceptible to the influences of human and environmental sources Apajalahti et a0al Stanley et a0al Until recently it was believed that chicks are sterile in ovo and that colonisation begins posthatch The application of recent technological advances has suggested that at least in some circumstances there may be very lowlevel bacterial colonisation in ovo Castaneda et a0al There have been attempts to deliver probiotics in ovo AbdelMoneim et a0al Wilson et a0al however bacterial load in ovo is very moderate and has production significance mainly in case of in ovo infection Allan et a0 al Bradbury and Howell Bacterial colonization of the gut is likely to be a competitive process whereby the initial bacterial colonizers inhibit or promote the establishment of subsequent bacterial invaders by modifying the gut environment eg pH andor crossfeeding metabolites that support or retard growth of other bacteria The formation of the microbial community in chickens is very rapid with and bacteria per gram of contents in the ileum and the ceca respectively oneday posthatch Numbers increase to and respectively by day three and remain high while continuously adapting and responding to environmental changes and host stressors Baldwin et a0al Lu et a0al Stanley et a0al This indicates that the first days posthatch are critical for controlled and pathogen restricted microbial exposure In this study we investigated the potential role of microbiota from poultry feed in the establishment and development of chicken gut microbiotaThe food consumed by an animal has an important impact on the composition of gut microbiota it supplies nutrients that the microbiota can use directly or which are derived from the host processing of the feed input Fujimura et a0 al Digestible and simple carbohydrates in the gut are quickly absorbed and used by both host and microbiota in the small intestine where they have the strongest influence on microbiota composition However dietary fibre in the form of nondigestible carbohydrates NDC nonstarch polysaccharides NSP resistant nondigestible oligosaccharides RO and resistant starch RS survive the passage through the small intestine largely unprocessed to reach the ceca and large intestine where they promote the growth of beneficial bacteria such as Bifidobacterium Costabile et a0 al Holscher et a0 al Koecher et a0 al Lecerf et a0al Whelan et a0al Lactobacillus sp Costabile et a0al Walton et a0al Akkermansia muciniphila Fruge et a0 al and Faecalibacterium prausnitzii Benus et a0al Roychowdhury et a0al Some of the metabolic products derived from the bacterial digestion of dietary fibre have beneficial effects on intestinal and general health Holscher et a0 al Koecher et a0al Unfortunately unlike fibredigesting bacteria which are beneficial to the host proteolytic intestinal bacteria in the chicken such as E coli are often pathogenic Tolckmitt King et a0 al identified Enterococcus faecalis Enterococcus gallinarum and Proteus mirabilis as frequently observed proteasesecreting bacterial species in chicken Bacterial metabolism of protein results in toxic metabolites High protein content used in poultry diets may contribute to gut damage and is a predisposing factor in necrotic enteritis Stanley et a0al Herring et a0al demonstrated that in humans high maternal dietary protein intake results in intrauterine growth reduction and embryonic death due to the toxicity of ammonia homocysteine and H2S that are generated from amino acid catabolism Recently proteolytic species like Bacillus subtilis Chen et a0 al have become popular protein digestion probiotics that aid proteolysis without pathogenic effects AbdelMoneim et a0al however the toxicity of the metabolites produced 0cHaberecht a0et a0al AMB Expr Page of requires careful consideration when deciding on the protein content in poultry feedMost of the current knowledge on the role of microbiota in fat metabolism comes from human studies on the effects of highfat diet and obesity Highfat diets have not yet been extensively researched in poultry nutrition from a microbiota perspective Highfat diet consumption generally leads to an increase in Firmicutes and causes microbiota alterations clearly associated with obesity and intestinal diseases A highfat diet increases the number of fatloving bacteria such as Verrucomicrobia Deltaproteobacteria Ruminococcus Lachnospiraceae and Bacteroidaceae Hussain et a0al Despite these bacterial groups being predominantly nonpathogenic or even beneficial to the host under normal diet circumstances under high fat intake conditions cumulative metabolic products of these bacteria can result in multiple negative effects High fat intake results in microbiota and host products that enhance gut permeability and result in chronic gut inflammation and predisposition to food allergy This effect is mediated by fatinduced changes in the gut microbiota Once a high fat diet increases fatloving bacteria the host retains the community with increased ability to extract energy from food as shown in human high fat diet and obesity studies reviewed in Murphy et a0al Many studies have investigated the role of particular micro and macronutrients on microbiota development in chickens and other animals as well as the role of exogenous enzymes and other metabolites found in the feed Other than the nutrientdriven influence of feed on microbiota development the role of feed in contributing to the colonization of the gut with indigenous feed bacteria is underexplored In this manuscript we present evidence of the presence of diverse bacterial population in poultry feed rations that is continuously from the first to the last day of life seeding the poultry intestine with bacteria that are naturally present in feed and thus already adapted to digest and utilise components from that feedMaterials and a0methodsAnimal trialThe study was approved by the Animal Ethics Committee of Central Queensland University under the approval number A1409318 The animal trial used in this study was performed with a range of treatments with different probiotic supplementations However here we present a subsection of that data obtained from the control birds and data from the feed that was provided to the birds from the hatchThe birds were hatched in the Central Queensland University research facility hatchery using Ross Broiler eggs provided by Bond Enterprises Hatchery Toowoomba Queensland The eggs were hatched under relatively clean conditions and immediately moved to the poultry room where the feed was provided immediately posthatch The feed used was a Chicken Starter Diet Red Hen Laucke Mills Australia with no antimicrobials or coccidiostats and was used throughout the trial for weeks The feed was formulated to meet or exceed the National Research Council standards for broiler chickens NRC All birds were fed ad a0libitum and had unrestricted access to drinking water Birds were individually tagged by leg bands and weekly weights demonstrated weights equal or above the Ross performance standards Birds were euthanised at day posthatch CO2 BOC Australia and dissected Ileum and caecum contents were collected for microbiota analysis flashfrozen in liquid nitrogen and stored at ˆ’ a0 a0 a0 a0°C until further processing Excreta samples were also collected by placing a specially made transparent divider into the pen without removing the bird from the pen The clean paper towel was placed under the bird and excreta collected immediately after voiding Fortyeight samples from birds were successfully collected and sequenced for microbiota phylogenetic analysisFeedstuff microbiota experimentAfter confirming the presence of microbial communities in the feed used in the animal trial we further investigated specific feedstuff components used in the poultry feed The original poultry feed ingredients that were sampled included meat and bone meal wheat corn canola barley soybean millrun shum poultry oil oats limestone bloodmeal acid oil and tallow Samples of each type of feed ingredient were sourced from four different suppliers from four distinct regions of Australia three from the state of Victoria which has a temperate climate and one supplier from Queensland a state with a warmer subtropical climateDNA extractionDNA was extracted from the feed feed ingredients ileal content caecal content and excreta samples using the same method previously described Bauer et a0 al 2019a b Briefly a0g of samples were lysed and purified using a DNA spin purification column Enzymax LLC Cat EZC101 Kentucky US The DNA quality and quantity were estimated using a NanoDrop spectrophotometer a0s rRNA gene sequencingThe V3V4 region of a0 s rRNA genes were amplified using ACT CCT ACG GGA GGC AGC AG forward and GGA CTA CHVGGG TWT CTAAT reverse primers containing barcodes spacers and Illumina sequencing linkers Fadrosh et a0al The sequencing library was 0cHaberecht a0et a0al AMB Expr Page of prepared following the manufacturer™s protocol Illumina Inc San Diego CA USA The a0 s rRNA amplicon sequencing was completed on the Illumina MiSeq platform using × a0bp pairedend sequencingThe data was analysed using Quantitative Insights Into Microbial Ecology QIIME v191 Caporaso et a0al The FastqJoin algorithm was used to combine the pairedend sequences allowing no mismatches within the region of overlap Phred quality threshold had a minimum score of The Uclust Edgar was used to pick the OTUs at similarity and chimeric sequences were filtered using Pintail Ashelford et a0 al Taxonomic assignments were performed against the GreenGenes database v2013_8 using QIIME default parameters DeSantis et a0al The OTU abundance table was rarefied to calculate a UniFrac matrix Calypso Zakrzewski et a0al was used for further downstream analysis and visualisation of the data using Hellinger transformed Legendre and Gallagher OTU tableThe sequence data is publicly available at the MGRAST database under a project ID mgp455839ResultsFeed microbiotaThe composition of microbiota in the feed was compared to the structure of the microbiota in the different gut sections in the birds fed the same feed Feed microbial composition comprised of phyla Actinobacteria of all reads Proteobacteria of reads Firmicutes and Bacteroidetes spread across genera including in alphabetic order Arthrobacter Acinetobacter Aerococcus Bacillus Bifidobacterium Blautia Brachybacterium Brevibacterium Clostridium Comamonas Coprococcus Corynebacterium Dietzia Enterobacter Enterococcus Facklamia Frigoribacterium Jeotgalicoccus Lactobacillus Lactococcus Leuconostoc Microbacterium Oscillospira Paenibacillus Proteus Pseudochrobactrum Pseudomonas Ruminococcus Sphingobacterium Sporosarcina Staphylococcus Streptococcus Trichococcus Turicibacter Wautersiella and unknown generaThe microbial composition of the feed was most similar to the microbiota of the ileum and excreta Fig a01a and most distant from the cecal community The core microbiota at genus level Fig a0 1b showed the overlap in the genera present in the feed and both ileum and cecum as well as with the excreta The linear discriminant analysis LDA effect size method LEfSe was used to determine the genera most likely to explain differences between the feed and gut sections microbiota via coupling standard tests for statistical significance with additional tests encoding biological consistency and effect relevance Fig a01c The Chao estimated richness of feed microbiota was very low compared to cecum and excreta samples Fig a01d but comparable to some ileal samplesIndividual feed ingredients carry distinctive microbial communityTo determine the potential origins of the microbiota found in the whole formulated feed ration the microbiota composition of the component ingredients of the feed were analysed Independentsamples of each of the ingredients were sourced from four different locations in Australia Fig a0 Figure a02a and b show clear differences in genus level composition of feed ingredients as well as in their estimated richness with bloodmeal and meat and bone meal showing more complex microbial richness compared to other feed ingredient groupsThe similarities and differences in microbiota compositions were further investigated by Discriminant Analysis of Principal Components DAPC and NonMetric Multidimensional Scaling NMDS multivariate analysis Fig a03ab This showed that bloodmeal meat and bone meal and limestone had the most distinct microbial communities followed by poultry oil whereas barley canola corn millrun oats shum soybean meal and wheat clustered together into almost entirely overlapping groupsBased on LEfSe the genera most likely to explain differences between each type of feed ingredient indicate that some groups of pathogens populating the bird GIT may originate from specific feedstuff with Clostridium and Streptococcus identified as representatives of bloodmeal Fig a0The influence of the feed ingredients supplier environmental differences andor climate conditions on the microbiota in feedstuffs is presented in a DAPC plot Fig a0 Surprisingly the differences in feedstuffs microbiota presented in the DAPC plot resemble the geographical position of suppliers and may be influenced by the differences between the processing facilities but also climate and geographic region from which the ingredients have been grown and producedDiscussionIt is now well established that an appropriate healthy microbiota provides individuals with numerous nutritional benefits intestinal mucosa development pathogen protection and immune system maturation Stecher and Hardt Young Zhu et a0al Although the mainstream knowledge on gut microbiota came from human research it is understood that intestinal bacterial inhabitants of chickens play both similar and poultryspecific influential roles Stecher and Hardt Young Zhu et a0al Although in some cases there may be small populations of bacteria in the gut 0cHaberecht a0et a0al AMB Expr Page of Fig Comparison of feed microbiota with ileum cecum and excreta microbiota The Fig presents 3D PCoA plot based on unweighted UniFrac distance a core microbiota at a genus level b LEfSe analysis of the top genera c and Chao microbial richness estimator d In all plots feed samples are represented in grey cecum in red ileum yellow and excreta in blueof embryos in ovo the main events of the establishment of the chicken intestinal microbiota community starts immediately posthatch Based on the current literature on poultry gut colonisation the environment rather than parental influence plays the major role in chicken gut maturation Stanley et a0al The microbiota in chickens is considered fully formed within the first weeks of life but it continues to mature and to respond to stressors and environmental challenges throughout the life of birdsThe type of the production system defines the environment and type of feed that birds are exposed to and hence has a major influence on the microbiota especially in freerange systems that replace strict biosecurity with exposure to the soil grass and other plant microbiota but most concerning microbiota of rodents wild birds and other animals and increases pathogen load in birds all presenting challenges for freerange production Biasato et a0al Islam et a0al Ocejo et a0al Until now the feed has been usually discussed in poultry gut maturation as a growth medium that will support a nutrient determined cohort of intestinal microbes This study now brings a new dimension to the role of feed in microbiota formation as a source of colonizing bacteria Fig a0The microbial community contained in poultry feed may play an important role in the colonisation of poultry gut microbiota as the study found a large overlap in the core microbiota of the feed and the birds™ ileum caecum and excreta Our results also indicate that there is a substantial overlap in the microbial composition of grains and seeds barley canola corn millrun oats shum soybean meal and wheat almost entirely overlap while blood meal meat and bone meal and limestone stand quite apart with more distinct microbiotas Each batch of feed ingredient differs in the composition of the microbiota it carries and hence each batch of formulated feed will also differ in microbiota composition Some of the ingredients carry bacteria which are potentially pathogenic These findings suggest that it would be prudent 0cHaberecht a0et a0al AMB Expr Page of Fig The feed ingredients microbiota composition and diversity Genus level barchart shows the top most abundant genera a and a Chao richness estimator b MBM meat and bone meal BM blood meal PO poultry oil Feed finished mixed feedFig Multivariate presentation of feed ingredients microbiota similarities via DAPC a and NMDS b plotsto consider and monitor feed microbial community particularly in starter diets used in the period when the core microbiota is formed and stabilisedIt is worth noting how poorly researched the field of microbial colonisation of feed ingredients used in poultry and human feed is A Google Scholar search on keywords 0cHaberecht a0et a0al AMB Expr Page of Fig Linear discriminant analysis effect size method LEfSe of the top genera in feedstuffsFig Discriminant Analysis of Principal Components DAPC of feedstuffs from the different suppliers a and a GoogleMap b view of their geographical regionœgrain and œmicrobiota January returned only manuscripts all investigating the role of grains consumed in the diet on microbiota structure Similar outputs are found when using specific grains On the other hand the feed can be easily contaminated with microbes as feed spoilage is not uncommon out in field situations 0cHaberecht a0et a0al AMB Expr Page of Fig Feed contains a diverse microbial community and is an active bacterial coloniser in poultryWhile biosecurity measures are implemented to variable degrees feed ingredients in milling environments are not always well protected from birds rodents insects and other potential microbial exposures It was believed that the process of pelletising will remove most of the bacteria However in addition to the fact that brief heating of feed during pelletising cannot remove microbial spores the pelletising process is far from sterilisation and will remove most but not all viable bacteria Its antimicrobial efficiency will be varying between the different processing systems It is also notable that feed can be easily colonized postpelletising from external and pelletising surviving microbiota This will strongly depend on the packaging humidity temperature and other finished feed storage conditionsAnother question that needs to be addressed is how much of the microbial signal identified by a0 s rRNA gene analysis represents viable bacteria DNA from the dead bacteria would be subjected to natural degradation but still depending on time frames and storage conditions significant amounts of amplifiable DNA from dead bacteria could be present Of course this can also be an issue with microbiota analysis of gut samples although in that case it is well established that DNA is efficiently digested and degraded by gastric juice and pepsin Liu et a0al We were able to grow bacteria on rich brainheart infusion media from all of the feed samples we investigated however culturing is strongly restricted by the media and anaerobicaerobic environment used Therefore it would certainly not be readily possible to identify culturable bacteria of all the genera identified in feed ingredients Although there are other methods capable of determining bacterial viability via sequencing and vital staining methodologies Young et a0al they are not commonly in use and face different challenges and methodological issuesThis study shows that further research into feed as a source of random or as the means of targeted colonisation of poultry gut could be productive Investigating feed microbiota will bring new challenges unlike the diverse and dense microbial populations present in intestinal samples many of the feed ingredients are hard to process for DNA isolation and for some only small amounts of bacterial DNA can be recovered Amplicon and whole metagenome sequencing methods are limited in resolution and cannot reliably detect bacteria that make up only a small fraction of the microbiota Resolution depends on sequencing depth but in most studies a presence at less than is unlikely to be reliably detected Therefore for some significant bacteria for example pathogens such as Clostridium perfringens or Salmonella it may be necessary to use more sensitive methods such as culturing or specific PCR to detect in feed ingredients Control of viable pathogens in feed 0cHaberecht a0et a0al AMB Expr Page of should be considered as a standard part of production system biosecurityAcknowledgementsThe data was analysed using the Marie Curie HighPerformance Computing System at Central Queensland University We wish to acknowledge and appreciate help from Jason Bell provided in all aspects of HighPerformance ComputingAuthors™ contributionsSH performed research YB performed research analysed data RM TTHV performed research DS conceived the study analysed data wrote the paper All authors edited the manuscript and agreed with its final form All authors read and approved the final manuscriptFundingThis research did not receive any specific grant from funding agencies or the commercial sector The project was funded internally by Central Queensland University Office of Research Merit Grant fund At our request undisclosed poultry feed company kindly provided annotated feedstuffs but had no further involvement Availability of data and materialsSequencing data is publically available on MGRAST Metagenomics Analysis Server Database https wwwmgrast with full sample annotation under project ID mgp455839Ethics approval and consent to participateAnimal ethics approvals were obtained from the Animal Ethics Committee at Central Queensland University with the Approval Number A1409318Competing interestsThe authors have no conflict of interest to declareAuthor details University of New England Armidale NSW Australia Institute for Future Farming Systems Central Queensland University Rockhampton QLD Australia School of Science RMIT University Bundoora VIC Australia Department of Microbiology Monash University Clayton VIC Australia Received May Accepted August ReferencesAbdelMoneim AE Elbaz AM Khidr RE Badri FB Effect of in ovo inoculation of Bifidobacterium spp on growth performance thyroid activity ileum histomorphometry and microbial enumeration of broilers Probiotics Antimicro doihttps doi101007s1260 xAbdelMoneim AE Selim DA Basuony HA Sabic EM Saleh AA Ebeid TA Effect of dietary supplementation of Bacillus subtilis spores on growth performance oxidative status and digestive enzyme activities in Japanese quail birds Trop Anim Health Prod “ https doi101007s1125 Allan B Wheler C Koster W Sarfraz M Potter A Gerdts V Dar A In ovo administration of innate immune stimulants and protection from early chick mortalities due to yolk sac infection Avian Dis “ doihttps doi10163711840 8Reg1Apajalahti J Kettunen A Graham H Characteristics of the gastrointestinal microbial communities with special reference to the chicken Worlds Poult Sci J “ doi 101079nVPS2004 Ashelford KE Chuzhanova NA Fry JC Jones AJ Weightman AJ At least in 16S rRNA sequence records currently held in public repositories is estimated to contain substantial anomalies Appl Environ Microbiol “ doi Doi https doi101128Aem7112772477362005Baldwin S Hughes RJ Hao Van TT Moore RJ Stanley D Athatch administration of probiotic to chickens can introduce beneficial changes in gut microbiota PloS One 133e0194825 doihttps doi101371journ alpone01948 Bauer BW Gangadoo S Bajagai YS Van TTH Moore RJ Stanley D 2019a Oregano powder reduces Streptococcus and increases SCFA concentration in a mixed bacterial culture assay PloS One 1412e0216853 doihttps doi101371journ alpone02168 Bauer BW Radovanovic A Willson NL Bajagai YS Hao Van TT Moore RJ Stanley D 2019b Oregano A potential prophylactic treatment for the intestinal microbiota Heliyon 510e02625 doihttps doi101016jheliy on2019e0262 Benus RF van der Werf TS Welling GW Judd PA Taylor MA Harmsen HJ Whelan K Association between Faecalibacterium prausnitzii and dietary fibre in colonic fermentation in healthy human subjects Br J Nutr “ doihttps doi101017S0007 Biasato I Ferrocino I Biasibetti E Grego E Dabbou S Sereno A Gai F Gasco L Schiavone A Cocolin L Capucchio MT Modulation of intestinal microbiota morphology and mucin composition by dietary insect meal inclusion in freerange chickens BMC Vet Res doihttps doi101186s1291 70181690yBradbury JM Howell LJ The response of chickens to experimental infection ˜em ovo™ with Mycoplasma synoviae Avian Pathol “ doihttps doi10108003079 Caporaso JG Kuczynski J Stombaugh J Bittinger K Bushman FD Costello EK Fierer N Pena AG Goodrich JK Gordon JI Huttley GA Kelley ST Knights D Koenig JE Ley RE Lozupone CA McDonald D Muegge BD Pirrung M Reeder J Sevinsky JR Tumbaugh PJ Walters WA Widmann J Yatsunenko T Zaneveld J Knight R QIIME allows analysis of highthroughput community sequencing data Nat Methods “ doihttps doi101038nmeth f303Castaneda CD McDaniel CD Abdelhamed H Karsi A Kiess AS Evaluating bacterial colonization of a developing broiler embryo after in ovo injection with a bioluminescent bacteria Poult Sci “ doihttps doi103382pspez05 Cebra JJ Influences of microbiot

Thyroid_Cancer

incidence and death rate of nonsmall cell lung cancer NSCLC in China ranks the first among the malignant tumors Circular RNA circRNA was reported to be involved in the progression of NSCLC Our study aimed to investigate the underlying mechanism of circ_0020123 in NSCLC progressionMethods Quantitative realtime polymerase chain reaction qRTPCR was used to detect the expression of circ_0020123 miR5905p and Thrombospondin THBS2 in NSCLC tissues and cells Cell proliferation and migration were examined by Cell Counting Kit8 CCK8 assay and Transwell assay respectively Flow cytometry assay was used to detect the apoptosis of NSCLC cells The protein levels of Ki67 matrix metalloprotein9 MMP9 Cleavedcaspase9 Cleavedcasp9 and THBS2 were detected by Western blot The targets of circ_0020123 and miR5905p were predicted by starBase and TargetScan and then confirmed by dualluciferase reporter assay and RNA immunoprecipitation RIP assay The animal experiment showed the effect of circ_0020123 on tumor growth in vivoResults The expression of circ_0020123 was upregulated in NSCLC tissues and cells Functionally circ_0020123 downregulation inhibited the proliferation and migration and promoted the apoptosis of NSCLC cells Interestingly circ_0020123 directly targeted miR5905p and inhibition of miR5905p reversed the knockdown effects of circ_0020123 on NSCLC cells More importantly THBS2 was a target of miR5905p and THBS2 overexpression reversed the effects of circ_0020123 knockdown on cell proliferation migration and apoptosis in NSCLC cells Finally suppression of circ_0020123 inhibited tumor growth in vivo through miR5905pTHBS2 axisConclusion Circular RNA circ_0020123 regulated THBS2 by sponging miR5905p to promote cell proliferation and migration and inhibit cell apoptosis in NSCLC cellsKeywords NSCLC Circ_0020123 miR5905p THBS2Highlights Circ_0020123 was upregulated and downregulation of circ_0020123 inhibited cell proliferation migration and promoted cell apoptosis in NSCLC cellsCorrespondence bskrju163comDepartment of Thoracic Surgery Lianyungang Second People™s Hospital No Hailian East Road Haizhou District Lianyungang Jiangsu China Circ_0020123 directly targeted miR5905p and miR5905p downregulation reversed the knockdown effects of circ_0020123 on NSCLC progression THBS2 acted as a target of miR5905p and overthe effects of expression of THBS2 reversed circ_0020123 knockdown on NSCLC progression Downregulation of circ_0020123 suppressed tumor growth in vivo through miR5905pTHBS2 axis 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 0cWang a0et a0al Cancer Cell Int Page of BackgroundLung cancer has the highest incidence of total cases and is the most common cause of cancer death of total cancer deaths in worldwide [] Lung cancer can be divided into several histological subtypes according to the location and the tendency of metastasis Small cell lung cancer SCLC accounts for about of all lung cancer cases [] However nonsmall cell lung cancer NSCLC accounts for of lung cancer and the a0years overall survival rate OS is only about [] Therefore it is important to find the effective treatment and potential molecular targets of NSCLC progressionCircular RNA circRNA is a single stranded RNA molecule with a closed circular structure Recently amounts of circular DNA have been discovered and most of which were thought to be the byproducts of typical splicing [ ] Previous reports indicated that the expression of circRNA was tissuespecific and the change of its expression intensity was associated with some diseases [“] Furthermore circRNA was involved in the occurrence and development of the disease and might be used as a potential biomarker in clinical diagnosis prognosis and treatment of diseases [ ] For example circ_0039569 facilitated cell proliferation and migration of renal cell carcinoma by sponging miR34a5p to regulate CC Chemokine ligand CCL22 [] Meanwhile hsa_circ_0043256 participated in the progression of NSCLC cells by mediating the cinnamaldehyde treatment [] A previous report suggested that circ_0020123 acted as an oncogene in NSCLC and circ_0020123 regulated zincfingerenhancer binding protein ZEB1 and enhancer of zeste homolog EZH2 by competitively binding with miR144 to induce cell progression and migration [] These reports suggested that circ_0020123 was a vital factor in the pathogenesis of NSCLC and its function and molecular mechanism need to be further studiedAs a small endogenous RNA microRNA miRNA is essential in regulating gene expression and plays a potential role in the exploitation of biomarkers [] Recently some aggregated miRNAs have been found in prostate cancer such as miR221222 miR143145 miR23b27b241 and miR1133a which were downregulated and had tumor inhibiting functions [] A previous study found that circulating miR5905p could be used as routine diagnostic tools for lung cancer and as a potential prognostic marker for liquid biopsy Besides overexpression of miR5905p reduced the development of NSCLC cells and regulated the expression of epithelialmesenchymal transformation EMTrelated proteins by targeting the signal transducers and activators of transcription STAT3 [] However the precise mechanism by which miR5905p affects NSCLC needs further investigationThrombospondin THBS2 as a secreted protein was confirmed to be highly expressed in different cancers including cervical cancer [] colorectal cancer [] and NSCLC [] A previous report suggested that THBS2 was involved in the proliferation apoptosis and antiautophagy regulation of cervical cancer cells by miR20a [] Tian et a0al found the expression and clinicopathological features of THBS2 in colorectal cancer were significantly correlated with the prognosis of cancer and might be used as a biomarker of prognosis [] However the molecular function of THBS2 in NSCLC remains poorly definedIn this study the targeting relationship between circ_0020123 and miR5905p was firstly detected The effects of circ_0020123 on cell proliferation migration apoptosis and tumor growth were performed by gain and lossoffunction experiments and molecular biology techniquesMaterials and a0methodsPatients and a0specimensNSCLC tissues and the adjacent healthy lung tissues were taken from NSCLC patients in the Lianyungang Second People™s Hospital All volunteers signed written informed consents NSCLC tissues and the adjacent tissues were immediately frozen in liquid nitrogen and kept at ˆ’ a0 °C for further experiments This research was approved by the Ethics Committee of Lianyungang Second People™s HospitalCell culture and a0cell transfectionTwo NSCLC cell lines A549 and H1299 and one normal lung cell line IMR90 were obtained from the Beijing Concorde Cell Library Beijing China A549 H1299 and IMR90 cells were cultivated in Dulbecco™s modified eagle medium DMEM HyClone Logan UT USA supplementing with fetal bovine serum FBS HyClone and cultured in an incubator at a0„ƒ with CO2Small interfering RNA siRNA targeting circ_00201231 sicirc_00201231 and sicirc_00201232 short hairpin RNA shRNA targeting circ_0020123 shcirc_0020123 miR5905pinhibitors siRNA negative control siNC shNC and NCinhibitors were all obtained from Biomics Biotech Jiangsu China Full length of THBS2 cDNA Sangon Biotech Shanghai China was subcloned into pcDNA31 plasmid EKBioscience Shanghai China Then cell transfection was performed by Lipofectamine Thermo Fisher Scientific Waltham MA USA 0cWang a0et a0al Cancer Cell Int Page of RNA isolation and a0quantitative real‘time polymerase chain reaction qRT‘PCRThe TRIzol reagent Invitrogen Carlsbad CA USA was used for extracting the total RNAs Next the reversed transcription was carried out by RTPCR kit Invitrogen The qRTPCR was performed using the ABI SYBR Green Master Mix Invitrogen The primers in our study were as follows F5²TTC GGA CGA CCG TCA AAC AT3² and R5²AGG ATC CCT GCA CCA CAA TG3² for circ_0020123 F5²TGA AAG ACG TGA TGG CAC AC3² and R5²CTT CCA TTT TGG for miR5905p F5²AGA AGG GGT TTT TGG3 ² CTG GGG CTC ATT TG3² R5²AGG GGC CAT CCA CAG TCT TC3² for glyceraldehyde3phosphate dehydrogenase GAPDH [] F5²GCG GCT GGG TCT ATT TGT C3² and R5²GCA GGA GGT GAA GAA CCA TC3² for THBS2 [] F5²ATT GGA ACG ATA CAG AGA AGATT3² and R5²GGA ACG CTT CAC GAA TTT G3² for U6 [] GAPDH and U6 were the internal parametersCell Counting Kit‘ CCK‘ assayThe proliferation viability of A549 and H1299 cells were detected by the CellCounting Kit8 MSK Wuhan China A549 and H1299 cells were cultivated into a 96well plate with a density of × a0cellswell and incubated in a0°C for or a0h Then a0μL fresh medium and CCK8 solution was added After incubation at a0°C for a0h the OD values were detected by the Multiskan Ascent microplate reader Abcam Cambridge MA USATranswell assayTranswell chamber Corning Life Sciences Corning NY USA was used to detect cell migration Firstly the serumfree DMEM Thermo Fisher Scientific was fixed with cell suspension cells and seeded into the upper chamber and the DMEM containing serum was put into the lower chamber After incubation for a0h the cells in lower surface of the upper chamber were treated with formaldehyde solution for a0 h and then thoroughly washed Finally the migrated cells were stained with crystal violet Corning Life Sciences and observed by using a microscopeFlow cytometryFirstly A549 and H1299 cells were cultured and PBS was used for washing cells Then the binding buffer was used to resuspend cells and the Annexin Vfluorescein isothiocyanate VFITCpropidium iodide PI Apoptosis Detection Kit Thermo Fisher Scientific was used to stain cells Finally cell apoptosis was detected by flow cytometry Thermo Fisher ScientificWestern blot analysisThe total proteins of NSCLC tumors or cells were collected by RIPA lysis buffer Sangon Biotech Then the proteins were separated by Sodium dodecyl sulphate polyacrylamide gel electrophoresis SDSPAGE and transferred to polyvinylidene fluoride PVDF membranes Thermo Fisher Scientific The skimmed milk was added and incubated with primary antiGAPDH antibody Invitrogen Carlsbad CA USA antiβactin antibody Invitrogen antiKi67 antibody Invitrogen antimatrix metalloprotein9 MMP9 antibody Invitrogen antiCleavedcaspase9 Cleavedcasp9 antibody Invitrogen or antiTHBS2 antibody Invitrogen at a0°C overnight Finally the membranes were incubated with the secondary antibody for a0 h at room temperature The results were viewed using Kodak film developer Fujifilm JapanDual‘luciferase reporter assaysThe wild type circ_0020123 sequences circ_0020123WT mutant circ_0020123 sequences circ_0020123MUT wild type THBS2 ²UTR sequences THBS2WT mutant THBS2 ²UTR sequences THBS2MUT were cloned into pGL3 luciferase reporter plasmid Promega Madison WI USA Then the plasmid and miR5905p or miRNC were cotransfected into A549 and H1299 cells by Lipofectamine Thermo Fisher Scientific After transfection for a0h the DualLuciferase Reporter Assay System Promega was performed to detect the luciferase activityRNA immunoprecipitation RIPFirstly the Magna RIP RNAbinding Protein Immunoprecipitation Kit gzscbio Guangzhou China was performed to verify the relationship between circ_0020123 and miR5905p In brief the magnetic beads and antiAgo2 antibody Abcam were added into cells and incubated for a0h Then the proteinase K and the phenol“chloroformisoamyl alcohol reagent were added for purifying RNAs Finally qRTPCR was used to measure circ_0020123 enrichmentAnimal experimentsThe 4weekold BALBc male nude mice Vitalriver Beijing China were raised in a sterile environment for 0cWang a0et a0al Cancer Cell Int Page of experiments Then PBS was used to suspend A549 cells × transfected with shcirc_0020123 or shNC Next the nude mice were divided into two groups n A549 cells transfected with shcirc_0020123 or shNC were shcirc_0020123 or shNC inoculated into the nude mice The tumor volume was detected every a0 days After a0days the nude mice were euthanatized and the tumor weight was detected Besides the tumor tissues from each group were collected to detect the expression of circ_0020123 miR5905p and THBS2 The animal experiment was approved by the Animal Experimentation Ethics Committee of Lianyungang Second People™s HospitalStatistical analysisThe software GraphPad Prism was performed for statistical analysis The data was displayed as mean ± standard deviation SD The significant difference was calculated by Student™s t test and oneway analysis of variance ANOVA P was considered as statistically significantResultsCirc_0020123 was a0upregulated in a0NSCLC tissues and a0cellsTo begin with qRTPCR was used to detect the expression of circ_0020123 the result showed that circ_0020123 was significantly upregulated in NSCLC tissues compared with the adjacent healthy tissues Fig a0 1a Similarly the expression of circ_0020123 in NSCLC cells A549 and H1299 was markedly higher than that in normal cells IMR90 Fig a01b From these data it is speculated that circ_0020123 might be acted as an oncogene in NSCLCFig Circ_0020123 was upregulated in NSCLC tissues and cells a QRTPCR was used to detect the expression of circ_0020123 in adjacent healthy tissues n and tumor tissues n b The expression of circ_0020123 in normal cell line IMR90 and NSCLC cell lines A549 and H1299 was detected by qRTPCR P Downregulation of a0circ_0020123 inhibited the a0proliferation migration and a0induced apoptosis of a0NSCLC cellsTo investigate the functional effects of circ_0020123 on NSCLC cells sicirc_00201231 and sicirc_00201232 were obtained and transfected into A549 and H1299 cells Firstly the transfection efficiency was detected by qRTPCR Fig a02a Next CCK8 was used to detect the proliferation and the results showed that the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was reduced Fig a0 2b Moreover the migration of A549 and H1299 cells was significantly downregulated by circ_0020123 knockdown Fig a02c In addition the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was obviously higher than transfected with siNC Fig a02d Finally the protein levels of cell proliferationrelated protein Ki67 and cell migrationrelated protein MMP9 were inhibited while cell apoptosisrelated protein Cleavedcasp9 was upregulated in NSCLC cells transfected with sicirc_00201231 or sicirc_00201232 Fig a02e These data suggested that inhibition of circ_0020123 suppressed cell proliferation migration and promoted apoptosis in NSCLC cellsCirc_0020123 directly targeted miR‘‘5pBy searching in the online software starBase the potential binding sites between circ_0020123 and miR5905p were detected Fig a0 3a To confirm that the dualluciferase reporter assay was performed the results showed that the luciferase activity of circ_0020123WT reporter plasmid was reduced by miR5905p mimic while the circ_0020123MUT reporter plasmid activity was not changed in A549 and H1299 cells Fig a03b Furthermore the expression of miR5905p was lower in A549 and H1299 cells compared with that in IMR90 cells Fig a0 3c In contrast miR5905p expression was elevated in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 Fig a0 3d Finally the RIP assay was also used to confirm the targeting relationship between circ_0020123 and miR5905p and the results showed that circ_0020123 and miR5905p were enriched in antiAgo2 group Fig a03eMiR‘‘5p downregulation reversed the a0inhibition effects of a0circ_0020123 on a0NSCLC cellsTo further explore the functional effects between circ_0020123 and miR5905p miR5905pinhibitor was established QRTPCR was used to detect the transfection efficiency Fig a0 4a Interestingly miR5905p was upregulated in A549 and H1299 cells transfected with sicirc_00201231 while the expression of miR5905p was recovered in cells transfected with 0cWang a0et a0al Cancer Cell Int Page of Fig Downregulation of circ_0020123 inhibited the proliferation and migration and induced the apoptosis of NSCLC cells a The transfection efficiency of sicirc_00201231 and sicirc_00201232 in A549 and H1299 cells was detected by qRTPCR b CCK8 assay was used to detect the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 c The migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was measured by Transwell assay d Flow cytolysis assay was used to detect the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 e The protein levels of cell proliferation related protein Ki67 cell migration related protein MMP9 and cell apoptosis related protein Cleavedcasp9 were detected by Western blot P Fig a0sicirc_00201231 miR5905pinhibitors 4b Moreover circ_00201231 knockdown inhibited cell proliferation and migration while the miR5905p inhibitor reversed these effects Fig a0 4c d In addition the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 was increased which was abolished by miR5905pinhibitor Fig a0 4e Similarly miR5905p inhibitors reversed the effects on the protein levels of Ki67 MMP9 and Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 Fig a0 4f These results that miR5905p downregulation reversed the effects of circ_0020123 downregulation on the proliferation migration and apoptosis of NSCLC cellsindicated MiR‘‘5p targeted THBS2 in a0NSCLC cellsThe THBS2 ²UTR was predicted to contain the binding sites of miR5905p through the online software TargetScan Fig a05a Then the dualluciferase reporter assay was used to confirm the targeting relationship The results showed that cotransfection of miR5905p and THBS2WT significantly limited the luciferase activity in both A549 and H1299 cells the luciferase activity was not altered in cells cotransfected with miR5905p and THBS2MUT Fig a05b Importantly the mRNA and protein level of THBS2 was enahnced in NSCLC cells Fig a05c d We further explored whether circ_0020123 affected the functions of THBS2 in NSCLC cells The mRNA and protein expression of THBS2 were repressed in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 Fig a05e f 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0020123 directly targeted miR5905p a The binding site between circ_0020123 and miR5905p was detected by the online software starBase b The luciferase activity of circ_0020123WT or circ_0020123MUT reporter plasmid in A549 and H1299 cells transfected with miRNC or miR5905p was detected by dualluciferase reporter assay c QRTPCR was used to detect the expression of miR5905p in A549 and H1299 cells d The expression of miR5905p in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qRTPCR e RIP assay was used to confirm the relationship between circ_0020123 and miR5905p P THBS2 overexpression reversed the a0effects of a0circ_0020123 knockdown on a0the a0proliferation migration and a0apoptosis of a0NSCLC cellsBased on the work ahead of us the pcDNA31THBS2 was constructed Then the qRTPCR and Western blot were used to detect the transfection efficiency and the THBS2 expression was increased in A549 and H1299 cells transfected with pcDNA31THBS2 Fig a0 6a b In addition the proliferation and migration rates of A549 and H1299 cells transfected with sicirc_00201231 pcDNA31THBS2 were higher than that transfected with sicirc_00201231 Fig a0 6c d Meanwhile a similarly phenomenon was also observed in cell apoptosis the pcDNA31THBS2 significantly reversed the promotion effect of circ_0020123 deletion on cell apoptosis Fig a0 6e Furthermore the effects of circ_0020123 deletion on Ki67 MMP9 and Cleavedcasp9 protein levels were also reversed by THBS2 overexpression Fig a0 6f These data suggested that overexpression of THBS2 could reverse the effects of circ_0020123 downregulation on cell proliferation migration and apoptosisReduction of a0circ_0020123 suppressed tumor growth in a0vivo through a0circ_0020123miR‘‘5pTHBS2 axisTo further explore the function of circ_0020123 in NSCLC cells the shcirc_0020123 was constructed and the xenograft tumor was established Then A549 cells transfected with shcirc_0020123 or shNC were injected into the nude mice The xenograft tumor volume was measured every a0 days after injection and the results showed that tumor volume was smaller shcirc_0020123 group than that in shNC group Fig a07a Moreover tumor weight was inhibited by circ_0020123 knockdown Fig a0 7b Furthermore the expression circ_0020123 and THBS2 was decreased while the miR5905p was increased in xenograft tumor transfected with shcirc_0020123 Fig a0 7c Western blot assay also revealed that the protein level of THBS2 was repressed by circ_0020123 knockdown Fig a07d Finally the digital tomosynthesis DTS was used to detect the number of lung metastatic nodules in xenograft tumor and it was reduced in shcirc_0020123 group Fig a07e The results suggested that downregulation of circ_0020123 inhibited tumor growth in a0vivoDiscussionClinically only a few NSCLC patients were diagnosed at an early stage and treated by surgical resection More than of NSCLC patients were diagnosed with the advanced stage or metastatic tumors [] Thus finding novel biomarkers and therapeutic targets were necessary for the effective diagnosis and treatment of NSCLCRecently circRNA was no longer considered as a random product in the RNA shearing process and its biological significance and function in malignant tumors 0cWang a0et a0al Cancer Cell Int Page of Fig MiR5905p downregulation reversed circ_0020123 knockdown effects in NSCLC cells a QRTPCR was used to detect the expression of miR5905p in A549 and H1299 cells transfected with miR5905pinhibitors b The expression of miR5905p in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors was detected by qRTPCR c The proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors was tested by CCK8 assay d Transwell assay was used to measure the migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors e Flow cytolysis assay was used to detect the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors f The protein levels of Ki67 MMP9 Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors were detected by Western blot P had received more and more attention Previous reports revealed that circ_0020123 was involved in the development of NSCLC [] Moreover the level of circ_0020123 was elevated in NSCLC cells [] Consistently we found that the expression of circ_0020123 was markedly higher in NSCLC tissues and cells Moreover this research indicated that inhibition of circ_0020123 suppressed the proliferation migration and induced apoptosis of NSCLC cells in a0 vitro Besides circ_0020123 promoted tumor growth in a0vivoEndogenous circRNAs could act as microRNA sponges to inhibit their function and some studies linked miRNA sponges to human diseases including cancer [] A previous study indicated that circRNA ctransferrin receptor cTFRC regulated TFRC by sponging miR107 to facilitate bladder carcinoma development [] MiR5905p was studied in different cells such as airway smooth muscle cells [] colon epithelial cells [] and NSCLC cells [] However the potential relationship between miR5905p and circRNA has not been researched In this study circ_0020123 directly targeted miR5905p and miR5905p inhibition reversed the effects of circ_0020123 knockdown on NSCLC progression These data provided a clue to the therapeutic strategy for NSCLC 0cWang a0et a0al Cancer Cell Int Page of Fig MiR5905p targeted THBS2 in NSCLC cells a The potential binding site between THBS2 ²UTR and miR5905p was predicted by the online software TargetScan b Dualluciferase reporter assay was used to measure the luciferase activity of THBS2WT or THBS2MUT reporter plasmid in A549 and H1299 cells transfected with miRNC or miR5905p c QRTPCR was used to detect the mRNA expression of THBS2 in NSCLC cells d The protein level of THBS2 in NSCLC cells was tested by Western blot e The mRNA expression of THBS2 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qRTPCR f Western blot was used to measure the protein level of THBS2 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 P Our study also confirmed that miR5905p could target THBS2 directly in NSCLC cells THBS2 is a calciumbinding protein that binds to and inactivates matrix metalloproteinase MMP genes involved in tissue formation and repair [ ] A previous document suggested that THBS2 acted as a target of miR2213p and participated in lymph node metastasis in cervical cancer [] The data in this research showed that the expression of THBS2 in NSCLC cells was markedly higher than normal healthy cells Furthermore overexpression of THBS2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of NSCLC cells suggesting that circ_0020123 promoted the progression of NSCLC cells through miR5905pTHBS2 axisConclusionIn conclusion our research showed that the expression of circ_0020123 was higher in NSCLC tissues and cells than control and downregulation of circ_0020123 inhibited the proliferation migration and promoted apoptosis of NSCLC cells and also suppressed tumor growth in a0 vivo Moreover circ_0020123 directly targeted miR5905p while miR5905p inhibition reversed the effects of circ_0020123 knockdown on NSCLC cells More importantly circ_0020123 regulated the expression of THBS2 by sponging miR5905p and upregulation of THBS2 reversed the effects of circ_0020123 knockdown on NSCLC cells Therefore our research demonstrated that circ_0020123 enhanced proliferation migration and inhibited 0cWang a0et a0al Cancer Cell Int Page of Fig Overexpression of THBS2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of NSCLC cells a b The mRNA and protein expression of THBS2 in A549 and H1299 cells transfected with pcDNA31THBS2 was detected by qRTPCR and Western blot c CCK8 assay indicated the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 d The migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 was measured by Transwell assay e The apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 was detected by Flow cytolysis assay f The protein levels of Ki67 MMP9 Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 were detected by Western blot P apoptosis of NSCLC cells by sponging miR5905p to regulate THBS2results and develop the manuscript All authors read and approved the final manuscriptAbbreviationsNSCLC Nonsmall cell lung cancer circRNA Circular RNA qRTPCR Quantitative realtime polymerase chain reaction CCK8 Cell Counting Kit8 MMP9 Matrix metalloprotein9 Cleavedcasp9 Cleavedcaspase9 Cleavedcasp9 Cleavedcaspase9 RIP RNA immunoprecipitation ZEB1 Zincfingerenhancer binding protein EZH2 Zeste homolog STAT3 Signal transducers and activators of transcription THBS2 Thrombospondin AcknowledgementsNot applicableAuthors™ contributionsLW collaborated to design the study LZ were responsible for experiments analyzed the data YW wrote the paper All authors collaborated to interpret FundingNoneAvailability of data and materialsPlease contact corresponding author for data requestsEthics approval and consent to participateThis research was approved by the Ethics Committee of Lianyungang Second People™s Hospital The animal experiment was approved by the Animal Experimentation Ethics Committee of Lianyungang Second People™s HospitalConsent for publicationAll listed authors have actively participated in the study and have read and approved the submitted manuscript 0cWang a0et a0al Cancer Cell Int Page of Fig Reduction of circ_0020123 suppressed the tumor growth in vivo through circ_0020123miR5905pTHBS2 axis a A total of × A549 cells transfected with shcirc_0020123 or shNC were injected into nude mice to establish the xenograft tumor Tumor volume was measured every d after injection b Tumor weight was measured on d c The expression of circ_0020123 miR5905p and THBS2 in xenograft tumor was measured by qRTPCR d The protein level of THBS2 in xenograft tumor was evaluated by Western blot e The number of lung metastatic nodules in xenograft tumor was detected by digital tomosynthesis DTS P Competing interestsThe authors declare that they have no competing interestsReceived April Accepted July References Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “ Abe H Takase Y Sadashima E Fukumitsu C Murata K Ito T Kawahara A Naito Y Akiba J Insulinomaassociated protein is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions validity and reliability with cutoff value Cancer Cytopathol “Li C Zhang L Meng G Wang Q Lv X Zhang J Li J Circular RNAs pivotal molecular regulators and novel diagnostic and prognostic biomarkers in nonsmall cell lung cancer J Cancer Res Clin Oncol “ Belousova EA Filipenko ML Kushlinskii NE Circular RNA new regulatory molecules Bull Exp Biol Med “ Zhang Z Xie Q He D Ling Y Li Y Li J Zhang H Circular RNA new star new hope in cancer BMC Cancer Li L Chen Y Nie L Ding X Zhang X Zhao W Xu X Kyei B Dai D Zhan S Guo J Zhong T Wang L Zhang H MyoDinduced circular RNA CDR1as promotes myogenic differentiation of skeletal muscle satellite cells Biochim Biophys Acta Gene Regul Mech “ Greco S Cardinali B Falcone G Martelli F Circular RNAs in muscle function and disease Int J Mol Sc

Thyroid_Cancer

"Accumulating evidence has revealed the critical role of long noncoding RNAs lncRNAs in cellularprocesses during tumor progression As documented in cancerrelated literatures LINC00992 expression isassociated with cancer progression whereas its function in tumors including prostate cancer has not beencharacterized yetMethods Data from GEPIA database suggested LINC00992 expression in prostate cancer tissues The expressionlevels of RNAs were monitored via qRTPCR Western blot evaluated the levels of proteins The proliferationapoptosis and migration of prostate cancer cells were assessed by CCK8 EdU TUNEL Transwell and woundhealing assays Luciferase reporter RNA pull down and RIP assays were applied to detect the interplays amongLINC00992 miR3935 and GOLM1Results Elevated levels of LINC00992 and GOLM1 were detected in prostate cancer tissues and cells LINC00992exerted facilitating functions in prostate cancer cell proliferation and migration Mechanically LINC00992 interactedwith and negatively regulated miR3935 to elevate GOLM1 expression in prostate cancer cells In addition thein vitro suppressive effect of silenced LINC00992 on prostate cancer cell proliferation and migration was reversed byGOLM1 upregulation Likewise LINC00992 depletion restrained tumor growth in vivo was offset by enhancedGOLM1 expressionConclusions LINC00992 competitively bound with miR3935 to elevate GOLM1 expression and therefore facilitatethe oncogenic phenotypes of prostate cancer cells implying a potential LINC00992targeted therapy for prostatecancerKeywords INC00992 miR3935 GOLM1 Prostate cancer Correspondence engineyangsinacom5Department of Urology the Second Affiliated Hospital of Bengbu MedicalCollege Hongye Road Bengbu Anhui 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 0cChen BMC Cancer Page of BackgroundClinically prostate cancer manifests as a dominatingcause of malerelated death worldwide and is characterized as the most continually occurred tumor amongmen in the United States [] The biggest challenge isthe detectable bone metastases in roughly advancedprostate cancer [] Virtually all prostate cancer patientsduring years™ androgen deprivation treatment inevitably undergo castrationresistance which contributes tothe poor clinical consequences in prostate cancer []However the mechanism underlaid prostate cancer remains mostly unknownThe widely studied long noncoding RNAs lncRNAsare transcribed from nonproteincoding human genomeand have more than nt in length [] LncRNAs are increasingly functionally identified and experimentally consolidated to be related to tumor neoplasia and progressionin diverse cancers [] Additionally lncRNAs with dysregulation can functionally modulate tumor developmentfrom multiple pathological aspects such as cell proliferation drugresistance and metastasis [“] For examplelncRNA A1BGAS1 inhibits cell proliferation and invasionin hepatocellular carcinoma via targeting miR216a5p []LncRNA LOC730100 sponges miR760 from FOXA1 toaccelerate cell proliferation and invasion in glioma []LncRNA SNHG16 functions as an oncogene in hepatocellular carcinoma [] Long intergenic nonprotein codingRNA LINC00992 is a novel lncRNA that has beenpreviously revealed to be elevated in tumors and substantiated as a master regulator for chemoresistance [] Besides LINC00992 has been uncovered as an elevatedlncRNA in prostate cancer [] which is consistent withthe detection from GEPIA database Despite that no previous study has given a comprehensive explanation aboutthe precise function or detailed mechanism of LINC00992in prostate cancerIn past decades the fact that lncRNAs function in tumors depending on their secondary or tertiary structureshas been reported in many cancerlinked studies For instance in the nucleus lncRNAs are entitled to work asmolecular scaffolds or alternative splicing assistants [] On the contrary lncRNAs dispersing in cytoplasm influence downstream mRNA translation or degradationthrough serving as miRNA sponges [ ] For exampleTNFαinduced lncRNA LOC105374902 promotes themalignant behaviors of cervical cancer cells by acting as asponge of miR12853p [] LncRNA TTNAS1 promotes papillary thyroid cancer tumorigenesis by regulatingmiR1533pZNRF2 axis[] Nevertheless whetherLINC00992 could exert its functions in prostate cancer viaits sponging role of certain miRNA remains unknownWe conducted this research aiming to explore thefunction or probable mechanism of LINC00992 in prostate tumor which might enrich the understanding interms of prostate tumor pathology and contribute to awider applied scopeMethodsTissue samplesThe prostate cancer tissue samples and matched peritumor tissue samples were collected from patientsdiagnosed with prostate cancer under the approval ofthe Ethics Committee of the First Affiliated Hospital ofKunming Medical University Each participant did notreceive radiotherapy and chemotherapy prior to tissuecollection and signed the written informed consentsbefore this study All samples were snapfrozen in liquidnitrogen and then stored at °C until required forfurther analysisCell cultureThe prostate epithelial cell line RWPE1 CRL11609and prostate cancer cellsincluding PC3 CRL1435LNCaP CRL1740 C4“ CRL3314 and DU145HTB81 were all purchased from American TypeCulture Collection ATCC Manassas VA USAinOctober All cells were cultured as recommendedin Dulbecco™s modified Eagle™s medium containing FBS GIBCO MA USA under the condition of a cellincubator with CO2 at °C Before using in thisstudy all cell lines were authenticated by STR profilingand tested for mycoplasma contamination in June Cell transfectionLINC00992 shRNA or negative control shRNA andpcDNA31LINC00992 pcDNA31GOLM1 or its emptycontrol pcDNA31 plasmid were chemically synthesizedand provided by Gene Pharma Shanghai China MiR mimics miR3935 inhibitor and theirrelatednegative controls NCmimics NCinhibitor were allpurchased for upregulating or downregulating miR3935from Ribobio Guangzhou China In line with the directions of LipofectamineTM RNAiMAX TransfectionReagent Thermo Fisher Scientific transfection of theseplasmids into DU145 PC3 and RWPE1 cells wasconducted and qRTPCR checked the transfection efficiency The sequences were as follows shNC ²CCGGTAGTAATTGACAACCATTATACTCGAGTATAATGGTTGTCAATTACTATTTTTG3²shLINC009921²CCGGATTATCCAAGAGTATTAACATCTCGAGATGTTAATACTCTTGGATAATTTTTTG3² shLINC0 ²CCGGTGTTAGATGATCATTGAGGTGCTCGAGCACCTCAATGATCATCTAACATTTTTG3² s²CCGGTTACCTAATCAGTAGAThLINC009923GCAGCTCGAGCTGCATCTACTGATTAGGTAATTTTTG3² NCmimics ²UCAGGUAGGGCUCAAACCAACC3² miR3935 mimics ²UGUAGAUACGAGCACCAGCCAC3² NCinhibitor²CUGGCUUUAG 0cChen BMC Cancer Page of GGUGCCACUUAG3² miR3935 inhibitor ²GUGGCUGGUGCUCGUAUCUACA3²Quantitative realtime PCR qRTPCROn the basis of the instructions of Trizol reagent Invitrogen USA RNA extraction was executed in prostatecancer cells After the examination of RNA purity withspectrophotometry cDNA was obtained from aboveRNA with reverse transcription kit ThermoFisher Scientific shanghai China qRTPCR analysiswas devised with the aid of a BioRad CFX96 system andSYBR green was applied for investigating the RNA levelsThe internal reference for LINC00992 and mRNAs wasGAPDH whereas that for miRNAs expression was U6Relative expression was assessed based on the method ofˆ’ΔΔCtab97779Western blotProtein content in cells was determined by western blotanalysis RIPA lysis buffer Beyotime Shanghai Chinawas adopted for cell lysing followed by the evaluation ofthe protein concentration with BCA Protein Assay KitP0011 Beyotime Tech SDSPAGE gel was applied for separating proteins μg protein per sampleand then proteins were transferred onto μm PVDFmembranes BioRad Hercules CA USA Antibodiesincluding antiGOLM1 ab109628 AbcamCambridge UK antiPCNA ab92552 AbcamantiCDK2 ab32147 Abcam antiCyclin D1ab40754 Abcam antiBax ab32503 Abcam antiBcl2 ab32124 Abcam antiMMP2antiMMP9ab38898 Abcam antipSrc ab40660 Abcam antiSrc ab47405 Abcam antipFAKab81298 Abcam antiFAK ab131435 Abcam antiGAPDH ab8245 Abcam andantiTubulin ab7291 Abcam were applied toprobe the membranes overnight at °C After that themembranes were further incubated for h with HRPconjugated secondary antibody Santa Cruz Co LtdSant Cruz CA USA atroom temperature ECLSubstrates Millipore Billerica MA USA was utilizedfor the visualization of signals followed by exposure toXfilm Kodak Rochester NY USA The quantificationof immunoblots was conducted with the aid of imageJsoftware National Institute of Health Bethesda MDUSA with GAPDH or Tubulin as the normalizer asneeded AbcamLuciferase reporter assayFragments of fulllength LINC00992 with wildtype ormutant binding sites for miR3935 and sequences ofGOLM1 ™UTR containing wildtype or mutated miR binding sites were inserted into the pmirGLOvectors Promega Madison WI USA for the construction of reporters LINC00992WT LINC00992MUTGOLM1WT GOLM1MUT Then the four reportersand miR3935 mimics or miR3935 inhibitor GenePharma were cotransfected into DU145 and PC3 cellsapplying lipofectamine2000 Invitrogen as neededFortyeight hours later DualLuciferase Reporter AssaySystem Promega was employed for the examination ofthe luciferase activity GloMax® Discover Multimode Microplate Reader Promega assessed the ratio of FireflyRenilla luciferase activity and the activity of Renilla wasthe normalized controlRNA immunoprecipitation RIP assayAccording to the direction for usage of Magna RIP„¢RNA Binding Protein Immunoprecipitation Kit “Millipore RIP assay was strictly performed RIP lysisbuffer was firstly applied to treat the transfected DU145and PC3 cells Afterwards the obtained cell lysates wereprocessed with magnetic beads integrated with humanantiAgo2 antibodies ab32381 Abcam MA USA orantiIgG AP162KC Millipore Following the recoveryof antibody by the protein AG beads qRTPCR detected the levels of LINC00992 miR3935 and GOLM1mRNA in the precipitates IgG worked as the negativecontrol for the normalization of RNAIPsRNA isolation of nuclear and cytoplasmic fractionsThe dispersion of LINC00992 in the prostate cancercells was assayed as described previously [] The isolation of cytosolic and nuclear sections was executed followingAM1921Invitrogen RNA levels of U1 nuclear control GAPDHcytoplasmic control and LINC00992 were all estimatedby qRTPCR analysisPARIS„¢ KittheprotocolofFluorescence in situ hybridization FISH assayIn line with the recommendation of Ribo„¢ FISH KitC10910 Ribobio Guangzhou China FISH analysiswas implemented for testing the presence of LINC00992in prostate tumor cells Ribobio Company synthesizedthe LINC00992 probes labeled by Cy3 fluorescent dyeFollowing the fixation by paraformaldehyde and Triton X100 permeabilization DU145 and PC3 cellswere subsequently blocked in prehybridization bufferblocking solution Then incubation of cells with probehybridization buffer was later performed Next day afterrinsing and Hoechst staining the fluorescence was measured under a confocal laser scanning microscope ZeissGermanyCell counting kit8 CCK8 assayFor the viability assessment in DU145 PC3 and RWPE cells CCK8 assay was implemented as described 0cChen BMC Cancer Page of previously [] Cell viability was monitored at and h In short after being seeded onto 96well platesand cultured for indicated times cells were processedwith μl of CCK8 solution Then a microplate readerexamined the absorbance values at the wavelength of nm²ethynyl2²deoxyuridine EdU incorporation assayCell proliferation was examined through EdU assay asdescribed previously [] by using ClickiT EdU AlexaFluor Imaging Kit C10086 Invitrogen After hoftransfection EdU staining was carried out asinstructed The observation and calculation of EdUpositive cells was proceeded under the fluorescencemicroscopyTransferasemediated dUTP nick end labeling TUNELstainingTUNEL assay was carried out as described previously[] for probing DU145 and PC3 cell apoptosis with theassistance of an In Situ Cell Death Detection Kit Roche Mannheim Germany TUNELpositivecells were recorded under a light microscope × from visual fields which were chosen at randomTranswell migration assayThe application of transwell chambers with pore size μm Corning Costar Cambridge MA USA was aimedfor detecting cell migration in strict line with the instructions Cells that were previously suspended inserumfree RPMI1640 media were seeded into theupper chamber RPMI1640 medium containing FBS was supplemented in lower chamber as a chemoattractant Cells in the filters following h incubationwere immobilized in methanol and went through crystal violet staining The images of cells migratedthrough the filters were obtained and counted under themicroscopeWound healing assayThe DU145 PC3 and RWPE1 cells × cellswellwere prepared on glass culture dishes and cultivated at °C for a whole night to allow cells adhered to theplates followed by the straight scratch made with a plastic pipette tip after cell samples reached confluenceLater cells were rinsed in PBS to clear the detachedcells Finally the wounds at and h were imaged viaa light microscopy Olympus Tokyo JapanIn vivo experimentSixteen sixweekold male BALBC athymic nude micewere commercially available from the National Laboratory Animal Center Beijing China and maintained inSPFgrade animal lab All animalrelated protocols wereapproved by the Animal Research Ethics Committee ofthe First Affiliated Hospital of Kunming Medical University The in vivo experiment was undertaken via subcutaneous injection of × DU145 cells into the nudemice while the DU145 cells injected into indicated fourshgroups of mice were transfected with shNCLINC009921shLINC009921 pcDNA31 orshLINC009921 pcDNA31GOLM1 Tumor volume wasmonitored every days 28day after injection nudemice were sacrificed via cervical dislocation and thentumor samples were carefully dissected for weight assessment and hematoxylin and eosin HE stainingImmunohistochemistry IHCThe tumor samples collected from in vivo experimentswere treated with PFA dehydrated and embedded inparaffin Afterwardsthe paraffinembedded sections μm were prepared for IHC assay as described previously [] by use of the antiKi67 and antiPCNA antibodies AbcamStatistical analysisSPSS statistical software SPSS Armonk NY USAwas employed in the processing of data from threebiological replicates and data were expressed as mean ±SD Significance of difference within two groups wasdetermined using Student™s ttest while that among noless than two groups was tested via oneway or twowayANOVA P was considered as the threshold ofsignificanceResultsLINC00992 is overexpressed in prostate cancer andregulates cell proliferation apoptosis and migrationLINC00992 expression pattern in prostate cancer wasacquired from online GEPIA database As a resultLINC00992 was considerably upregulated in PRADprostate adenocarcinomatissues relative to normalones Fig 1a After detecting LINC00992 expression intissue samples obtained from patients with prostate cancer we observed that LINC00992 expression was higherin prostate cancer tissues than that in peritumor tissuesFigure S1A Moreover clinical data showed that higherexpression of LINC00992 in prostate cancer patientswas associated with lower survival rate Figure S1BFurthermore LINC00992 expression in the prostate cancer cells and RWPE1 cells was evaluated by qRTPCRConsequently higher level of LINC00992 was exhibitedin prostate cancer cells than that in RWPE1 cells Fig1b which was completely consistent with the result presented in previous discovery [] Particularly DU145and PC3 cells expressed the highest level of LINC00992and was thereby chosen for the later assays For silencingLINC00992 special shRNAs targeting LINC00992 was 0cChen BMC Cancer Page of Fig LINC00992 was overexpressed in prostate cancer and regulates cell proliferation apoptosis and migration a GEPIA database demonstratedthe overexpression of LINC00992 in tumor tissues in contrast to adjacent normal ones b LINC00992 expression was detected by qRTPCR in fourprostate cancer cell lines and control RWPE1 cells c LINC00992 expression was monitored by qRTPCR in DU145 and PC3 cells after transfectionwith shRNAs targeting LINC00992 shNC was used as the negative control d The viability of DU145 and PC3 cells was estimated through CCK8assay following LINC00992 depletion e The proliferation of DU145 and PC3 cells was investigated after LINC00992 depletion via EdU assay Scalebar μm f The apoptosis of DU145 and PC3 cells transfected with shLINC0099212 or shNC was estimated via TUNEL assay Scale bar μm g Western blot analysis was applied to examine the expression of apoptosisrelated proteins hi The migration of DU145 and PC3 cellswas analyzed via Transwell migration assay scale bar μm and wound healing assay scale bar μm after inhibiting LINC00992expression The fulllength images for blots in Fig 1g were presented in Supplementary figure P p transfected into DU145 and PC3 cells and the efficiencywas corroborated in qRTPCR Fig 1c And then thedata from CCK8 assay revealed that LINC00992 depletion suppressed the proliferation of DU145 and PC3cells Fig 1d As expected a declined proportion ofEdU positive cells was observed after knocking downLINC00992 Fig 1e suggesting the suppressive effect ofLINC00992 deficiency on prostate cancer cell proliferation Additionally the expression levels of proliferationrelated proteins PCNA CDK2 and Cyclin D1 were allreduced by silenced LINC00992 Figure S1C On thecontrary TUNEL assay uncovered that LINC00992knockdown facilitated cell apoptosis Fig 1f Meanwhile western blot analysis revealed that LINC00992knockdown promoted the apoptosis of DU145 and PC3cells as Bax protein level was increased whereas Bcl2protein level was decreased after LINC00992 was silenced in these two cells Figs 1g Figure S1D FurtherTranswell and wound healing assays indicated that themigration of DU145 and PC3 cells was retarded byLINC00992 depletion Fig 1hi Likewise the expression of migrationrelated molecular markers MMP2MMP9 pSrc and pFAK was decreased by LINC00992inhibition Figure S1E To further verify the biological 0cChen BMC Cancer Page of role of LINC00992 in prostate cancer we carried outgainoffunction assays in RWPE1 cells After overexpressing LINC00992 in RWPE1 cells Figure S2A cellproliferation was promoted Figure S2BC As expectedthe expression of PCNA CDK2 and Cyclin D1 wasdecreased by upregulation of LINC00992 Figure S2DSimilarly LINC00992cellIn addition upregulatingmigration Figure S2EFLINC00992 resulted in the elevated protein levels ofMMP2 MMP9 pSrc and pFAK Figure S2G All thesedata elucidated that LINC00992 could facilitate cell proliferation and migration whereas suppress cell apoptosisin prostate cancerupregulationfacilitatedMiR3935 is targeted by LINC00992Given the high correlation of the sublocalization ofLINC00992 with its functional mechanism the predication of LINC00992 presence in cells was performed viaLncLocatorhttpwwwcsbiosjtueducnbioinflncLocator Result predicted that LINC00992 located mainlyin cytoplasm Fig 2a Likewise FISH assay and RNAisolation of nuclear and cytoplasmic fractions furtherverified the abundance of LINC00992 in the cytoplasmof prostate cancer cells Fig 2bc highlighting a posttranscriptional control of LINC00992 in such cellsHence we speculated that LINC00992 might act as aceRNA in prostate cancer regulation According toDIANAlncBase the top three potential miRNAs possessing the binding capacity with LINC00992 were listedFig 2d To targetthe highlymatched miRNA toLINC00992 qRTPCR analysis was conducted to testthe expression changes of these miRNAs following eitherLINC00992 depletion or augmentation The resultsdemonstrated that only miR3935 expression was increased by LINC00992 depletion Fig 2e but reducedby LINC00992 overexpression in the meantime Fig 2fThus miR3935 was chosen for further analysis Afterwards RNA pull down assay was implemented and theresult depicted that LINC00992 was pulled down byBiomiR3935WT Fig 2g which indicated the bindingof LINC00992 and miR3935 Later we observed thesatisfactory efficiency of miR3935 overexpression andmiR3935 inhibition through qRTPCR analysis Fig2h Thereafter RIP assay applying antiAgo2 was executed Results illustrated that LINC00992 and miR3935were highly enriched in antiAgo2 group in comparisonwith control antibody Fig 2i certifying the associationof LINC00992 with miR3935 in the RNAinduced silencing complexes RISCs To further explore the interaction between LINC00992 and miR3935 the bindingsites between LINC00992 and miR3935 were predictedat first and then data from luciferase reporter assayrevealed that miR3935 upregulation decreased theluciferase activity of LINC00992WT reporter whereasmiR3935 inhibition increased the luciferase activity ofLINC00992WT reporter Fig 2j Altogether LINC0 combined with miR3935 to act as a miRNA decoyin prostate cancerLINC00992 regulates the expression of GOLM1 a targetof miR3935Present evidence has suggested that miRNAs can bindwith downstream target genes to inhibit their expressionHerein we searched the miR3935 target genes andeight mRNAs were found out Subsequently we detectedtheir expression in prostate cancer cells and normalcells Interestingly we found that only Golgi membraneprotein GOLM1 was highly expressed in four prostate cancer cell lines relative to normal controls Fig 3aFurther we discovered that GOLM1 expression wasmarkedly upregulated in prostate cancer tissues according to data from GEPIA database Fig 3b SimilarlyGOLM1 expression was much higher in prostate cancertissue samples than in peritumor samples Figure S3AIn addition the mRNA and protein levels of GOLM1were overexpressed in prostate cancer cells in contrastto RWPE1 cells Fig 3c Figure S3B Besides GOLM1has been previously revealed as a prostate cancer facilitator and was metastasisrelated in prostate tumor [“] Thus we hypothesized that GOLM1 might act asthe downstream of LINC00992miR3935 signaling inprostate cancer Through TargetScan httpwwwtargetscanvert_72 the binding site between GOLM1and miR3935 was predicted Fig 3d After conductingluciferase reporter assay in DU145 and PC3 cells weobserved that upregulation of miR3935 specifically decreased the luciferase activity of GOLM1WT reporterFig 3e confirming the interaction between miR3935and GOLM1 relied on the putative binding sites Thenwe unveiled that GOLM1 mRNA and protein levels wereboth reduced by LINC00992 inhibition or miR3935upregulation according to qRTPCR and western blotanalyses Fig 3fg Figure S3C Moreover data fromRIP assay unveiled the binding of miR3935 to GOLM1in the RISCs Fig 3h Further we demonstrated thatthe decreased mRNA and protein levels of GOLM1 induced by LINC00992 depletion could be restored afterinhibiting miR3935 expression Fig 3ij Figure S3DAll the results showed that LINC00992 upregulatedGOLM1 expression via directly binding to miR3935LINC00992 promotes prostate cancer cell proliferationand migration via elevating GOLM1 expressionTo test whether LINC00992 affected prostate cancer cellproliferation apoptosis and migration via regulatingmiR3935targeted GOLM1 we executed the rescue experiments with the upregulation of GOLM1 To beginwiththe efficiency of overexpressing GOLM1 was 0cChen BMC Cancer Page of Fig MiR3935 was targeted by LINC00992 a LncLocator predicted LINC00992 subcellular location b FISH analysis of LINC00992 distribution inprostate cancer cells Scale bar μm c RNA isolation of nuclear and cytoplasmic fractions assayed the subcellular distribution of LIN00992 inprostate cancer cells d Top three miRNAs which might interact with LINC00992 were predicted by DIANAlncBase e After transfection ofLINC00992silencing plasmids the expression of miR31575p miR11783p and miR3935 was examined via qRTPCR f Following LINC00992upregulation qRTPCR tested the levels of miR31575p miR11783p and miR3935 in DU145 and PC3 cells g RNA pull down assay wasimplemented to testify the binding capacity between LINC00992 and miR3935 h miR3935 overexpression efficiency and inhibition efficiencywere examined by qRTPCR i RIP assay disclosed the binding of miR3935 to LINC00992 in the antiAgo2 group j The potential binding sitebetween LINC00992 and miR3935 was shown And the luciferase activity of LINC00992WT or LINC00992MUT reporter was assessed vialuciferase reporter assay in DU145 and PC3 cells after transfection with miR3935mimics miR3935inhibitor NCinhibitor or NCmimics P p p analyzed through qRTPCR and western blot analysesand the outcome turned out to be satisfactory Fig 4abFigure S3E Then we observed that overexpression ofGOLM1 could significantly elevate the mRNA and protein expression of GOLM1 in shLINC009921transfected cells Figure S3F Afterwards data from CCK8revealed that the viability of DU145 cells was firstly hindered due to LINC00992 depletion while subsequentGOLM1 elevation reversed the inhibitory trend onDU145 cell viability Fig 4c Results from EdU assayalso exposed similar trends that GOLM1 upregulationimpactposedthesuppressivecountervailedbyLINC00992 downregulation on DU145 cell proliferationFig 4d Similarly the restraining effect of silencedLINC00992 on the expression of proliferationrelatedproteins could be reversed by GOLM1 upregulationFigure S3G Later TUNEL assay revealed that cellapoptosis rate was elevated by LINC00992 depletion andthen overexpressing GOLM1 reduced the increasedapoptosis rate of LINC00992depleted cells Fig 4eLikewise western blot analysis uncovered that overexpressing GOLM1 could offset the effect of LINC00992 0cChen BMC Cancer Page of Fig LINC00992 regulated the expression of GOLM1 a target of miR3935 a The expression of eight mRNAs in four prostate cancer cell linesand RWPE1 cells was detected by qRTPCR b GOLM1 was overexpressed in prostate cancer tissues according to GEPIA database c The mRNAand protein levels of GOLM1 were evaluated in prostate cancer cell lines and RWPE1 cell line by qRTPCR and western blot respectively d Thebinding sites between GOLM1 and miR3935 were predicted via TargetScan e Luciferase reporter assay presented the inhibited luciferase activityof GOLM1WT reporter in the presence of miR3935 mimics not NCmimics fg GOLM1 expression in transfected cells was tested by qRTPCR andwestern blot analyses h The combination of GOLM1 with miR3935 in the antiAgo2 group was validated by RIP assay ij The mRNA and proteinlevels of GOLM1 in different groups were examined via qRTPCR and western blot The fulllength gels for western blot data in Fig 3c g and jwere presented in Supplementary Figure P p p downregulation on the expression of apoptosisrelatedproteins Fig 4f Figure S3H Moreover Transwellmigration and wound healing assays illuminated thatthe retarding influence of silenced LINC00992 on cellmigration could be rescued by GOLM1 overexpression Fig 4gh As expected the inhibitory effect ofLINC00992 depletion on the expression of migrationrelated molecular markers MMP2 MMP9 pSrc andpFAK could be countervailed by GOLM1 overexpression Figure S3I Collectively GOLM1 was requiredcancercellular processesLINC00992regulatedinprostateLINC00992 contributes to tumor growth via upregulatingGOLM1 expressionAfter the in vitro exploration of LINC00992 performance in prostate cancer we applied the in vivo assays tofurther validate above findings As shown in Fig 5a tumors derived from LINC00992silenced DU145 cellswere smaller with the growth rate quite slower thanthose from control cells more importantly such blockage on tumor growth was obviously countervailed afterGOLM1 overexpression Besides elevating GOLM1 expression could recover the lessened tumor volume anddeclined tumor weight induced by LINC00992 deficiency 0cChen BMC Cancer Page of Fig LINC00992 promoted prostate cancer cell proliferation and migration via elevating GOLM1 expression ab GOLM1 mRNA and proteinlevels in DU145 cells transfected with pcDNA31 or pcDNA31GOLM1 were detected via qRTPCR and western blot pcDNA31 served as thenegative control c The viability of DU145 cells was determined via CCK8 following transfection of different plasmids d The proliferation oftransfected cells was evaluated via EdU assay e The apoptosis of transfected cells was monitored via TUNEL assay Scale bar μm f Theprotein levels of Bax and Bcl2 in different groups were detected via western blot gh The migration of transfected cells was measured viaTranswell migration assay scale bar μm and wound healing assay scale bar μm The fulllength gels for western blot data in Fig 4band f were presented in Supplementary Figure P p Fig 5bc Of note we discovered decreased level ofLINC00992 and enhanced level of miR3935 in tumorsfrom latter three groups compared to control group whilethe lowered expression of GOLM1 in tumors withLINC00992 inhibition was normalized under GOLM1overexpression Fig 5d In addition the inhibitory impactof silenced LINC00992 on the positivity of proliferationassociated proteins PCNA and Ki67 could be reversedby upregulation of GOLM1 Fig 5e Taken togetherLINC00992 promoted the tumorigenesis of prostate cancer through upregulating GOLM1 expressionDiscussionAs documented the aberrant regulation of lncRNAs is afrequent event in diversified tumor types Besides thecorrelation between abnormallncRNA expression andprostate cancer oncogenesis has also been extensivelyexplored For example lncRNA SNHG7 facilitates prostate cancer carcinogenesis via cyclin D1 by spongingmiR503 [] LncRNA SChLAP1 aggravates prostatecancer cell proliferation and metastasis by targetingmiR198 [] LncRNA PCAT1 contributes to prostatecancer tumorigenesis through modulating FSCN1 andsponging miR1455p [] In our work LINC00992 wasrevealed to be highly expressed in prostate cancer tissuesand cells but unlike former investigations our studygave a precise explanation about its role in prostate cancer Our study unveiled that LINC00992 promoted cellproliferation and migration whereas suppressed cellapoptosis in prostate cancer Abovementioned data 0cChen BMC Cancer Page of Fig LINC00992 contributes to tumor growth via upregulating GOLM1 expression a Representative images and the growth curves of tumorsfrom indicated groups bc The volume and weight of tumors from above groups d The expression of LINC00992 miR3935 and GOLM1 intumors from different groups was detected via qRTPCR analysis e The staining of PCNA and Ki67 in different groups was measured via IHCScale bar μm p 0cChen BMC Cancer Page of validated that LINC00992 elicited a tumorpromotingfunction in prostate cancerPresently accumulating evidence has indicated thatcytoplasmic lncRNAs assisted the expression of downstream miRNAtargeted mRNAs via sponging the specific miRNAs Before exploring LINC00992mediatedmechanism in prostate cancer herein we firstly discovered its subcellular distribution in prostate cancer cellswith both aids from online prediction tool LncLocatorand experimental data FISH and RNA isolation of nuclear and cytoplasmic fractions Our study for the firsttime uncovered that LINC00992 located mainly in thecytoplasm of prostate cancer cells Besides our studyalso completed LINC00992modulated mechanism bydisclosing the downstream target miR3935 The directinteraction between LINC00992 and miR3935

Thyroid_Cancer

"Oral cancer is one of the most common noncommunicable diseases worldwide This paper presentsan evaluation of the trends and geographical distributions of oral cancers in the Saudi Arabian populationMethods Data from Saudi Cancer Registry reports were used in this analysis which assessed the period between and All cancer cases are recorded in these reports as well as the age gender region and histologicalcancer sites for each patient Agestandardised and agespecific incidence rates were calculated in these reportsFor the purposes of this paper only cancers of the lips tongue and mouth were considered oral cancersResults Between and the Saudi Cancer Registry identified cancer cases in total Of these were oral cancer The mean agestandardised rate of oral cancer for the study period was per peoplefor females it was and for males it was The incidence of oral cancer varied by region with Jazan displayingthe highest agestandardised rate and Hail displaying the lowest A positive correlation was observed between oralcancer incidence and ageConclusion The overall trend of the agestandardised rate for both sexes remained constant from to However the oral cancer incidence in Saudi Arabia varies by region Studying this variation in more detail will helpto guide awareness programmes in the regions that are most in needKeywords Cancer epidemiology Cancer prevention and control Oral neoplasmBackgroundCancer is an intractable global health problem and theleading cause of death in the developed world in the developing worldit is the secondleading cause [] In the most recent year for which information fromthe International Agency for Research on Cancer IARCis available approximately million new cancer caseswere diagnosed and million people died from cancerworldwide [] In this same year new cases oflip and oral cavity cancers were reported representing of all cancer casesA review of the global prevalence of oral cancer revealsa wide variation in distribution among countries []Correspondence bmalshehrinuedusaDepartment of Clinical Laboratory Faculty of applied Medical SciencesNajran University PO Box Najran Kingdom of Saudi ArabiaTwothirds of the estimated incidence of oral cancer occurred in developing countries with up to of allnew oral cancer cases in Sri Lanka India Pakistan andBangladesh [] Converselyin France which has thehighest rate of oral cancer incidence in the EuropeanUnion only oral cancer cases were reported in representing just of all cancer cases [] Inthe USA the American Cancer Society estimated that in approximately people were diagnosed withoral cavity or oropharyngeal cancer and will dieof these cancers [] In Arab countries the prevalence oforal cancer is concentrated between western and southeast Asia [] While this type of cancer is relatively uncommon across Arab gulf countries Saudi Arabia andYemen are notable exceptions [] No studies have beenpublished discussing the epidemiological parameters andgeographic distribution of oral cancer cases or any of its 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 0cAlshehri World Journal of Surgical Oncology Page of subtypes in the Saudi Arabian population Thereforethis study analysed and discussed oral cancer trends inthe Saudi population by using the most recent dataavailableAccording to the International Classification of Diseases 10th revision ICD10 oral cancer is classifiedinto six sites mucosal lip ICD10 C00 tongue ICD C02 gum ICD10 C03 mouth floor ICD10C04 palate ICD10 C05 and mouth ICD10 C06However examining trends in oral cancer incidencerates that include all oral sites can be misleading Thedata analysed in this study only include cancers of thelip tongue and mouth ICD10C00“C06 which formthe majority of oral cancers moreover they have severalrisk factors in common and share a similar biology []Thus those accounting for a minority of oral cancercases were excludedMaterials and methodsDataThis retrospective descriptive epidemiological study analysed oral cancer cases in a Saudi population that hadbeen diagnosed from January through December The study used a method of analysis similar tothat used by Alshehri [] Their analyses incorporated male and female data on lip tongue and mouthICD10C00“C06 cancer cases to evaluate disease patterns in the Saudi population Data for the present studywere obtained from the Saudi Cancer Registry SCR apopulationbased registry established in by theMinistry of Health in Saudi Arabia This data can onlybe obtained from the reports published by the SCRSince the SCR has been publishing reports oncancer in Saudi Arabia with the primary objective of defining populationbased cancer incidences The presentstudy was conducted using these reports to derive a descriptive epidemiology of oral cancer in Saudi Arabia InSCR reports agestandardised ASR and agespecificAIR incidence rates were calculated with a focus ongenderspecific and regional differencesThe analysis included cases recorded in the SCR filesfrom January to December totalling cancer cases overall approximately of which wereoral cancerData analysisThe GraphPad Prism6 software was used to analyse thedata Descriptive analyses of epidemiological data wereconducted by calculating the mean of the percentagesand ASR stratified by age sex region and year of diagnosis The ASR was calculated in the SCR reports byadjusting all Saudi regions™ populations mathematicallyto have the same age structure On the other hand theAIR was calculated by summation of the number ofcancer cases occurring during the year in a region™spopulation among specific age and sex groups dividedby the midyear population of these age and sex groupsUsing these two standardised rates is important because age is a basic element of the risk of developingcancer globally [] Using summary measure tools suchas the ASR and AIR which represent the schedule ofagespecific rates in different regions and across timewill give us a more representative picture of the characteristics in question and enable comparisons of cancerincidences between several populations of Saudi regionsthat differ with respect to ageResultsIncrease in the number of oral cancer casesThe total number of cancer cases identified by the SCRfrom to was with males and females Of this total cases were oral cancer The number of registeredoral cancer cases increased gradually from MF in to a peak of MF in however only cases were reported in MF Table Table Number of oral cancer cases in Saudi Arabia for theperiod from to YearNumber of female casesNumber of male casesTotal 0cAlshehri World Journal of Surgical Oncology Page of The percentage of cases representing oral cancers was for females and for males Fig in These percentages decreased to for females and for males in Fig The percentage curve fororal cancer out of all cancer types for males and femalescorrelated with increases and decreases over the studyperiod apart from the years and Fig ASR of oral cancer fluctuated over the study periodBetween and the ASR per male casesfluctuated in it was trending downwards to alow of in and peaking at in beforedropping again to in Fig The female ASRper increased from in to a peak of in decreasing again to in Fig For bothsexes ASR curves like oral cancer percentages correlateto increases and decreases over the study period apartfrom the years and Fig and generally remained constant from to ASR of oral cancer varies by regionThe ASR data for oral cancer cases of all persons demonstrated a wide variation across Saudi regions TheASR means per people for the period from to ranged from in Hail to in Jazan with anational average of per Fig The Jazan region had the highest male ASR mean at followed by the Najran and Tabuk regions at each Fig Conversely Qassim Baha Hail and theNorthern province reported the lowest ASR averages at and per respectively Fig Male and female ASR data were generally equivalentin terms of region rankings with the Jazan region posting the highest overall ASR of as an average valueof both genders followed by the Makkah region at and the Najran region at Fig Similarly the HailBaha Qassim and Madinah regions posted the lowestASR averages at and respectively FigAIR of oral cancer increases with ageThe AIR data from to showed a positive correlation between oral cancer incidence and age withmost cancer cases occurring in the older age groups Figure shows the AIR of oral cancer increasing noticeablywith age up until age More than of cases werediagnosed after the age of Some AIR differences were found between the sexesacross age groups From ages to rates of oral cancer were higher in females than in males however thistrend had reversed to favour males in the 75andoverage group The overall AIR per showed onlyslight differences between the sexes at for femalesand for males Fig DiscussionA review of oral cancer data in Saudi Arabia for theperiod from to showed an overall increasingtrend in the numbers of oral cancer patients Despitethis rise ASR data trends for oral cancer remained constant from to Fig This curve stabilised inthe face of a substantial Saudi Arabian population increase from million in to million in [] Many accumulative factors could be contributed tothis stability First the significant increased access tohealth services in Saudi regions has contributed to thedissemination of oral health awareness and early diagnosis of some cases of metaplasia that were discovered before they could develop into cancerous tumours SecondFig Consistency in percentage curves for oral cancer out of all cancer types from to The percentage curve for oral cancer out of allcancer types for males and females are correlated with overall increases and decreases over the period from to with the exception ofyears and 0cAlshehri World Journal of Surgical Oncology Page of Fig Agestandardised incidence rates ASR of oral cancer fluctuated over the study period Between and the male ASR was per in and dropped to in The female ASR fluctuated between and per the increased level of public health in the Kingdom isusually linked to an increase in the economic level of thecountry and individuals may have contributed to thisconstancy as many infectious factors such as virusesand fungi have been linked to oral cancers Third SaudiArabia is a majority Islamic country wherein many oralcancer risk factors such as alcohol consumption andcigarette smoking are forbidden by Islamic law Islamiclaw may thus mediate the lower number of oral cancercases in Saudi Arabia compared to the rest of the world[] Thus based on the IARC data for eight ofthe nine world regions whose ASR of oral cancer isabove the global rate [ ] were located in nonMuslimcountries [] with Melanesian regions having the highest rate [] In contrast most of the regions locatedwithin Muslim countries were ranked below the globalASR [] Further investigation of this aspect could therefore be valuable to cancer prevention effortsThe ASR data revealed that more females than maleswere diagnosed with oral cancer in Saudi Arabia at for men and for women This finding is in contrastwith global data showing that men are more likely to develop oral cancer than women [] In the most recent year for which IARC information is available theglobal ASR of oral cancer was for men and forwomen [] While these rates do not match the globalFig Agespecific incidence rates AIR of oral cancer increases with age The total AIR of oral cancer increased noticeably with up until patientswere and over More than of the cases were diagnosed after the age of 0cAlshehri World Journal of Surgical Oncology Page of Fig Agestandardised incidence rates ASR of oral cancer varies by region in Saudi Arabia For all persons the ASR means per peoplefor the period from to ranged from in the Hail region to in the Jazan regionsex distribution oral cancer in Saudi Arabia has a relatively low overall ASR when compared to the globalaverage as discussed aboveResults also revealed consistency in the ASR oral cancer curve for both sexes Fig potentially due to thepresence of common risk factors for oral cancer in malesand females This finding could be used as a startingthreshold for studying the risk factors of oral cancer inthe Saudi population through studying the common factors between the sexesAs with many other types of cancer the present studyfound a correlation between the occurrence of oral cancer and age with of cases diagnosed after the age of years In the USA the average age at diagnosis of oralcancer is years and twothirds of individuals with thisdisease are over the age of [] Ageing is accompaniedby increased susceptibility to cancercausing geneticmaturations due to accumulated exposures to environmental and behavioural risk factors Avoiding these riskfactors could greatly reduce the role that ageing plays incancerThis study found a wide variation in the incidence oforal cancers among Saudi regions Such differencescould indicate that regional environmental factors andlifestyle habits affect oral cancer incidence The resultsreviewed above found that the Jazan region possessedthe highest ASR of people with oral cancer In contrastthe Northern province presented the lowest ASR Severalstudies have focused on investigating why the Jazan region has such a high incidence of oral cancer [“]Ibrahim and others focused on the association ofcertain eating habits and lifestyle behaviours with the development of oral cancer especially the abuse ofshamma a form of smokeless tobacco and the chewingof khat Catha edulis leaves These substances havebeen classified as carcinogens especially in relation tooral cancer Studies by these researchers found that consuming shamma increased the odds of developing oralcancer 29fold suggesting a strong link between oralcancer and diet and lifestyle choicesAccording to the above poor dietary habits related totobacco use and its derivatives are one of the main reasons for the high incidence of oral cancer in some citiesand not others Other factors such as variations in thegenetic background of the Saudi regions™ citizens cannotbe excluded especially because most of the populationin the Kingdom™s regions is tribal so consanguineousmarriages are highly common Thus genomic sequencing can provide information on genetic variants thatmay be present in citizens of these regions and that maybe linked with increased or decreased rates of oral cancer development Populationbased genetic testing issuggestedConclusionDespite the presence of yeartoyear changes in the incidence of oral cancer in the Saudi population there wasoverall no noticeable change in the incidence of oralcancer in the Saudi Arabian population for the periodbetween and In contrast to some international findings females were somewhat more likelythan males to be diagnosed with oral cancer in SaudiArabia The positive correlation between ageing and theincidence of oral cancer for both males and femalesdemonstrates that oral cancer is mainly a disease of theelderly both in Saudi Arabia and across the globe Thewide variation in the incidence rates among Saudi regions raises an important research question concerning 0cAlshehri World Journal of Surgical Oncology Page of World Bank World Development Indicators Washington DC WorldBank Access online via httpwwwirieduarpublicaciones_irianuariocd_anuario_2014Economia4bpdf Albar MA Islamic teachings and cancer prevention J Family CommunityMed “Ibrahim EM Satti MB Al Idrissi HY Higazi MM Magbool GM Al QA Oralcancer in Saudi Arabia the role of alqat and alshammah Cancer DetectPrev “ Alsanosy RM Mahfouz MS Gaffar AM Khat chewing among students ofhigher education in Jazan region Saudi Arabia prevalence pattern andrelated factors Biomed Res Int Quadri MFA Alharbi F Bajonaid AMS Moafa IHY Al Sharwani A AlamirAHA Oral squamous cell carcinoma and associated risk factors in JazanSaudi Arabia a hospital based case control study Asian Pacific J CancerPrev “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationspotential causes that need to be investigated furtherThe knowledge produced by this study must be translated into interventions by performing indepth analysesof regional differences This will contribute to the effortsof preventing oral cancer in Saudi ArabiaAbbreviationsAIR Agestandardised incidence rates ASR Agestandardised specific ratesIARC International Agency for Research on Cancer SCR Saudi CancerRegistry ICD10 International Classification of Diseases 10th revisionAcknowledgementsI express my thanks and gratitude to the Saudi Ministry of Health forproviding me with the Saudi Cancer Registry reportsAuthor™s contributionsI certify that I have participated sufficiently in the intellectual contentconception and design of this work analysis and interpretation of the dataas well as the writing of the manuscript to take public responsibility for itand have agreed to have my name listed as a contributor The author readand approved the final manuscriptFundingThis research received no specific grant from any funding agency in thepublic commercial or notforprofit sectorsAvailability of data and materialsThe data that support the findings of this study are available from SaudiMinistry of Health but restrictions apply to the availability of these datawhich were used under authorization for the current studyEthics approval and consent to participateThis study was approved by the Research Ethics Committee at NajranUniversity The ethical document reference No ETConsent for publicationA secondary data analysis was conducted in this retrospective study byusing a published dataCompeting interestsThe author declares that he is the only author for this work No other authorcontributed to this work He is also in agreement with the content of themanuscript He declares no conflict of interestReceived June Accepted August ReferencesJemal A Bray F Center MM Ferlay J Ward E Forman D Global cancerstatistics CA Cancer J Clin “Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin “ Warnakulasuriya S GloWarnakulasuriya S Global epidemiology of oral andoropharyngeal cancer Oral Oncology ““ httpsdoi101016joraloncology200806002bal epidemiology of oral andoropharyngeal cancer Oral Oncol Rick A Afsaneh B Cancer Facts Figures American Cancer Society Access online via httpswwwcancercontentdamcancerresearchcancerfactsandstatisticsannualcancerfactsandfigures2017cancerfactsandfigures2017pdfAlJaber A AlNasser L ElMetwally A Epidemiology of oral cancer in Arabcountries Saudi Med J “Ariyawardana A Johnson NW Trends of lip oral cavity and oropharyngealcancers in Australia overall good news but with rising rates inthe oropharynx BMC Cancer Alshehri B Descriptive epidemiological analysis of thyroid cancer in the Saudipopulation Asian Pacific J Cancer Prev “Armitage P Doll R The age distribution of cancer and a multistage theoryof carcinogenesis Br J Cancer “ 0c"

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

"diagnostic performance of intravoxel incoherent motion diffusionweightedimaging IVIMDWI in the differential diagnosis of pulmonary tumors remained debatable among published studiesThis study aimed to pool and summary the relevant results to provide more robust evidence in this issue using ametaanalysis methodMaterials and methods The researches regarding the differential diagnosis of lung lesions using IVIMDWI weresystemically searched in Pubmed Embase Web of science and Wangfang database without time limitation ReviewManager was used to calculate the standardized mean difference SMD and confidence intervals ofapparent diffusion coefficient ADC tissue diffusivity D pseudodiffusivity D and perfusion fraction f Stata was used to pool the sensitivity specificity and area under the curve AUC as well as publication bias andheterogeneity Fagan™s nomogram was used to predict the posttest probabilitiesResults Eleven studies with malignant and benign lung lesions were included Most include studies showed alow to unclear risk of bias and low concerns regarding applicability Lung cancer demonstrated a significant lower ADCSMD P D SMD P and f values SMD P than benign lesions except Dvalue SMD P D value demonstrated the best diagnostic performance sensitivity specificity AUC and highest posttest probability and for D ADC f and D values in the differential diagnosisof lung tumors followed by ADC sensitivity specificity AUC f sensitivity specificity AUC and D values sensitivity specificity AUC Continued on next page Correspondence 849049724qqcom wuypsysucccnhenisysucccn Jianye Liang Jing Li and Zhipeng Li contributed equally to this work2Department of Radiology Maoming People™s Hospital Maoming Guangdong China1Department of Medical Imaging Sun Yatsen University Cancer Center StateKey Laboratory of Oncology in South China Collaborative Innovation Centerfor Cancer Medicine No651 Dongfeng Road East Guangzhou Guangdong China 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 0cLiang BMC Cancer Page of Continued from previous pageConclusion IVIMDWI parameters show potentially strong diagnostic capabilities in the differential diagnosis of lungtumors based on the tumor cellularity and perfusion characteristics and D value demonstrated better diagnosticperformance compared to monoexponential ADCKeywords IVIMDWI Posttest probability Diagnostic performance Lung neoplasm Magnetic resonance imaging MetaanalysisIntroductionLung cancer is the most commonly diagnosed cancer of the total cases and the leading cause of cancerdeath of the total cancer deaths in aroundthe world [] The incidence and mortality of lung cancer still increased in recent years Accurate and earlydiagnosis is help to select optimal treatment strategy andimprove the outcome of patients with lung cancerlungtumorsandefficacyComputed tomography CT is the main imaging modality for lung lesions largely based on morphologicaland enhanced characteristics However the relativelylow specificity and administration of contrast agent limitits wide use in clinical practice Magnetic resonance imaging MRI was rarely used in detecting lung lesionspreviously due to the obvious cardiac and respiratorymotionlow signaltonoise ratio from the inherentlylow lungproton density and magnetic susceptibilityartifact of airfilled pulmonary tissue subjected to highfield strength [] With the development of MRI hardwares and various rapid imaging technologies such asimproved gradient performance parallel imaging techniques and freebreathing acquisition MRI has been inidentification of benign andcreasingly used formalignantevaluationDiffusionweighted imaging DWI is a radiationfreeand contrastfree functional imaging sequence which allows measurement of water molecular movement usingapparent diffusion coefficient ADC and demonstratespotential to differentiate malignant from benign lung lesions A previous metaanalysis even reported a higherdiagnostic performance with a pooled sensitivity specificity and areas under the curve AUC of and in DWI compared to PETCT whose sensitivityspecificity and AUC were and respectivelyThe monoexponential model is expressed as SI SI0 expˆ’b·ADC where SI0 refers to the mean signal intensity SI of the region of interest for b smm2 while SIrefers to the signal intensity for higher b values However the monoexponential model cannot separate thepseudodiffusion from pure molecular diffusion andADC calculated from the monoexponential modelmixesthe conventionalmonoexponential model cannot accurately reflect thetrue diffusivity owing to the influence of microcirculation perfusion []the two effects Thereforechangesthe microenvironmentIntravoxel incoherent motion IVIM is an advancedimaging technique which was first proposed by Le Bihan [] It can separate the incoherent motion of watermolecules within the capillaries from molecular diffusionin the extravascular space [] The true diffusion coefficient D value pseudodiffusion coefficient D valueand perfusion fraction f value were generated using abiexponential model with multiple bvalues expressed asSI SI0 f · expˆ’bD f · expˆ’bD The IVIMmodel can separate the pseudodiffusion from pure molecular diffusion and independently reflect the microcirculation perfusion D and tumor cellularity D basedon that equation [] This model provides more detailedand accurate information and can make a better interpretation forandcharacterization of tumor grades As such these parameters are important to be analyzed Several studies hadapplied IVIMDWI to discriminate lung cancer from benign lesions and demonstrated better or comparablediagnostic performance compared with traditional ADCvalue [“] However the diagnostic performances ofIVIMDWI derived parameters in the differentiation oflung tumors were not consistent and the application stillremained debatable in the lung For example severalstudies indicated that lung cancer had a higher D valuethan benign lesion [“] while some studies reportedadverse [ ] or insignificant results [ ] Theoretically the true diffusitivity should have better diagnostic performance than ADC in distinguishing lunglesions but some studies indicated a much lower areaunder the curve AUC or accuracy in D value comparedto ADC [ ] Cancerous tissue generally has activeangiogenesis and rich blood supply compared to benignlesions but most studies indicated a lower f value inlung cancer the results of which should be further confirmed The sample sizes in most studies were still notenough to draw a robust for its performancethe application of IVIMDWI in the lung has not yetformed a clinical guideline or become a routine sequence in the MRI protocol Therefore we attempted topool all the published results about the diagnostic performance of IVIMDWI in the differentiation of malignant and benign lung lesions using a metaanalysismethod Besides the diagnostic performance of IVIMDWI was compared to conventional DWIderived ADC 0cLiang BMC Cancer Page of this study provides additionalvalue to determine the suitability for clinical applicationThe controversialissues between different researcheswill also be addressed with more reliable evidence Furthermoreinformationabout technical feasibility on lung MRI and the functional changes oflung lesions with IVIMDWI Thisstudy may further attract the researchers to perform thelung studies using noninvasive MR imaging by solvingthe technical issues on Lung MRIMaterials and methodsData sourcesThe studies regarding the differential diagnosis of lungtumors using IVIMDWI parameters were systemicallyretrieved by two senior librarians in PubMed EmbaseWeb of science and Wangfang database without timelimitation A searching formula was formed with different combinations of the medical subject headings or keywords from IVIM intravoxel incoherent motion multiple bvalue DWI biexponential and lung or pulmonarylesion cancer carcinoma neoplasm The primarysearches were limited in the titles and abstracts We alsoperformed a manual retrieval of the reference lists fromincluded studiesbStudies selectionStudies met the following criteria were included a theresearch purpose was to differentiate lung cancer frombenign lesions using IVIMDWI parametersthemean and standard deviation SD of each parameterwas provided c their diagnostic performance aboutsensitivity and specificity or truepositive TP falsenegative FN falsepositive FP and truenegative TNwere reported d the lung cancer should be confirmedby pathology after initial MRI examination Exclusioncriteria mainly included a duplication from the sameauthors or institutions b metaanalysis conference abstract review or any unpublished results and c animalexperiments or nonlung researchesData extractionA spreadsheet was used to extract the mean values andSD as well as the diagnostic performance of ADC D Dand f values with threshold value AUC sensitivityand specificity in respective study by one author andreviewed by another one Other information includedthe first author publication years field strength and vendors b values patient ages tumor sizes and numbers ofmalignant and benign lesions TP FN FP and TN canbe calculated when only the amount of malignant andbenign lesions as well as sensitivity and specificity or receiver operating curve was providedQuality assessmentThe quality of studies and likelihood of bias were evaluated using Review Manager software Cochrane Collaboration Oxford UKreferring to the QualityAssessment of Diagnostic Accuracy Studies [] Weassessed the risk of bias and applicability in four domains including patient selection index tests referencestandard flow and timing []Publication bias and heterogeneity evaluationAs two parts of data were pooled in our study includingquantitative values and diagnostic performance of eachparameter funnel plots and Begg™s test were used tovisually and quantitatively assess the publication bias forthe continuous variables and Deek™s plot assessed thepublication bias of sensitivity and specificity using Stataversion StataCorp LP College Station TX Anasymmetric or skewed funnel plot P of Begg™s testor Deek™s test indicated the potential of publication bias[] Inconsistency index I2 and Cochran™s Q tests wereused to explore the heterogeneity of included studieswith I2 or P for Cochran Q test suggestedstatistically significant heterogeneity and a randomeffect model was applied in subsequent pooling or afixedeffect model when I2 []Evidence synthesisWe constructed the forest plots for continuous variablesand calculated the standardized mean difference SMDbetween lung cancer and benign lesions using ReviewManager software We used the bivariate regressionmodel to pool the diagnostic performance with sensitivity specificity positive likelihood ratio PLR negativelikelihood ratio NLR diagnostic odds ratio DOR andAUC using Stata version The summary receiveroperating characteristic curves and Fagan™s nomogramswere also plotted to determine the diagnostic values andpredict the posttest probabilities of ADC D D and fvalues in the differential diagnosis of lung tumorsResultsLiterature search and selectionBy searching the key words in the titles and abstracts atotal of potential studies were obtained from multiple databases A total of studies regarding metaanalysis conference abstract case report and reviewwere excluded after screening the titles and abstractsAnimal studies nonlung researches and duplicationfrom the same authors or institutions led to further exclude studies We scrutinized the fulltexts of theremaining studies in detail and excluded an additional studies for the following reasons a lack ofsufficient data to be pooled b low quality assessmentcIVIMDWI was interfered by treatment and d 0cLiang BMC Cancer Page of cancer was not confirmed by pathology Eventually eligible studies with malignant and benign lunglesions were included for analysis The flowchart detailing the process of study selection was provided in Fig Basic information and diagnostic performance for eachincluded study was detailed in Table and Table Inother to include every potential we did not set acriterion on the field strength T or T FromTable there are three studies using T and eightstudies using T for imaging Although field strengthof T is better for image quality the results from Tscanner are also acceptable Therefore studies with either of field strengths are included for analysisQuality assessmentThe distribution of Quality Assessment of DiagnosticAccuracy Studies“ scores for risk of bias and applicability concerns were shown in Fig The overall qualityof included studies was acceptable Regarding patient selection four studies were marked unclear risk of bias dueto ambiguity for consecutive enrollment and prospectivedesign or not The applicability concerns remainedunclear concern as the tumor types were inconsistentbetween malignant and benign tumors from two studiesTwo studies were marked unclear and high risk of biaswith unclear concern of applicability for index test asthe threshold values for D and f values were not provided Three studies showed unclear risks of bias for reference standard because some of the benign lesionswere diagnosed through a long time followup Threestudies were marked unclear and high risk of bias in patient flow and timing domain because the time intervalbetween MR examination and pathological confirmationwas not reportedQuantitative analysisADC used for diagnosis of lung tumorNine studies regarding ADC used in differentiating lungtumors were included for analysis The χ2 andP of heterogeneity test with I2 suggestedmoderate heterogeneity among included studies Theforest plot in Fig showed the distribution of ADC between lung cancer and benign lesions A randomeffectsmodel generated a SMD of ˆ’ ˆ’ ˆ’ P Fig Flowchart detailing the study selection process Eleven studies that met the inclusion criteria were included FN false negative FP falsepositive TN true negative TP true positive 0cLiang BMC Cancer Page of Table Basic information for each included studyAuthorDeng []Machine type T PhilipsYearb values smm2Huang []Jiang []Jiao []Wan []Wang LL []Wang Y []Yuan []Zhou []Wang XH []Koyama []NA Not available T GE T Siemens T GE T Philips T Siemens T Philips T Siemens T GE T GE T PhilipsAge years ± ± ““ “ ± “NA ± ± ± Tumor size cm Malignant ± BenignNA ± NA “ ± NA “ ± “Table The diagnostic performance for each included studyIndicatorADCAuthorDeng []ThresholdYearHuang []Jiang []Wan []Wang Y []Yuan []Zhou []Huang []Jiang []Jiao []Wan []Wang LL []Wang Y []Yuan []Zhou []Wang XH []Deng []Huang []Jiang []Wan []Yuan []Zhou []Wang XH []Deng []Huang []Wan []Wang LL []Yuan []NANANADDfAUCNANANANANANASensitivitySpecificityTPFPFNTNWang XH []NA Not available ADC Apparent diffusion coefficient D Tissue diffusivity D pseudodiffusivity f Perfusion fraction AUC Area under the curve FNFalse negative FP False positive TN True negative TP True positive Threshold values of ADC D and D are factors of ˆ’ mm2s 0cLiang BMC Cancer Page of Fig The distribution of risk of bias and applicability concerns for each included study using QUADAS2 a and a summary methodologicalquality b between lung cancer and benign lesions forADC A basically symmetric funnel plot in Fig andP of Begg™s Test suggested no publication biasin ADCD value used for diagnosis of lung tumorEleven studies regarding D value used in differentiatinglung tumors were included for analysis The χ2 and P of heterogeneity test with I2 suggested moderate heterogeneity among included studies Theforest plot in Fig showed the distribution of D value between lung cancer and benign lesions A randomeffectsmodel generated a SMD of ˆ’ ˆ’ ˆ’ P between lung cancer and benign lesions for D value A basically symmetric funnel plot in Fig and P of Begg™sTest suggested no publication bias in D value 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of apparent diffusion coefficient ADC between lung cancer and benign lesions The standardized meandifferences indicated that lung cancers had a significantly lower ADC than benign lesionsFig Funnel plot of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction f Thebasically symmetric funnel plots indicated no publication bias in these parameters 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of tissue diffusivity D between lung cancer and benign lesions The standardized mean differencesindicated that lung cancer had a significantly lower D value than benign lesionsD value used for diagnosis of lung tumorTen studies regarding D value used in differentiatinglung tumors were included for analysis The χ2 and P of heterogeneity test with I2 suggested obvious heterogeneity among included studiesThe forest plot in Fig showed the distribution of Dbetween lung cancer and benign lesions A randomeffects model generated a SMD of ˆ’ P between lung cancer and benign lesions forD A basically symmetric funnel plot in Fig and P of Begg™s Test suggested no publication bias in Df value used for diagnosis of lung tumorEleven studies regarding f value used in differentiatinglung tumors were included for analysis The χ2 and P of heterogeneity test with I2 suggested moderate heterogeneity among included studiesThe forest plot in Fig showed the distribution off value between lung cancer and benign lesions Arandomeffects model generated a SMD of ˆ’ ˆ’ ˆ’ P between lung cancer andbenign lesions for f value A basically symmetricfunnel plot in Fig and P of Begg™s Testsuggested no publication bias in f valueDiagnostic performanceThe Diagnostic performance with pooled sensitivity specificity PLR NLR DOR and AUC of ADC D D and fvalues were listed in Table Deek™s funnel plots in Fig and asymmetry tests indicated no obvious publicationbias in ADC D D and f values P and for ADC D D and f values respectively Fig plotted the summary receiver operating characteristiccurves of ADC D D and f values D value demonstrated the best diagnostic performance sensitivity specificity AUC in the differentialdiagnosis of lung tumors followed by ADC sensitivity specificity AUC f sensitivity Fig Forest plot of the mean value of pseudodiffusivity D between lung cancer and benign lesions The standardized mean differencesindicated that the difference of D value between lung cancers and benign lesions were insignificant 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of perfusion fraction f between lung cancer and benign lesions The standardized mean differencesindicated that lung cancer had a significantly lower f value than benign lesionsspecificity AUC and D values sensitivity specificity AUC Posttest probabilitiesLikelihood ratio and posttest probability were also important for diagnosing a disease [] which provided alikelihood that a patient was diagnosed with a certaindisease or not using the MRI parameters Fig plottedthe Fagan™s nomograms of ADC D D and f values forpredicting posttest probabilities All the pretest probabilities were set at by default We regarded thediagnosis of lung cancer as a positive event corresponding to a lower ADC D and f values Similarly the noncancerous tissues with a higher ADC D and f valueswere regarded as a negative event The posttest probability increased to from a pretest probability of with a PLR of and decreased to with a NLRof with the prompt of ADC This indicated that thediagnostic preference to lung cancer will be obviouslyenhanced with the help of ADC a lower ADC compared with the condition without the prompt of ADCwhose diagnostic probability was set at beforehandIn contrast the probability of diagnosing lung cancerwill significantly drop from to when a negativeevent occurs a higher ADC Similarly the posttestprobability of diagnosing lung cancer will reach to with a PLR of and drop to with a NLR of using D for guiding The posttest probability of diagnosing lung cancer will reach to with a PLR of and drop to with a NLR of in the help of fvalue These data indicated that both ADC and IVIMparameters helped to enhance the accuracy for diagnosing lung cancerDiscussionIVIMDWI is a noninvasive technique that shows superiority in reflecting tumor cellularity and perfusion without the need of contrast agent It had already beenapplied in the differentiation of thyroid nodules []breast [] liver [] and brain tumors [] with gooddiagnostic performance To our best knowledge there isstill no pulmonary study with large sample size to settledown the value of IVIM for quantitatively distinguishinglung cancer from benign tissues in the background ofIVIM becoming a research hotspot in the wholebodytumors Our study provided a timely summary in thisissue through pooling all published evidence with strictinclusion criteria and quality assessment The resultsdemonstrated IVIM model had a good diagnostic performance in distinguishing lung lesionsTable Pooled estimates and heterogeneity measures for ADC D D and f valuesDORIndexSpecificitySensitivityNLRPLRAUCADC DD I2 SensitivitySpecificity fADC Apparent diffusion coefficient D Tissue diffusivity D Pseudodiffusivity f Perfusion fraction PLR Positive likelihood ratio NLR Negative likelihood ratio DORDiagnostic odds ratio AUC Area under the curve I2 inconsistency index 0cLiang BMC Cancer Page of Fig Deeks™ funnel plots regarding diagnostic performance for a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivityD and d perfusion fraction f No publication bias was indicated in the four parameters P In this metaanalysis the SMDs suggested that lungcancer demonstrated a lower ADC and D values thanbenign lesions The lung cancer usually has dense cellularity and nucleoplasm ratio with active proliferativecapacity which may reduce the extracellular space andrestrict the movement of water molecules causing a reduction in diffusion coefficient The pooled results alsosuggested an excellent diagnostic performance with ahigh sensitivity specificity AUC and increased posttestprobability in both ADC and D values followed by fvalue Monoexponential modelancannot provideindependent perfusionrelated parameter and may miscalculate the water molecule movement due to a mixwith microcirculation perfusion and therefore resultedin an overestimated ADC value in a certain extent []Therefore the best diagnostic performance was observedin D value instead of ADC valueInterestinglylung cancer demonstrated a significantlower f value but insignificant D value compared withbenign lesions F value refers to vascular volume ratioand reflects the microcirculation perfusion in the capillaries F value increases with increased tissue perfusion 0cLiang BMC Cancer Page of Fig Summary receiver operating characteristic SROC curve of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction f in the diagnosis of lung lesions D value demonstrated the highest area under the curve followed byADC f and D valuesinflammatoryconsistHigher f value is supposed to be observed in malignanttumors due to neovascularization compared to benignlesions However these results are not unreasonable because the benign lesions occurring in the lung are generallyoftuberculosisinfectiongranuloma or bloodrich tumor such as inflammatorypseudotumor They are usually featured by marked vascular changesincreased bloodflow and enhanced vessel permeability which generallyincluding vasodilationinfections whichanic pneumoniafungaloccur at the capillary network [] A perfusion studyusing CT with exogenous contrast indicated active infectious nodules had comparable or even higher perfusionpeak enhancement increment and blood volume withsteeper time to peak than malignant nodules [] Theresults were in good agreement with our study in another aspect However the diagnostic performance of fvalue was relatively low with the sensitivity specificityand AUC of and only F value is also associated with echo time relaxation effects and T2 0cLiang BMC Cancer Page of Fig Fagan™s nomogram of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction fD and ADC demonstrated similar and highest posttest probability among the four parameterscontribution [] which may reduce its diagnostic accuracyperformance to a certain extentD value is proportional to the average blood velocityand mean capillary segment length [] D value wasnot statistically significant in differentiating benign andmalignant lung lesions in this metaanalysis A poormeasurement reproducibility of D was indicated by thehuge standard deviations in the included studies Theoretically the more bvalues are selected the higher theaccuracy of model fitting will be Besides measurementat lower bvalue had been reported to be less reproducible and stable compared with measurement at higherbvalue and previous studies suggested measurements ata larger number of lower bvalue should be obtained forreducing measurement errors and signalto noise variation [ ] However a larger number of bvalue applied in IVIM model will significantly prolong thescanning times and introduce obvious motion and susceptibility artifacts especially in the pulmonary MRITherefore D value is still not adequate to differentiatelung lesions due to the low reliability stability and accuracy as indicated in our metaanalysisADC D D and f values all demonstrated moderate toobvious heterogeneity which should be explored Firstboth T and T MR scanners with various combinations of bvalue were used to perform IVIMDWI inthese studies which may influence the accurate calculations of diffusion and perfusion coefficients and decrease the diagnostic performance compared to monoexponential ADC Second the lesion sizes and density oflung cancer such as ground glass opacity on initial CTvaried from studies to studies which may perform different biological characteristics and also lead to themeasurement variability in ADC and IVIM parametersindicated by Weller [] and Jiang []Third the benign lesions consisted of a variety of inflammatory infections and benign tumors which mayintroduce significant heterogeneity in these parameterswhen compared with lung cancer Last most studies delineated the regions of interest on the largest slice instead of the entire tumors which may lead to someselection bias owing to tumor heterogeneity Histogramanalyses for the whole lesions which can reduce themeasurement variability may be a more promisingmethod for assessing lung nodules in the future studyThere were several limitations First as the sensitivityof detecting pure ground glass opacity or small lesionsare quite low on conventional DWI or IVIMDWI theselesions were inevitably excluded from the original studies which may decrease the availability of IVIM in theclinical application to a certain extent Second we hadnot performed a direct comparison with dynamic contrast enhancedCTMRI or Fluorine 18FDG PETCTwhich was also commonly used in the diagnosis of lungcancer The issue about whether IVIMDWI addedvalues to multiparametric MRI or CT in a large samplesize was still not clearConclusionsIVIMDWI parameters show potentially strong diagnostic capabilities in the differential diagnosis of lung tumors and D value demonstrated better diagnosticperformance compared to monoexponential ADC Fvalue can differentiate the perfusion difference betweenlung cancer and benign lesions The application ofIVIMDWI will further help the clinicians make a bettermanagement for cancer treatment and prognosis evaluation based on the tumor cellularity and perfusion characteristics detected by IVIM technique 0cLiang BMC Cancer Page of AbbreviationsAUC Area under the curve ADC Apparent diffusion coefficient D Tissuediffusivity D Pseudodiffusivity IVIMDWI Intravoxel incoherent motiondiffusionweighted imaging SMD Standardized mean differenceI2 Inconsistency index PLR Positive likelihood ratio NLR Negative likelihoodratio DOR Diagnostic odds ratioAcknowledgementsNot applicableAuthors™ contributionsNH was the guarantor of this metaanalysis and had full access to all the datain the study and took responsibility for the integrity of the data and the accuracy of the data analysis NH YW and XL conceived the study and revisedthe manuscript JL ZL and TM drafted the manuscript JC and WM searchedthe databases and acquired the data WM and SC performed data analysisand interpretation Jing Li substantively revises the manuscript based on thecomments and provides language proofreading for the revised version Allauthors had read and approved the manuscriptAuthors™ informationNot applicableFundingThe Highlevel Hospital Construction Research Project of Maoming People™sHospital supported the consultation fee from a statistician for checking thecorrectness of the statistical methods the National Key Research and Development Program of China grant no 2017YFC0112605 and the Medical Science Research Foundation of Guangdong Province of China grant no supported the fee for language editing and processingcharge for accessAvailability of data and materialsAll the original data were provided in the main document as well as thetables and figures They can also be obtained from the Internet databasesEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors have stated explicitly that there are no conflicts of interest inconnection with this Received May Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin “httpsdoi103322caac21492Koyama H Ohno Y Seki S Nishio M Yoshikawa T Matsumoto S Maniwa YItoh T Nishimura Y Sugimura K Value of diffusionweighted MR imagingusing various parameters for assessment and characterization of solitarypulmonary nodules Eur J Radiol “ httpsdoi101016jejrad201411024Le Bihan D Turner R The capillary network a link between IVIM andclassical perfusion Magn Reson Med “ httpsdoi101002mrm1910270116Le Bihan D Breton E Lallemand D Grenier P Cabanis E LavalJeantet MMR imaging of intravoxel incoherent motions application to diffusion andperfusion in neurologic disorders Radiology “ httpsdoi101148radiology16123763909Liang J Ma R Chen H Zhang D Ye W Shi C Luo L Detection ofHyperacute reactions of Desacetylvinblastine Monohydrazide in a Xenograftmodel using Intravoxel incoherent motion DWI and R2 mapping AJR Am JRoentgenol “ httpsdoi102214AJR1820517Liang J Cheng Q Huang J Ma M Zhang D Lei X Xiao Z Zhang D Shi CLuo L Monitoring tumour microenvironment changes during antiangiogenesis therapy using functional MRI Angiogenesis “httpsdoi101007s10456019096704Deng Y Li X Lei Y Liang C L

Thyroid_Cancer

" The adopted strategy was the same as that used in prior years [] and is based on four exclusive queries that return four disjoint citation subsets The first query QPub_plain is based on a plaintext search in PubMed titles and s using keywords The second query QPub_indexed relies on the PubMed indexing scheme using MeSH terms and results are made exclusive of the previous set The third one QWoS_restricted is based on a plaintext search in WoS restricted to the two research areas œMedical Informatics and œHealth Care Sciences Services The fourth query QWoS_filtered is based on the same plaintext search used in WoS but filtered by nonrelevant research areas eg Archeology Dance Zoology etc and the two research areas of the previous query It is of note that the two WoS queries select only nonPubMedindexed papers that are supposed to be caught by the two PubMed queriesA first review of the four subsets of retrieved citations was performed by the two section editors to select candidate best papers Following the IMIA Yearbook protocol these candidate best papers were then individually reviewed and rated by both section editors the chief editor of the Decision Support section and external reviewers from the international Medical Informatics community Based on the reviewers™ ratings and comments the Yearbook editorial committee then selected the best papers of the year in the decision support domainIMIA Yearbook of Medical Informatics IMIA and Ge Thieme Verlag KG 0cReview Results The literature search has been performed on January A total of unique references were obtained distributed as follows for QPub_plain for QPub_indexed for QWoS_restricted and for QWoS_filtered yielding subtotals of references from PubMed and from WoS Compared to the previous year the global query retrieved more papers After a first individual screening independently performed by both section editors based on the title and of papers not rejected by both section editors were discussed by the two editors to achieve a final selection of candidate best papers After the external review of these s the editorial committee finally selected three of them as best papers for [“] Table They are discussed in the next section and summaries of their contents are available in the AppendixDiscussion and OutlookIn the first paper Hendriks [] propose an approach to the modeling of clinical practice guidelines which certainly builds on already existing approaches but which is systematically conducted in order to be scalable and used to represent complex guidelines They promote the formalism of clinical decision trees CDTs as they are both clinically interpretable by healthcare professionals and computerinterpretable thus suitable for implementation in datadriven CDSSs The disambiguation of textual guidelines is supported first by the formal unequivocal specification of data items used as decision criteria using international coding systems to enforce interoperability and second by the representation of guideline knowledge as CDTs The method is applied to the Dutch breast cancer guidelines Sixty CDTs were built involving a total of data items among which could not be linked to standard terminologies The authors report the ambiguity of certain criteria which could be subjective or had multiple definitions The resulting knowledge base was implemented in a decision support application where it can be interactively browsed or automatically executed By modeling guidelines in such a way this work is a step forward in the sharing of encoded knowledgeIn the second paper KamiÅ¡alić [] tackled the issues linked to the formalization of the medical processes used for managing chronic diseases and their execution in CDSSs They analyzed the decisionmaking dimensions of the therapeutic management of chronic diseases like those known to increase the cardiovascular risk and identified three basic levels therapy strategy dosage adaptation and intolerance management To handle these different aspects consistently they propose a formalism called extended Timed Transition Diagram eTTD With eTTDs they illustrate the multilevel and finegrained modeling required to capture the contents of arterial hypertension management guidelines This detailed demonstration on how procedural knowledge for hypertension management can be formalized to develop a CDSS could certainly be used in other medical domainsThe third paper by Khalifa [] presents a conceptual and practical framework to help assess confidence in predictive tools GRASP for Grade and Assess Predictive Tools is both a method to look for evidence from the published literature and an analysis grid It standardizes the assessment of the available literature associated to a predictive tool and the grading of its level of proof Three phases of evaluation are considered i before the implementation of the tool to assess both its internal and external validity ii during the implementation to assess its potential effect and usability and iii after the implementation to assess its effectiveness and safety In each phase the level of evidence can be assessed from the study design A qualitative summarizes the direction of evidence positive negative mixed This grid can be considered as similar to existing grids for instance the CONSORT statement for clinical trials However it gives a rigorous methodology for a critical appraisal of predictive tools and could be extended to all kind of CDSSs It might be a useful tool to extend the evidencebased culture in the field of medical informaticsBesides the three best papers selected for the Decision Support section of the edition of the IMIA Yearbook several other works retrieved from the literature review deserve to be cited Some of them deal with the personalization of decisions Laleci [] propose a scientific and technical approach to develop personalized care plans that comply with clinical practice guidelines for the management of complex polypathology situations Jafarpour [] propose a solution to dynamically manage the conflicts that can rise in this type of complex contexts Ben Souissi [] introduce the use of health information technology involving multiple criteria decision to support the choice between antibiotics alternatives Interestingly other works promote the creation and sharing of operational knowledge bases as exemplified by Hendriks [] Thus Huibers [] transform the textual STOPPSTART criteria into unambiguous definitions mapped to medical terminologies Canovas et al [] formalize EUCAST expert rules as an ontology and production rules to detect antimicrobial therapies at risk of failure M¼ller [] propose an diagnostic knowledge base that can compete with commercial ones Replacing humans is another topic of research and Spnig [] work on two aspects to virtualize a doctor the automatic acquisition of data through sensors and speech recognition and the automation of diagnostic reasoning Rozenblum et al[] propose a machine learning method to generate clinically valid alerts to detect errors in prescriptions Acceptability of CDSS is another key point Kannan [] propose a method for a CDSS design to best meet a precisely specified and assessable user purpose Design alerts may also avoid rejection of CDSSs by caregivers Fernandes [] created algorithms able to aggregate filter and reduce the notifications delivered to healthcare professionals Amrose et al [] tried to understand in real life the impact of alerts on users and to find the actions they triggered Finally it is always interesting to obtain varied evaluation results of controversial CDSSs In this respect Kim [] evaluated Watson for Oncology in thyroid carcinoma and reported a concordance rate with local practices considered as too low to adopt the tool As evidenced by the number and the variety of works around decision support research in the field is very active This year™s selection highlighted pragmatic works that promote the transparency and sharing of the IMIA Yearbook of Medical Informatics 2020Duclos 0cTable Best paper selection of s for the IMIA Yearbook of Medical Informatics in the section 'Decision Support' The s are listed in alphabetical order of the first author™s surname Section Decision Support\uf0a7 Hendriks MP Verbeek XAAM van Vegchel T van der Sangen MJC Strobbe LJA Merkus JWS Zonderland HM Smorenburg CH Jager A Siesling S Transformation of the National Breast Cancer Guideline Into DataDriven Clinical Decision Trees JCO Clin Cancer Inform \uf0a7\t KamiÅ¡alić\tA\tRia±o\tD\tKert\tS\tWelzer\tT\tNemec\tZlatolas\tL\tMultilevel\tmedical\tknowledge\tformalization\tto\tsupport\tmedical\tpractice for chronic diseases Data Knowledge Engineering “\uf0a7 Khalifa M Magrabi F Gallego B Developing a framework for evidencebased grading and assessment of predictive tools for clinical decision support BMC Med Inform Decis Mak knowledge bases used by decision support tools as well as the grading of their utility The ultimate goal is that users could trust such tools to then use themAcknowledgementWe would like to thank all the present and past editorial boards of the IMIA Yearbook especially Martina Hutter and Adrien Ugon for their support as well as the reviewers for their participation to the selection of the best papers for the Decision Support section We cannot end this synopsis without a meaningful thought for our colleague and friend Vassilis Koutkias who started this year again to tackle the tasks of a Decision Support section coeditor but passed away in last December and unfortunately could not finishReferences Jankovic I Chen JH Clinical Decision Support and Implications for the Clinician Burnout Crisis Yearb Med Inform Koutkias V Bouaud J Contributions on Clinical Decision Support from the Literature Yearb Med Inform Aug2811357 Hendriks MP Verbeek XAAM van Vegchel T van der Sangen MJC Strobbe LJA Merkus JWS Transformation of the National Breast Cancer Guideline Into DataDriven Clinical Decision Trees JCO Clin Cancer Inform KamiÅ¡alić A Ria±o D Kert S Welzer T Nemec Zlatolas L Multilevel medical knowledge formalization to support medical practice for chronic diseases Data Knowledge Engineering “ Khalifa M Magrabi F Gallego B Developing a framework for evidencebased grading and assessment of predictive tools for clinical decision support BMC Med Inform Decis Mak Laleci GB Yuksel M Sarigul B Arvanitis TN Lindman P Chen R A Collaborative Platform for Management of Chronic Diseases via GuidelineDriven Individualized Care Plans Comput Struct Biotechnol J “ Jafarpour B Raza Abidi S Van Woensel W Raza Abidi SS Executiontime integration of clinical practice guidelines to provide decision support for comorbid conditions Artif Intell Med Ben Souissi S Abed M El Hiki L Fortemps P Pirlot M PARS a system combining semantic technologies with multiple criteria decision aiding for supporting antibiotic prescriptions J Biomed Inform Huibers CJA Sallevelt BTGM de Groot DA Boer MJ van Campen JPCM Davids CJ Conversion of STOPPSTART version into coded algorithms for software implementation A multidisciplinary consensus procedure Int J Med Inform C¡novasSegura B Morales A Juarez JM Campos M Palacios F Impact of expert knowledge on the detection of patients at risk of antimicrobial therapy failure by clinical decision support systems J Biomed Inform M¼ller L Gangadharaiah R Klein SC Perry J Bernstein G Nurkse D An access medical knowledge base for community driven diagnostic decision support system development BMC Med Inform Decis Mak Spnig S EmbergerKlein A Sowa JP Canbay A Menrad K Heider D The virtual doctor An interactive clinicaldecisionsupport system based on deep learning for noninvasive prediction of diabetes Artif Intell Med Rozenblum R RodriguezMonguio R Volk LA Forsythe KJ Myers S McGurrin M Using a Machine Learning System to Identify and Prevent Medication Prescribing Errors A Clinical and Cost Analysis Evaluation Jt Comm J Qual Patient Saf Kannan V Basit MA Bajaj P Carrington AR Donahue IB Flahaven EL User stories as lightweight requirements for agile clinical decision support development J Am Med Inform Assoc Fernandes CO Miles S Lucena CJP Cowan D Artificial Intelligence Technologies for Coping with Alarm Fatigue in Hospital Environments Because of Sensory Overload Algorithm Development and Validation J Med Internet Res 20192111e15406 Amroze A Field TS Fouayzi H Sundaresan D Burns L Garber L et al Use of Electronic Health Record Access and Audit Logs to Identify Physician Actions Following Noninterruptive Alert ing Descriptive Study JMIR Med Inform 201971e12650 Kim M Kim BH Kim JM Kim EH Kim K Pak K Concordance in postsurgical radioactive iodine therapy recommendations between Watson for Oncology and clinical practice in patients with differentiated thyroid carcinoma Cancer Correspondence toPr Catherine DuclosLIMICS INSERM Facult© L©onard de Vinci rue Marcel Cachin Bobigny FranceEmail catherineduclosaphpfr IMIA Yearbook of Medical Informatics 2020Pragmatic Considerations on Clinical Decision Support from the Literature 0cAppendix Content Summaries of Best Papers for the Decision Support Section of the IMIA YearbookHendriks MP Verbeek XAAM van Vegchel T van der Sangen MJC Strobbe LJA Merkus JWS Zonderland HM Smorenburg CH Jager A Siesling STransformation of the National Breast Cancer Guideline into datadriven clinical decision treesJCO Clin Cancer Inform May3114Since clinical practice guidelines are still narrative and described in large textual documents the aim of this work was to model complex guidelines as datadriven clinical decision trees CDTs that could be still humaninterpretable while computerinterpretable for implementation in decision support systems The Dutch national breast cancer guidelines were translated into CDTs Data items which characterize the patient and the tumor and represent decisional criteria were encoded unambiguously using existing classifications and coding systems related to breast cancer when feasible In total CDTs were necessary to cover the whole guidelines driven by data items Of all data items could be coded using existing classification and coding systems All CDTs represented unique patient subpopulations Complex guidelines could be transformed as systematically constructed modular datadriven CDTs that are clinically interpretable and executable in a decision support applicationKamiÅ¡alić A Ria±o D Kert S Welzer T Nemec Zlatolas LMultilevel medical knowledge formalization to support medical practice for chronic diseasesData Knowledge Engineering “This research is focused on knowledge representation to support the medical processes involved in chronic diseases management which can be viewed as a procedural and sequential application of knowledge An intuitive easy and effective mechanism for medical knowledge formalization is proposed through a formalism called extended Timed Transition Diagram eTTD This formalism allows for the consistent representation of three basic levels of decision making that should be taken into account in the prescription and adaptation of longterm treatment therapy strategy dosage and intolerances The methodology can be manually applied to build eTTDs from clinical practice guidelines eTTDs implementation is demonstrated by modeling clinical practice guidelines for the therapeutic management of arterial hypertension The obtained models can be used as a baseline framework for the development of decision support systems involving medical proceduresKhalifa M Magrabi F Gallego BDeveloping a framework for evidencebased grading and assessment of predictive tools for clinical decision supportBMC Med Inform Decis Mak Oct Deciding to choose a clinical predictive tool in clinical practice should be guided by its correctly assessed effectiveness The objective of this work is to developp a conceptual and practical framework to Grade and Assess Predictive tools GRASP and provide clinicians with a standardised evidencebased system to support their search for and selection of efficient predictive tools The GRASP framework grades predictive tools based on published evidence across three dimensions phase of evaluation level of evidence and direction of evidence The final grade of the tool is based on the phase of evaluation that gets the hightest grade supported by the highest level of positive or mixed evidence that supports a positive This framework was successfully applied to five predictive tools GRASP report updates could be a way to maintain a data base that documents the evidence of predictive tools IMIA Yearbook of Medical Informatics 2020Duclos 0c"

Thyroid_Cancer

"Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedAURKA a cell cycleregulated kinase is associated with malignant transformation and progression in many cancer types Weanalyzed the expression change of AURKA in pancancer and its eï¬ect on the prognosis of cancer patients using the TCGAdataset We revealed that AURKA was extensively elevated and predicted a poor prognosis in most of the detected cancer typeswith an exception in colon cancer AURKA was elevated in colon cancer but the upregulation of AURKA indicated betteroutcomes of colon cancer patients Then we revealed that undermethylation of the AURKA gene and several transcriptionfactors contributed to the upregulation of AURKA in colon cancer Moreover we demonstrated that AURKA overexpressionpromoted the death of colon cancer cells induced by Oxaliplatin whereas knockdown of AURKA significantly weakened thechemosensitivity of colon cancer cells to Oxaliplatin Mechanistically AURKA inhibited DNA damage response by suppressingthe expression of various DNA damage repair genes in a TP53dependent manner which can partly explain that ARUKA isassociated with a beneficial outcome of colon cancer This study provided a possibility to use AURKA as a biomarker to predictthe chemosensitivity of colon cancer to platinum in the clinic IntroductionAurora Kinase A AURKA is a cell cycleregulated kinaseinvolved in centrosome maturation mitotic entry bipolarspindle assembly and chromosome separation [] The elevated expression of AURKA is frequently reported in manycancer types [] AURKA alone or combined with otherfactors can trigger cell malignant transformation [ ] andpromote the malignant phenotype of cancer cells [ ]AURKA shows oncogenic activity by regulating multipleoncogenic and tumorsuppressive proteins [] Of theseproteinstumor suppressor TP53 has been intensivelystudied Phosphorylation of TP53 at Ser215 and Ser315 byAURKA results in TP53 degradation by MDM2mediatedubiquitination and abrogation of TP53 DNA bindingtransactivation activity respectively [ ] In turn TP53downregulation increases the expression of ARUKA atboth transcriptional and posttranslationallevels [ ]Negative feedback regulation between AURKA and TP53greatly promotes carcinogenesis and progressionMaintaining genome stability by transactivating theDNA damage response DDR genes is the critical mediatorof TP53dependent tumor suppression [ ] thus TP53deficiency causes the loss of various DDR mechanisms and 0cBioMed Research Internationalthereby facilitates genome instability and cancer development [] Meanwhile platinuminduced DNA damage cantrigger the DDR which is a major contributor to chemoresistance to platinum [] In view of the association betweenAURKA TP53 and DDR the upregulated AURKA in cancer might promote the cancer progression but meanwhileenhance the chemosensitivity of DNA damageinducingdrugs in the clinicIn this study we analyzed the expression state and regulation mechanism of AURKA in colon cancer We also testedthe eï¬ect of dysregulated AURKA on chemosensitivity to theplatinum drug and explored the underlying molecular mechanism in colon cancer These results provided a novel insightinto the function of AURKA in cancer Material and Methods Dataset and Processing The data of AURKA mRNAexpression in types of cancers and matched normal tissueswere downloaded from The Cancer Genome Atlas TCGAdatabase including Bladder Urothelial Carcinoma BLCABreast Invasive Carcinoma BRCA Cervical Squamous CellCarcinoma and Endocervical Adenocarcinoma CESCColon Adenocarcinoma COAD Head and Neck SquamousCell Carcinoma HNSC Kidney Renal Clear Cell Carcinoma KIRC Kidney Renal Papillary Cell CarcinomaKIRP Liver Hepatocellular Carcinoma LIHC Lung Adenocarcinoma LUAD Lung Squamous Cell CarcinomaLUSC Pancreatic Adenocarcinoma PAAD Prostate Adenocarcinoma PRAD Rectum Adenocarcinoma READSarcoma SARC Skin Cutaneous Melanoma SKCM Stomach Adenocarcinoma STAD Thyroid Carcinoma THCAand Uterine Corpus Endometrial Carcinoma UCEC Thecorrelation between the AURKA level and the overall survivalOS of cancer patients was also analyzed through the GEPIAhttpgepiacancerpkucnindexhtml The mRNA expression data are shown in Supplementary We compared theexpression level of AURKA mRNA by calculating the meanvalue and standard deviation The eï¬ect of AURKA copynumber variant CNV on AURKA expression level was alsoanalyzed based on the Colon Adenocarcinoma COAD datafrom the TCGA database The eï¬ect of methylation on theexpression of AURKA was assessed using the MEXPRESS datamexpressbe The transcription factor TF targeting ARUKA was screened based on the ChipSeq data in theUCSC databank httpgenomeucsceduENCODE Thetargeted regulatory capacity of TP53 on DDR genes wasassessed using the Cistrome Data Browser httpcistromedb Meanwhile the correlation between AURKAand TF genes was analyzed in colon cancer through theGEPIA httpgepiacancerpkucnindexhtml Cell Culture Two colon cancer cell lines SW1116 andHCT116 and 293TN cell line were used in this study Missense mutation presents in TP53 in SW1116 whereasHCT116 has a wildtype TP53 according to the Cancer CellLine Encyclopedia CCLEportalsbroadinstituteccleabout Additionally all the DDR genes involvedin our study are the wildtype but for BRCA2 which has aframeshift in HCT116 They were cultured using Dulbecco™sModified Eagle Medium DMEM HyClone Logan UTUSA with fetal bovine serum FBSInvitrogenCarlsbad CA USA μgml streptomycin and IUml°penicillin at C in a humidified atmosphere containing CO2 Cells used to detect phosphorylated TP53 were treatedwith a specific proteasome inhibitor MG132 Sigma StLouis MO USA μM for six hours The Construction of Stable Cell Lines Overexpression orknockdown of AURKA was achieved by using lentivirus ps to infect colon cancer cells described as before [] Inbrief the ORFs of AURKA cloned by PCR and synthesizedshRNA against AURKA were inserted into PlvxPuro andSHC201 vectors respectively The scramble sequences wereinserted into these vectors to be used as control Thesevectors were transfected into 293TN cells with the packingvectors System Bioscience Mountain View CA USA toget pseudo lentiviral ps After being filtered andconcentrated by PEG precipitation System Biosciencelentiviral ps were added to the culture medium toinfect colon cancer cells for h After routine culture for h the stable cells were selected and purified by puromycin μgml MTT Assay Colon cancer cells were seeded in 96wellplates at a density of cells per well and incubated overnight The culture medium was replaced with fresh culturemedium containing a diï¬erent concentration of Oxaliplatin and μgml with replicates each After h of incubation μl MTT gl was added to each wellfor h in the incubator The supernatant was removed and μl DMSO was added to each well After being vibratedfor min the plate was read on a microplate reader at nm to calculate the cell viability rate All assays werereplicated three times The result was analyzed using the cellviability percentage the total number of viable cells at eachdrug concentration relative to the number of viable cellstreated with the solvent control Western Blot Total proteins were extracted from coloncancer cells using the RIPA buï¬er Beyotime Institute ofBiotechnology Shanghai China μg protein was separated in SDSPAGE gel by electrophoresis and transferredonto PVDF membrane The blots were blocked by BSA°at C overnight The membrane was incubated with primaryantibodies AURKA rabbit polyclonal antibody ProteinTech Wuhan China No 102971AP diluted at TP53 rabbit polyclonal antibody Proteintech WuhanChina No104421AP diluted at phosphoTP53Ser315 mouse monoclonal antibody Santa Cruz Biotechnology Nosc135772 MDM2 rabbit polyclonal antibodyProteintech Wuhan China No 190581AP and GAPDHmouse monoclonal antibody ProteinTech Wuhan ChinaNo 600041Ig diluted at After washingthemembranes were incubated with peroxidaseconjugatedsecondary antibody Santa Cruz Biotechnology for h at°C The ECL system Thermo Scientific Rockford IL 0cBioMed Research InternationalUSA was used to visualize the blots All assays were replicated three times RealTime PCR Total RNA was extracted from coloncancer cells using TRIzol Invitrogen Carlsbad CA USAEasyScript® Reverse Transcriptase TransGen Biotech CoBeijing China was used to reverse RNA into cDNA Thelevel of DDR gene ATR XLF XRCC1 RPA1 BRCA2 andRAD51 was quantified using the SYBR Green PCR mixBioresearcher Beijing China through CFX96TM RealTime System BioRad The reaction mixture underwent° cycles consisting of denaturation for s at C and°annealing and prolongation for s each at C GAPDHwas used as the endogenous controls All assays were replicated three times The primers used for PCR are shown inSupplementary Statistics Analysis The expression of AURKA in a diï¬erent type of tumors and the diï¬erential expression of genesbetween two groups were analyzed by a twosided Student™sttest Survival analyses were conducted with the KaplanMeier method using the logrank test and the median valueseparation model based on the AURKA expression is presented The hazard ratio was calculated based on the CoxPH model The correlation between methylation statusand AURKA expression was analyzed using the Pearsoncorrelation and Wilcoxon ranksum test Pearson™s correlation and Z test were used to analyze the correlationbetween AURKA and TFs The eï¬ect of CNV on AURKAexpression was assessed by the KruskalWallis test MTTresults were analyzed using variance analysis ˆ—p ˆ—ˆ—p ˆ—ˆ—ˆ— p Results AURKA Was Upregulated and Predicted a BeneficialOutcome in Colon Cancer To explore the eï¬ect of AURKAon cancer progression and prognosis we firstly employedthe TCGA dataset to analyze the mRNA expression ofAURKA in types of tumors Compared with the matchednormal tissues AURKA was significantly upregulated incancer tissues in out of cancer types Figure 1a Nextwe assessed the correlation between the AURKA level andoverall survival OS in cancer types using the GEPIAWe showed that the AURKA level was adversely correlatedwith OS in of cancers including LUAD KIRP PAADSKCM and LIHC However a high level of AURKA wasassociated with a longer OS in COAD Figure 1b Theseresults suggested that AURKA overexpression might playan important role during the carcinogenesis and progressionof cancer however the elevated expression of AURKA predicted a beneficial outcome only in colorectal cancer DNA Undermethylation and Several TranscriptionFactors Might Contribute to the Elevated Expression ofAURKA in Colon Cancer To explore the mechanism bywhich AURKA was upregulated in colon cancer we firstlyanalyzed the eï¬ect of methylation status on AURKA expression By using the MEXPRESS there were methylationsites in the AURKA gene identified Of them methylationsites were significantly adverse correlated with the level ofAURKA Figure 2a Meanwhile we screened the potentialTFs activating AURKA expression based on the ChipSeqdata using the UCSC database and found that a total of TFs potentially regulate AURKA transcription Of themthe expression of TFs was positively correlated with thelevel of AURKA in colon cancer tissues according to theGEPIA correlation analysis Moreover of them have beenidentified to be overexpressed in colon cancer tissues compared with the matched normal tissues through the GEPIAexpression analysis Figure 2b The top four TFs highlycorrelated with AURKA r p were E2F1MYBL2 MYC and BRCA1 The expression and correlationwith AURKA of these four TFs are shown in Figures 2cand 2d We also analyzed the eï¬ect of AURKA CNV onthe expression level of AURKA The result indicated thatthe expression level of AURKA in the AURKA CNV gaingroup was much higher than that in the AURKA CNV neutral group in COAD whereas there was no diï¬erencebetween the AURKA CNV loss and CNV neutral groupFigure 2e But the incidence of CNV gain was lower incolon cancer patients These results indicated that undermethylation the elevated TFs and gene amplification mightcontribute to the elevated expression of AURKA in coloncancer AURKA Increased the Chemosensitivity of Colon CancerCells to Oxaliplatin We found that upregulated AURKAwas associated with the improved prognosis of colon cancerpatients thus we speculated that if AURKA increases chemosensitivity of platinum by increasing the genomic instability in colon cancer We firstly constructed the stable cell lineswith AURKA overexpression or knockdown Figure 3aand then assessed the eï¬ect of AURKA on the chemosensitivity of colon cancer cells The resultindicated thatAURKA overexpression promoted the death of HCT116and SW1116 colon cancer cells induced by Oxaliplatinwhereas knockdown of AURKA significantly weakened theresponse of colon cancer cells to Oxaliplatin Figures 3band 3c These results showed that AURKA may improvethe prognosis of colon cancer patients by increasing the chemosensitivity of colon cancer cells to the DNAdamagingdrug AURKA Downregulated the Expression of DDR Genes byInhibiting TP53 Previous research showed that AURKAinhibits the expression of TP53 which mediates the expression of DDR genes at the transcriptional level We detectedthe eï¬ect of AURKA on TP53 expression by immunoblotin colon cancer cells The result indicated that TP53 wasdownregulated when AURKA was overexpressed whereasupregulated when AURKA was knocked down in coloncancer cells Figures 4a and 4b Next we screened a setof DDR genes that play an important role in DNA damageinduced by chemotherapeutics Meanwhile most of themfunction after the activation of TP53 [] Using theCistrome Data Browser we assessed the transcriptionalregulatory potential of TP53 on these genes and found someof them had higher scores in two sets of data with high 0c AKRUA fo level evitaler ecid2ŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽŽBioMed Research InternationalŽŽŽŽŽŽŽŽŽCESCCOADHNSCKIRCKIRPLIHCLUADLUSCPAADPRADREADSARCSKCMSTADTHCAUCECTNBLCABRCATumorNormalCOAD Overall survivalLogrank p0034HRhigh06pHR0036nhigh135nlow135lavivrus tnecrePaLUAD Overall survivalLogrank p0047HRhigh13pHR0049nhigh238nlow239lavivrus tnecrePKIRP Overall survivalLogrank p0007HRhigh23pHR00088nhigh141nlow141MonthsMonthsMonthsLow AURKA TPMHigh AURKA TPMPAAD Overall survivalLogrank p00059HRhigh18pHR00068nhigh89nlow89Low AURKA TPMHigh AURKA TPMSKCM Overall survivalLogrank p0014HRhigh14pHR0014nhigh229nlow229lavivrus tnecrePlavivrus tnecrePLow AURKA TPMHigh AURKA TPMLIHC Overall survivalLogrank p000022HRhigh19pHR000028nhigh181nlow181lavivrus tnecrePlavivrus tnecrePMonthsLow AURKA TPMHigh AURKA TPMMonthsMonths Low AURKA TPMHigh AURKA TPMbLow AURKA TPMHigh AURKA TPMFigure AURKA was upregulated in colon cancer and predicted a benefit outcome a Compared with the matched normal tissuesAURKA was significantly upregulated in cancer tissues in out of cancer types b AURKA expression level was adversely correlatedwith OS in of cancers but positively correlated with OS in COADquality control Supplementary We then applied realtime PCR to verify the expression of six representative genesATR XLF XRCC1 RPA1 BRCA2 and RAD51 The resultsindicated that the six DDR genes were downregulated incolon cancer cells with AURKA overexpression but upregulated when knocking down AURKA in colon cancer cellsFigures 4c and 4d which implied that AURKAincreased the chemosensitivity of colon cancer cells to DNAdamageinducing drugs by inducing the degradation ofTP53 and then decreasing the expression of DDR genes 0cBioMed Research InternationalGpG islandAURKA chr2054967393chr2054944445 bpchr2054966401 chr2054967165 chr2054967495 chr2054967671 chr2054967718 r r r r r p p p p p TFE2F1MYBL2MYCBRCA1CBX3rapTFZNF217ELK1EZH2ZBTB33SMARCC1MYBLŽbrMYCŽpTFARID3ATCF3YY1FOXM1TEAD4rpBRCA1ŽE2F1ŽCOADnumT275 numN349COADnumT275 numN349COADnumT275 numN349COADnumT275 numN349cFigure Continued 0cBioMed Research International MPTFEgol MPTCYMgolp value r p value r MPTLBYMgollog2AURKA TPMlog2AURKA TPMp value r log2AURKA TPMŽŽp value r MPTACRBgollog2AURKA TPMdnoisserpxe AKRUACNV loss CNV neutral CNV gainCNV_TypeCNV_TypeCNV LossCNV NeutralCNV gaineFigure Undermethylation upregulation of TFs and gene amplification potentially contributed to the elevated expression of AURKAa Five methylation sites in AURKA DNA were significantly adversely correlated with the level of AURKA b Based on the public data analysisa total of TFs potentially regulated AURKA transcription The expression of TFs was positively correlated with the level of AURKAMoreover of them have been identified to be overexpressed in colon cancer tissues compared with the matched normal tissues c d Theexpression of the top four TFs highly correlated with AURKA was higher in colon cancer tissues compared with normal tissues e Theexpression level of AURKA in the AURKA CNV gain group was significantly higher than that in the AURKA CNV neutral group in COAD 0cBioMed Research InternationalCtrol AURKACtrolCtrol AURKACtrolshAURKAshAURKAAURKAGAPDH ytilibaiv lleC ytilibaiv lleCAURKAGAPDHSW1116aHCT116p00009Drug concentration 𝜇gmlControlAURKAIC50 𝜇gml 𝜇gml ytilibaiv lleCSW1116p00427Drug concentration 𝜇gmlControlAURKAIC50 𝜇gml 𝜇gmlb ytilibaiv lleCcHCT116HCT116p00029Drug concentration 𝜇gmlControlshAURKA1037shAURKA1184IC50 𝜇gml 𝜇gml 𝜇gmlSW1116p00043Drug concentration 𝜇gmlControlshAURKA1037shAURKA1124IC50 𝜇gml 𝜇gml 𝜇gmlFigure AURKA increased the chemosensitivity of colon cancer cells to Oxaliplatin a AURKA was upregulated or knocked down in twocancer cell lines b c AURKA overexpression promoted the death of HCT116 and SW1116 colon cancer cells induced by Oxaliplatinwhereas knockdown of AURKA significantly weakened the response of colon cancer cells to Oxaliplatin DiscussionIn this study we evaluated the expression of AURKA in types of tumor tissues and matched normal tissues Theresult indicated that AURKA was upregulated in most testedcancer types compared with their normal tissues OS analysisshowed that higher AURKA was correlated with a worse outcome of most of the cancer types whereas it only indicated afavorable outcome in colon cancer The prognostic role ofAURKA has ever been assessed in colorectal cancer patientsby a research team in [] Despite the lack of statisticalsignificance they still put forward that AURKA may have a 0cBioMed Research InternationalCtrolAURKACtrolCtrolAURKA CtrolshAURKAshAURKAAURKAMDM2TP53GAPDHpTP53GAPDHAURKAMDM2TP53GAPDHpTP53GAPDHGMGMANRm fo level evitaler ecid2ANRm fo level evitaler ecid2SW1116aŽŽŽŽŽŽŽATRBRCA2 RPA1 XRCC1 RAD51 NHEJ1SW1116 CtrolSW1116 AURKAŽŽŽŽŽŽATRBRCA2 RPA1 XRCC1 RAD51 NHEJ1HCT116 CtrolHCT116 AURKAANRm fo level evitaler ecid2cANRm fo level evitaler ecid2dGMGMHCT116bŽŽŽŽŽŽŽŽŽŽŽATRRPA1BRCA2SW1116 CtrolSW1116 shAURKA1024SW1116 shAURKA1037ŽŽŽŽXRCC1RAD51NHEJ1ŽŽŽŽŽŽŽ ŽŽŽŽŽŽŽATRRPA1BRCA2HCT116 CtrolHCT116 shAURKA1024HCT116 shAURKA1037XRCC1RAD51NHEJ1Figure AURKA downregulated the expression of DDR genes by inhibiting TP53 a b Overexpression of AURKA promoted thephosphorylation of TP53 and decreased the level of total TP53 whereas knockdown of AURKA reduced the phosphorylation of TP53 andincreased the level of total TP53 in colon cancer cells by immunoblot AURKA had no eï¬ect on the expression of MDM2 c d Sixrepresentative DDR genes were downregulated in colon cancer cells with AURKA overexpression but upregulated when knocking downAURKA in colon cancer cells by realtime PCRpositive eï¬ect on survival and emphasized the necessity tostudy the eï¬ect of AURKA on response to treatment []Further study showed that undermethylation and upregulation of TFs potentially contribute to the elevated expressionof AURKA in colon cancer at least partly Some studiesindicated that gene amplification is another contributor tothe elevated AURKA [ ] We also identified that geneamplification in colon cancer patients can result in AURKA 0cBioMed Research Internationalupregulation however its incidence rate was very low incolon cancer patients Finally we demonstrated that AURKAmight improve the prognosis of colon cancer patients byincreasing the chemosensitivity of colon cancer to Oxaliplatin via inhibiting the DDR Our results uncovered thedoubleedged sword eï¬ects of AURKA by inhibiting TP53in colon cancerThe genomic instability has been recognized as a hallmark of cancer and it is associated with carcinogenesis andprogression of cancer [ ] AURKA functions as an oncogene during the development of multiple malignant tumorsby inducing centrosome amplification and genomic instability [ ] In colon cancer the overexpressed AURKA is thecontributor to chromosomal instability [ ] MoreoverAURKA has been revealed to impair the function of DNAdamage repair through inhibiting the expression of DDRgenes such as RAD51 and BRCA12 [“] In additionto the DDR genes involved in Homologous RecombinationRepair HRR TP53 showed the transcriptional regulatorypotential on Mismatch Repair MMR genes according tothe binding scores from the ChipSeq data Supplementary The inhibitory eï¬ect of AURKA on TP53 which has beendemonstrated to transcriptionally activate many DDR genesenlarges the potential of AURKA facilitating DNA damage[] Some studies also indicate that TP53 is essential for chemoresistance rendered by AURKA [ ] In order to verifythe function of TP53 during this process we respectivelyassessed the correlation between AURKA level and OS inpatients with wildtype or mutant TP53 The results indicated that the patients with the higher AURKA had a longerOS time in TP53 wildtype groups although only a marginalsignificance was achieved due to the reduced number ofsamples But no diï¬erence was found in TP53 mutant groupsSupplementary The current research supports that AURKA is involved incolon carcinogenesis through promoting genomic instabilitybut the increased AURKA provides a good chance forenhancing the sensitivity of chemotherapy based on DNAdamageinducing drugs The eï¬ect of AURKA on chemosensitivity has been studied in diï¬erent cancer types Up to nowthey all concluded that AURKA impaired the chemosensitivity which is the exact opposite of our finding Forexample it was reported that inhibiting AURKA enhancesthe chemosensitivity of cancer cells to the taxane and paclitaxel [ ] cisplatin [] doxorubicin [ ] and5fluorouracil 5Fu [] In particular platinum chemosensitivity is inhibited by AURKA in various cancers includingovarian cancer [] hepatocellularcarcinoma [] medulloblastoma [] acute myeloid leukemia []as well as headand neck cancer [] Our finding that AURKA increasedthe platinum chemosensitivity in colon cancer was diï¬erentfrom the previous studies in other cancer types which coincided with our finding that higher AURKA indicated betterprognosis only in colon cancer but not in other cancersThough cancer stem cell is a small subpopulation of cancercells AURKA silencing sensitized the response of colorectalcancer stem cell CRCSC to Oxaliplatin by upregulatingantiapoptotic factors [] which is diï¬erent from our findings in colon cancer cells The diï¬erence might be associatedwith heterogeneity induced by tumor microenvironment andgenomic instability [] AURKAmediated TP53 inhibitionmight result in diï¬erent consequence in diï¬erent geneticcontexts However this hypothesis might be determined byfurther experiments ConclusionAURKA was upregulated in various cancer types but onlypositively correlated with the prognosis of colon cancerpatients The mechanism might be that AURKA improvesthe chemosensitivity of colon cancer cells to Oxaliplatin byinhibiting the expression of TP53regulated DDR genes andthen facilitating DNA damage This study provides a possibility to use AURKA as a biomarker to predict the chemosensitivity of colon cancer to platinum in the clinicData AvailabilityAll the data used to support the findings of this study areincluded within the Conflicts of InterestThe authors declare that there is no conflict of interestregarding the publication of this paperAuthors™ ContributionsBaocong Shan Ran Zhao Jian Zhou and Minghui Zhangcontributed equally to this workAcknowledgmentsThis work was partly supported by the National NaturalScience Foundation of China to Xiaobo Li Natural Science Foundation of Heilongjiang Province H2018009to Tianzhen Wang H2018010 to Yiqi Wu the FundamentalResearch Funds for the Provincial Universities to Ran Zhao to Yuanyuan Zhu and KYYWF0289 to Weiwei Yang and Heilongjiang Postdoctoral Fund LBHZ17136 to Weiwei YangSupplementary MaterialsSupplementary Figure The correlation of AURKA leveland OS in patients with wildtype TP53 or mutant TP53 AThe patients with the higher AURKA had a longer OS timein TP53 wildtype groups although only a marginal significance was achieved due to the reduced number of samplesB No diï¬erence was found in TP53 mutant groups C Mostof the mutations in TP53 were missense variant followed bystop gained and frameshift variant Supplementary Table We compared the expression level of AURKA mRNA by calculating the mean value and standard deviation The eï¬ect ofAURKA copy number variant CNV on AURKA expressionlevel was also analyzed based on the Colon adenocarcinomaCOAD data from the TCGA database SupplementaryTable The primers for PCRSupplementary Table Theassessment of transcriptional regulatory potential of p53 on 0cBioMed Research InternationalDDR genes based on ChipSeq data from cell lines Supplementary Table The assessment of transcriptional regulatory potential of p53 on Mismatch Repair MMR genesbased on ChipSeq data from celllines SupplementarymaterialsReferences[] B Goldenson and J D Crispino œThe aurora kinases in cellcycle and leukemia Oncogene vol no pp “[] A S Nikonova I Astsaturov I G Serebriiskii R L DunbrackJr and E A Golemis œAurora A kinase AURKA in normaland pathological cell division Cellular and Molecular Life Sciences vol no pp “ [] H Zhou J Kuang L Zhong œTumour amplified kinase_STK15_ _BTAK_ induces centrosome amplification aneuploidy and transformation Nature Genetics vol no pp “ [] A H SillarsHardebol B Carvalho M Tijssen œTPX2and AURKA promote 20q amplicondriven colorectal adenoma to carcinoma progression Gut vol no pp “ [] G Vader and S M A Lens œThe Aurora kinase family in celldivision and cancer Biochimica et Biophysica Acta BBA Reviews on Cancer vol no pp “ [] M Yan C Wang B He œAuroraA Kinase a potentoncogene and target for cancer therapy Medicinal ResearchReviews vol no pp “ [] H Katayama and S Sen œAurora kinase inhibitors as anticancer molecules Biochimica et Biophysica Acta BBA GeneRegulatory Mechanisms vol no pp “[] Q Liu S Kaneko L Yang œAuroraA abrogation of p53DNA binding and transactivation activity by phosphorylationof serine  Journal of Biological Chemistry vol no pp “ [] H Katayama K Sasai H Kawai œPhosphorylation byaurora kinase A induces Mdm2mediated destabilization andinhibition of p53 Nature Genetics vol no pp “[] C C Wu T Y Yang C T R Yu œp53 negatively regulates Aurora A via both transcriptional and posttranslationalregulation Cell Cycle vol no pp “ [] S S Chen P C Chang Y W Cheng F M Tang and Y S LinœSuppression of the STK15 oncogenic activity requires atransactivationindependent p53 function The EMBO journal vol no pp “ [] A Janic L J Valente M J Wakefield œDNA repairprocesses are critical mediators of p53dependent tumor suppression Nature Medicine vol no pp “ [] A B Williams and B Schumacher œp53 in the DNAdamagerepair process Cold Spring Harbor Perspectives in Medicinevol no [] L Galluzzi L Senovilla I Vitale œMolecular mechanismsof cisplatin resistance Oncogene vol no pp “ [] X Li J Zhang L Gao œMiR181 mediates cell diï¬erentiation by interrupting the Lin28 and let7 feedback circuitCell Death Diï¬erentiation vol no pp “ [] N Hosoya and K Miyagawa œTargeting DNA damageresponse in cancer therapy Cancer science vol no pp “ [] S Goktas M Yildirim D Suren œPrognostic role ofAuroraA expression in metastatic colorectal cancer patientsJournal of BUON official journal of the Balkan Union ofOncology vol no pp “ [] Synnöve Staï¬ J Isola M Jumppanen and M TannerœAuroraA gene is frequently amplified in basallike breastcancer Oncology Reports vol no pp “ [] S Yamamoto M YamamotoIbusuki Y YamamotoS Fujiwara and H Iwase œA comprehensive analysis ofAurora A transcript levels are the most reliable in associationwith proliferation and prognosis in breast cancer BMC cancer vol no [] A Janssen and R H Medema œGenetic instability tipping thebalance Oncogene vol no pp “ [] W M Grady œGenomic instability and colon cancer CancerMetastasis Reviews vol no pp “ [] X Wang Y X Zhou W Qiao œOverexpression of aurorakinase A in mouse mammary epithelium induces geneticinstability preceding mammary tumor formation Oncogenevol no pp “ [] N Nishida T Nagasaka K Kashiwagi C R Boland andA Goel œHigh copy amplification of the AuroraA gene isassociated with chromosomal instability phenotype in humancolorectal cancers Cancer Biology Therapy vol no pp “ [] Y Baba K Nosho K Shima œAuroraa expression isindependently associated with chromosomalinstability incolorectal cancer Neoplasia vol no pp “[] T Sourisseau D Maniotis A McCarthy œAuroraAexpressing tumour cells are deficient for homologydirectedDNA double strandbreak repair and sensitive to PARP inhibition EMBO Molecular Medicine vol no pp “ [] S Sankaran D E Crone R E Palazzo and J D ParvinœAuroraA kinase regulates breast cancer associated gene inhibition of centrosomedependent microtubule nucleationCancer Research vol no pp “ [] G Yang B Chang F Yang œAurora kinase A promotesovarian tumorigenesis through dysregulation of the cell cycleand suppression of BRCA2 Clinical Cancer Researchvol no pp “ [] H Yang L He P Kruk S V Nicosia and J Q ChengœAuroraA induces cell survival and chemoresistance by activation of Akt through a p53dependent manner in ovariancancer cells International journal of cancer vol no pp “ [] P Cammareri A Scopelliti M Todaro œAuroraA isessential for the tumorigenic capacity and chemoresistance ofcolorectal cancer stem cells Cancer Research vol no pp “ [] T Hata T Furukawa M Sunamura œRNA interferencetargeting aurora kinase a suppresses tumor growth andenhances the taxane chemosensitivity in human pancreaticcancer cells Cancer Research vol no pp “[] Y Lin F M Richards B F Krippendorï¬ œPaclitaxel andCYC3 an aurora kinase A inhibitor synergise in pancreatic 0cBioMed Research Internationalcancer cells but not bone marrow precursor cells British Journal of Cancer vol no pp “ [] M A Miller C Palaniswamy D Sharma and V Y ReddyœInappropriate shock from a subcutaneousimplantablecardioverterdefibrillator due to transcutaneous electricalnerve stimulation Heart Rhythm vol no pp [] Q Zhu X Yu Z W Zhou C Zhou X W Chen and S FZhou œInhibition of Aurora A Kinase by alisertib inducesautophagy and cell cycle arrest and increases chemosensitivityin human hepatocellular carcinoma HepG2 cells CurrentCancer Drug Targets vol no pp “ [] J Wang K Nikhil K Viccaro L Chang J White andK Shah œPhosphorylationdependent regulation of ALDH1A1by Aurora kinase A insights on their synergistic relationship inpancreatic cancer BMC Biology vol no p [] Y Shionome W H Lin H Y Shiao H P Hsieh J T A Hsuand T Ouchi œA novel auroraA inhibitor BPR1K0609S1sensitizes colorectal tumor cells to 5fluorofracil 5FU treatment International Journal of Biological Sciences vol no pp “ [] M Li H Li F Liu œCharacterization of ovarian clear cellcarcinoma using target drugbased molecular biomarkersimplications for personalized cancer therapy Journal ofOvarian Research v

Thyroid_Cancer

"Gastroenterol Res Primary Localized Amyloidosis of the Intestine A Pathologist ViewpointSaeed Ali Alshehria c Mahmoud Rezk Abdelwahed HusseinbAbstractBackground Localized amyloidosis of the intestine is a rare entity which can clinically masquerade several conditions such as colitis polyps and malignant tumors This study aims to evaluate the clinicopathological features of this entityMethods To evaluate the clinicopathological features of this entity a comprehensive search of the literature to was done using the following keywords œamyloidosis and œsmall intestine or œduodenum or œileum or œjejunum or œcolon We identified studies about gastrointestinal amyloidosis Data were examined for studies about localized intestinal amyloidosis The clinicopathological features were describedResults The age at presentation ranged from to years The male to female ratio was The jejunum and sigmoid colon were the most commonly involved sites Abdominal pain and intestinal obstruction small intestine or rectal bleeding sigmoid region were the most common clinical presentations Colonoscopic findings included wall thickening mucosal ulcerations small intestine and tumorlike masses colonConclusions The clinical presentations of localized intestinal amyloidosis depend on the site of the deposition of the amyloid In most cases amyloid deposits consisted of light chain proteinKeywords Amyloidosis Amyloid Colon Small intestine Rectum Gastrointestinal tractIntroductionThe term œamyloid was initially coined to describe materials Manuscript submitted June accepted July Published online August aDepartment of Pathology Armed Forces Hospitals Southern Region King Fahd Hospital Saudi ArabiabDepartment of Pathology Faculty of Medicine Assuit University Hospitals Assuit EgyptcCorresponding Author Saeed Ali Alshehri Department of Pathology Chemistry Section Armed Forces Hospital Southern Region Khamis Mushyte Saudi Arabia Email syd46118gmailcom 1014740gr1303found in plants and has been thought to represent carbohydrate Greek œamylon starch In medical literature the term œAmyloidosis has been known for more than years It was named after the deposition of the hallmark protein called crosssheet amyloid fibrils which represents either misfolded or misassembled proteins The nomenclature of the amyloid protein uses two letters The first letter œA refers to amyloid whereas the second refers to the precursor proteins [] The amyloid proteins include several types such as immunoglobulin light chains AL in primary amyloidosis transthyretin TTR beta2microglobulin in hemodialysisassociated amyloidosis and serum amyloid A SAA protein in secondary AA amyloidosis [] There are some associations between AL amyloid deposition and several hematological conditions including myeloma lymphoplasmacytic disorders and plasma cell dyscrasia Moreover AL amyloidosis and myeloma may show similar genetic abnormalities such as 14q translocations and 13q deletion []Amyloidoses are degenerative conditions associated with significant suffering not only for patients but also for their families as well [] They are characterized by the deposition of extracellular protein fibrils in the ans such as the kidneys nerves gastrointestinal tract heart and skin Amyloidoses are associated with significant morbidities such as malabsorption renal insufficiency and hematological disorders or even lifethreatening events such as intestinal obstruction renal failure and fatal arrhythmia The clinicopathological features of amyloidosis are nonspecific They depend on the type of the precursor protein site and severity of the amyloid deposition The diagnosis of amyloidosis depends on the examination of the tissue biopsies from the lesions It is established by the detection of amyloid deposits with its characteristic birefringence under polarized light following staining with Congo red or thioflavinT or immunohistochemical stains Management of amyloidosis includes reduction of the supply of amyloid fibril precursor proteins stabilization of the precursor protein formation administration of antiplasma therapies general care systemic amyloidosis or surgical resection in localized amyloidosis [ ]Amyloidosis includes both primary and secondary types In the former also known as AL for amyloid light chain there is no evidence of coexisting disease and it has an immunoglobulin light chain as a major protein component In secondary amyloidosis also known as AA for amyloidassociated there is evidence of coexisting disease resulting in the production of inherently amyloidogenic proteins or overproduction of potentially amyloidogenic normal proteins and it has serum amyloidassociated protein SAA The deposition of amys The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastroresThis is distributed under the terms of the Creative Commons Attribution NonCommercial International License which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited 0cLocalized Amyloidosis of the Intestine Gastroenterol Res loid may be either widespread systemic amyloidosis [ ] or restricted to a specific an localized amyloidosis In systemic amyloidosis the amyloidogenic proteins are synthesized at sites far away from the sites of amyloid deposition The amyloid protein is deposited at several sites and therefore the patients have disparate clinical manifestations such as arrhythmia neuropathies malabsorption and proteinuria due to cardiac neural intestinal and renal involvement respectively [] Currently the classification of amyloidosis is based on the type of precursor proteins []Localized amyloidosis refers to the infiltration of the tissues by amyloid where the precursor proteins are synthesized at or close to the site of deposition It includes both anspecific amyloidoses where the amyloid is produced by the an itself and nonan specific amyloidosis where the fibrillar protein is not specific to the affected an [] Localized amyloidosis of the intestine is a rare condition that involves the duodenum jejunum and ileum appendix or colorectum The clinicopathological features are nonspecific and therefore they can masquerade several conditions such as collagenous colitis ischemic colitis bowel infarction perforation polyps and malignant tumors [] The colonoscopic features include mucosal granularity friability erosions ulcerations petechial hemorrhage and polypoid lesions [] The radiological findings include ulcerations thickening of the mucosal folds narrowing of the colonic lumen effacement of haustrations and nonspecific mucosal nodularity []To date few cases of localized intestinal amyloidosis have been reported [ ] In Tada examined patients with small intestinal amyloidosis Clinical findings included malabsorption mass lesions diarrhea and hematochezia Endoscopy of the jejunum revealed polypoid lesions wall thickening erosions ulcerations and fine granular appearance The amyloid proteins included amyloid A protein AA light chain protein AL beta 2microglobulin and prealbumin Histologically amyloid deposits were seen in the submucosa and muscle layer [] Chen reported a case of localized amyloidosis of the transverse colon in a 52yearold male patient The clinical presentations included abdominal pain bloody stool and weight loss Colonoscopy revealed a marked narrowing of the transverse colon associated with the presence of multiple polypoid growths The clinical and radiological findings supported the initial impression of carcinoma and therefore the patient underwent right hemicolectomy with lymph node dissection [] Grossly there was an ulcerative mass with marked narrowing of the lumen Histologically amyloid deposits were seen in the lamina propria []To date our knowledge about the clinicopathological features of the localized intestinal amyloidosis is limited To gain insights into these issues we conducted a comprehensive literature search and metaanalysis of the relevant published literature Also we presented a rare case of primary isolated intestinal amyloidosisMaterials and MethodsThis is a viewpoints study not an original research investigation that did not include any interaction or intervention with human subjects or any access to any identifiable private information Therefore the study did not require institutional review board review [] This study was performed in accordance with the principles of the Declaration of Helsinki []Search strategy and selection criteriaThe authors adhered to Preferred Reporting Items for Systematic Reviews and MetaAnalyses PRISMA guidelines [] The literature was reviewed by searching the PubMed electronic database using the key term œamyloidosis and the following words œintestine or œsmall intestine or œduodenum or œileum or œjejunum or œappendix or œcolon or œsigmoid or œrectum andor œcecum to identify the eligible studies The search results were considered based on their titles abstracts and publication date Their full texts were examined to confirm their eligibility and then were included in the study A summary of the search strategy and selection criteria is shown in Figure [ ]Data extraction a methodological assessment and statistical analysisThe clinicopathological findings were extracted including the family name of the first author year of publication patient age gender symptoms endoscopic features histology special stains and presence or absence of systemic involvement The authors independently read and analyzed each study They defined the final diagnosis as œthe histopathological diagnosis to ensure homogeneous evaluation of the findings Statistical computations were performed using IBMSPSS IBMSPSS Inc Chicago IL USA Descriptive statistics mean standard error of mean and ranges were calculatedResultsFlow trialOur search yielded previous studies about gastrointestinal amyloidosis of which were excluded The remaining s have undergone œabstract review These studies covered a period of years to This relatively long period allowed proper evaluation of a good number of cases in the literature Twentyseven studies with the final diagnosis of localized intestinal amyloidosis were included in the present study In all cases the workup for monoclonal gammopathy and systemic amyloidosis serum protein electrophoresis and pathological examination of the tissue specimens was performed The diagnosis of localized amyloidosis was substantiated by the use of ancillary studies including radiological examination such as abdominal ultrasound barium enema []wholebody computed tomography [] magnetic resonance imaging positron emission tomography [] skeletal surveys electrocardiography and echocardiography [ ] s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cAlshehri Gastroenterol Res Figure Flow chart of literature search and study selection for cases of localized intestinal amyloidosis The inclusion criteria included patients who had amyloid deposition in the intestine without evidence of systemic involvement or an associated condition leading to secondary amyloidosis [] The exclusion criteria included patients with one or more of the followings Evidence of chronic diseases such as collagen vascular disease that may have led to secondary amyloidosis [] Additional an involvement by amyloidosis Intestinal symptoms suggestive of amyloidosis but without confirmatory diagnostic tissue biopsy Plasma cell dyscrasia myeloma lymphoplasmacytic disorders or other Bcell malignancies such as Waldenstrom™s macroglobulinemia [] Systemic AL type [] Laboratory findings revealing positive detection of monoclonal light chains in serum andor urine []The reported cases did not have any evidence of generalized metabolic complications or extraintestinal complications of accumulated amyloidosis [ ] A summary of the flow chart of the literature search and study selection criteria is shown in Figure [ ]Clinical presentations and treatment modalities in localized intestinal amyloidosisThe colorectum jejunum transverse colon ileum and duodenum were the most commonly affected sites The lesions were more common in males than in females The mean age at presentation was ± years small intestine and ± years colorectum [ ] In the small intestine the main symptoms were abdominal pain [ ] and intestinal obstruction [ ] followed by diarrhea and vomiting [ ] bleeding [ ] malabsorption [] and constipation [] In the colorectum the main symptoms were rectal bleeding [ ] followed by abdominal pain [ ] anemia [] intestinal obstruction [] and weight loss [] Chen described a case of localized amyloidosis of the transverse colon in a 52yearold male patient who presented with a periumbilical pain rectal bleeding and weight loss Colonoscopic examination revealed mucosal ulcerations and luminal narrowing by multiple polypoid lesions [] Treatment modalities included in these cases included drug therapy prednisolone azathioprine melphalan and colchicines [ ] intestinal resection partial jejunectomy endoscopic submucosal resection [ ] and colonic resection [ ] with close followup of the patients [ ] A summary of these results is presented in Tables [ ]s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cLocalized Amyloidosis of the Intestine Gastroenterol Res Table Data From Individual Studies of Primary Localized Amyloidosis of the Small IntestineCase Sex Age years SitesIleumClinical manifestationsColonoscopic findingsStudies[][][][][][][][][][][][][][]FMFMFMMMMMMFMiddle ageAgedAcute abdominal pain diarrhea and vomitingPseuntestinal obstructionMelena and anemiaJejunumJejunumSmall intestineIntestinal obstructionJejunum Chronic diarrhea and and ileummalabsorptionSmall intestine Acute abdomenDuodenum and jejunumSmall intestineAbdominal pain nausea and constipationSmall bowel obstructionSmall intestine Abdominal pains and hematemesisThickening of the wall of the ileum and ascending colonThickening of the mucosal folds and multiple polypoid protrusionsDiffuse thickening of the mucosa with friability nodularity erosions and polypoid protrusionsDiffuse thickening of the wall of the small intestineThickening of the jejunal and proximal ileal mucosal foldsPerforation of the wallMultiple shallow ulcers and several erosionsBand of amyloid and connective tissue surrounding the small intestineMucosal ulcerations with discrete nodulesSmall intestine Nonspecific digestive symptoms Multiple mucosal polypsJejunumSmall intestine Nonspecific digestive symptoms Thickening of the wall with rough and Nonspecific digestive symptoms Polypoid pseudotumoral mucosal formationsHeartburn and constipationpolypoid intestinal mucosal foldsF M MSmall intestine three casesDuodenumScreening for cancer stomachA localized depressed lesionEndoscopic and histological findings in localized intestinal amyloidosisIn the small intestine the most common endoscopic findings were thickening of the intestinal wall [ ] and polypoid mucosal protrusions [] followed by mucosal ulceration [ ] friability and nodularity of the mucosa [ ] and wall perforations [] In the colorectum polypoid masses with luminal narrowing were the most common endoscopic finding [ ] followed by mucosal ulcerations solitary or multiple ulcers [ ] or and thickening of the wall [ ] nodularity and friability of the mucosa [ ] and perforation [] In all cases deposition of pale eosinophilic amorphous amyloid material with the prominent cracking artifact in the mucosa submucosa muscle layer or walls of the blood vessels represented the main histological feature [ ] The further Congo red staining revealed the characteristic salmonpink color confirming the presence of amyloid deposits The deposits showed the characteristic applegreen birefringence under polarized light Immunohistochemical stains were also performed in some cases to substantiate the diagnosis Other histological features included mucosal ulceration amyloid angiopathy necrotizing angiitis [] foreign body giant cell reaction [] ischemic changes focal active colitis and cryptitis a personal observation The amyloid type was AL light chain protein in the majority of the cases of the small intestine [ ] and colorectum [ ] Other amyloid protein types included AAamyloid [] and high serum amyloid SAA in some cases [] Summary of clinicopathological findings in patients with localized intestinal amyloidosis is presented in Tables [ ]Case reportPrimary localized amyloidosis of the intestineA 55yearold female patient presented with anorexia diarrhea rectal bleeding and weight loss Colonoscopy revealed picture reminiscent of active pancolitis friable mucosa with mucosal erosions ulcerations and hemorrhage and six biopsies from the ileum cecum and colon ascending transverse descending colons and rectum were obtained On histology all biopsies showed deposition of pale eosinophilic amorphous material with the prominent cracking artifact characteristic of amyloid in the lamina propria and submucosa In some areas s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cAlshehri Gastroenterol Res Table Data From Individual Studies of Primary Localized Amyloidosis of the ColonCase SexSitesClinical presentationColonoscopic findingsStudiesAge yearsMMMMMFMMMFMFTransverse colonbleeding and weight lossIntestinal obstructionTransverse colon A periumbilical pain rectal Descending colon AnemiaSigmoid colonUlcerations and luminal narrowing by multiple polypoid lesionsElderlyA stenosing massMiddle age Transverse colon Nonspecific digestive symptoms Submucosal massUlcerative lesionsMucosal ulceration perforation and thickening of the bowel wallA submucosal tumorlike massMucosal ulcerA single friable rounded mucosal lesionA 15cm shallow depressed mucosal lesionAcute abdominal pain rectal bleeding and fecal peritonitisHematocheziaHemepositive stoolsRectal bleedingRoutine colonoscopyNonspecific digestive symptoms Several irregularlyshaped discrete ulcerationsSigmoid colonSigmoid colonSigmoid colonSigmoid colonSigmoidDescending colon Abdominal pain bloating flatulence and hematocheziaRectal bleedingHemorrhagic mucosa and amass lesionDiffuse nodular friable lesions ascending colon and several irregular large ulcers with nodularity descending colonThickening of the wall and mucosal ulcerationsAscending and descending colonsColon seven casesAscending colonF M Middle age ColonF M Rectal bleeding two casesHeartburn and constipationFAcute abdominal pain diarrhea and vomitingThickening of the wall of the ileum and ascending colon[][][][][][][][][][][][][][][]Table Clinical Colonoscopic and Histological Findings in the Primary Localized Amyloidosis of the IntestineAspectsAge mean ± SEMMale to female ratioSite of involvementClinical presentationsColonoscopic findingsHistological featuresSmall intestine duodenum jejunum and ilium ± years [ ]Jejunum Ileum Colorectum ± years [ ]The left colon Transverse colon Abdominal pain and intestinal obstruction [ ]Rectal bleeding [ ] followed by abdominal pain [ ] and anemia []Thickening of the wall [ ] polyps and polypoid mucosal protrusions [] ulcerations [ ] friability and nodularity of the mucosa [ ] and perforation []Mass lesions tumorlike lesion polypoid protrusions and polyps with narrowing were the most common [ ] Ulcerations with solitary or multiple ulcers [ ] thickening of the wall [ ] nodularity and friability of the mucosa [ ] and perforation []Amyloid deposits increased density of mixed inflammatory cells in the lamina propria light chain protein [ ]Amyloid deposits increased density of mixed inflammatory cells in the lamina propria foreign body giant cell reaction amyloid angiopathy necrotizing angiitis focal active colitis and immunoglobulin lightchain AL [ ]s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cLocalized Amyloidosis of the Intestine Gastroenterol Res Figure Isolated primary amyloidosis of the intestine case report a e Colonic mucosa with deposition of pale eosinophilic amorphous material star in the submucosa with abundant deposits of pink amorphous materials with some cracking artifacts characteristic of amyloid Focal surface mucosal ulceration is noted The background mucosa shows focal active inflammation including cryptitis arrow and there is patchy crypt distortion No granulomas are seen hematoxylin and eosin a × b × c d × and e × f g Congo red stain which reveals the characteristic salmonpink color confirming the presence of amyloid deposits Congo red f × and g × h i The deposits exhibit characteristic applegreen birefringence under polarized light Congo red under polarized light h × and i × the lamina propria was entirely replaced by these deposits The background mucosa showed focal active inflammation focal ulceration cryptitis occasional crypt abscess This focal chronic active inflammatory change may be secondary to erosionulceration associated with amyloid deposits [] Congo red stain confirmed the presence of amyloid deposits with applegreen birefringence under polarized light Fig Immunohistochemical analysis revealed that the amyloid deposits were positive for amyloid lambda but negative for amyloid A These findings were consistent with amyloid lightchain AL amyloidosis We performed several laboratory and radiological investigations to rule out the possibility of systemic involvement The hematological indices red blood cells RBCs count hemoglobin mean corpuscular volume MCV mean corpuscular hemoglobin MCH platelets and white blood cells count and the biochemical tests total protein albumin globulin alkaline phosphatase aspartate aminotransferase AST total bilirubin ferritin vitamin B12 folic acid and prostatespecific antigen PSA urea creatinine electrolytes and spot urine proteincreatinine ratio protein electrophoresis and immunoelectrophoresis of serum and urine were within normal limits Thyroid function tests were within normal limits Skeletal surveys electrocardiograms and radiological findings liver spleen and kidneys were unremarkable Bone marrow biopsy showed no abnormalities Therefore the diagnosis of localized primary intestinal AL amyloidosis involving terminal ileum cecum colon and rectum associated ulceration and inflammatoryregenerative changes was establishedThe study presents a representative retrospective case in which the archival formalinfixed paraffinembedded tissues from the consultation files of the authors were used [] All the materials paraffinembedded tissue block slides and pathology report were coded The information obtained was analyzed and reported in such a way that the identity of the case presented cannot absolutely be ascertained The patient consent was obtainedDiscussionLocalized amyloidosis of the intestine is of interest to the pathologists and gastroenterologists because some cases may s The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cAlshehri Gastroenterol Res mimic tumors Here we present a comprehensive review of the literature about this entity In agreement with previous reports [ ] our analysis indicates that the primary localized intestinal amyloidosis is a rare condition with disparate and nonspecific clinicopathological and endoscopic features Also its outcome depends on the site of the deposition of the amyloid small intestine versus colorectum [ ] In Cowan examined the clinicopathological features of patients with amyloidosis Seventysix patients had gastrointestinal amyloidosis The age range of the patients was years The patients include cases with localized gastrointestinal amyloidosis and cases with gastrointestinal involvement as a part of systemic amyloidosis In the latter mutations of the transthyretin gene were found Gastrointestinal bleeding and weight loss were the most common presenting symptoms []Threlkeld and Nguyen reported as a case of isolated colonic amyloidosis in a 46yearold male patient who presented with hematochezia and abdominal pain Colonoscopic studies revealed the presence of a tumorlike mass in the descending colon and therefore partial colectomy was performed Grossly the mucosa was necrotic but no mass lesion was identified There were light chainspositive amyloid deposits in the muscle layers submucosa and submucosal blood vessels Laboratory investigations for inflammatory condition and plasma cell disorders were negative and therefore the diagnosis of isolated gastrointestinal amyloidosis was established The patient received colchicine with marked clinical improvement [] Similarly Diaz Del Arco reported a case of globular amyloid deposits involving the left colon in a 74yearold male patient presenting with anemia and colitis Histologically there were Congo red positive deposits in the lamina propria of the colon []The duodenum is an important site for the deposition of amyloid protein Patients with primary duodenal amyloidosis may present with epigastric pain nausea constipation steatorrhea protein loss malabsorption hemorrhage and vomiting Endoscopic findings include the thickening of the duodenal wall and the presence of polypoid lesions [ ] Deguchi described a case of an isolated duodenal and jejunal amyloidosis in a 47yearold male patient who presented with features of intestinal pseudoobstruction Endoscopy revealed thickening and polypoid lesions of the duodenal mucosal folds The further histological evaluation confirmed the presence of amyloid deposits in the duodenal and jejunal walls [] Similarly Choi reported a case of localized amyloidosis of the small intestine duodenum and jejunum in a 62yearold male patient who presented with abdominal pain nausea and constipation of several years duration Endoscopy revealed mucosal erosions ulceration and tumorlike masses Immunohistological analysis revealed the presence of lambda light chain protein therefore establishing the diagnosis of amyloidosis []In the primary localized amyloidosis the isolated deposition of amyloid proteins may be due to the local tissue damage with the synthesis and deposition of amyloid substances in the individual ans [] The synthesis of amyloid results from plasma cell reaction to the inflammatory antigens The otherwise benign polyclonal plasma cells produce monoclonal immunoglobulin light chains L that are deposited as AL amyloid [] It is still possible that the deposition of amyloid results from the inability of the body to clear light chains produced by plasma cells [] Amyloid deposits in the intestine are usually irreversible but can sometimes be reversible with chemotherapy treatment especially with melphalan and steroid therapy []In primary localized intestinal amyloidosis we found variations in the site of deposition of amyloid proteins There was the deposition of the amyloid proteins in the walls of the blood vessels mucosa submucosa muscularis mucosae muscularis propria and the neural elements [] The destructive effects of amyloid usually resulted from the local replacement of these important histological compartments For instance amyloid deposition in the wall of the blood vessels usually leads to ischemia bowel infarction [] or intestinal perforation [] The involvement of the mucosa by amyloid deposits usually produces mucosal ulcerations leading to malabsorption [] hematochezia and anemia [ ] The deposition of amyloid in the submucosa and the muscle layers usually leads to the formation of the mass lesions amyloidoma or amyloid tumor resulting in intestinal obstruction [] The involvement of the neural elements results in altered bowel motility and chronic ileus [] Hemorrhage melena and bleeding per rectum is reasoned to bowel ischemia hemorrhagic necrosis of the mucosa damage of the vessel walls and abnormal platelet aggregation []The variations in the gross findings of intestinal amyloidosis may be reasoned to the type and the site of deposition of the amyloid protein When the deposition of the amyloid protein occurs predominantly in the perivascular and interstitial spaces it usually produces diffuse thickening of the intestinal wall rather than polypoid or tumorlike lesions [ ] Elevations or localized tumorlike lesions may reflect the presence of nodular aggregates of plasma cells locally producing abundant amyloid proteins which usually originate from AL protein produced by local aggregates of plasma cells involving the mucosa submucosa or the muscle layer [ ] Fine granular lesions or mucosal erosion or ulcerations are commonly associated with the deposition of AA amyloidosis [ ]To conclude the data presented here provides an indepth characterization of the clinicopathological features of primary localized intestinal amyloidosis It indicates that this entity is rare and its presenting features are generally nonspecific and therefore the diagnosis is challenging and can be easily missed Knowledge of this type of amyloidosis is warranted to avoid aggressive management such as treatment with an alkylating agent A high index of suspicion should be maintained both on the part of the gastroenterologists and the histopathologistsAcknowledgmentsNone to declareFinancial DisclosureNone to declares The authors Journal compilation Gastroenterol Res and Elmer Press Inc„¢ wwwgastrores 0cLocalized Amyloidosis of the Intestine Gastroenterol Res Conflict of InterestNone to declareInformed ConsentInformed consent was obtainedAuthor ContributionsSaeed Ali Alshehri conceived and designed the analyses collected the data biochemical and clinicopathologic aspects of amyloidosis performed the analyses tools including the statistical data analysis and wrote the paper Mahmoud Rezk Abdelwahed Hussein supervised the data analysis and contributed to the conception of the idea of the manuscript data collection Histological aspects of amyloidosis writing up and revising of the manuscriptData AvailabilityThe authors declare that data supporting the findings of this study are available within the References Westermark P Benson MD Buxbaum JN Cohen AS Frangione B Ikeda S Masters CL Amyloid fibril protein nomenclature Amyloid Monge M Chauveau D Cordonnier C Noel LH Presne C Makdassi R Jaureguy M Localized amyloidosis of the genitourinary tract report of new cases and review of the literature Medicine Baltimore Mathis H [Rokitansky Virchow and Heschl on the problem of amyloidosis a historical study] Zentralbl Allg Pathol Sack GH Jr Serum amyloid A a review Mol Med Gillmore JD Wechalekar A Bird J Cavenagh J Hawkins S Kazmi M Lachmann HJ Guidelines on the diagnosis and investigation of AL amyloidosis Br J Haematol Cowan AJ Skinner M Seldin DC Berk JL Lichtenstein DR O'Hara CJ Doros G Amyloidosis of the gastrointestinal tract a 13year singlecenter referral experience Haematologica Ankarcrona M Winblad B Monteiro C Fearns C Powers ET Johansson J Westermark GT Current and future treatment of amyloid diseases J Intern Med Selkoe DJ Folding proteins in fatal ways Nature Otaka Y Goda F Nakazato Y Tsutsui T Systemic heavy and lightchain amyloidosis presenting nephrotic syndrome and congestive heart failure a case presentation and literature review Amyloid 201926sup19596 Picken MM The pathology of amyloidosis in classification a review Acta Haematol Trinh TD Jones B Fishman EK Amyloidosis of the colon presenting as ischemic colitis a case report and review of the literature Gastrointest Radiol Threlkeld C Nguyen TH Isolated amyloidosis of the colon J Am Osteopath Assoc GarciaGonzalez R Fernandez FA Garijo MF Fernando ValBernal J Amyloidosis of the rectum mimicking collagenous colitis Pathol Res Pract Tada S Iida M Yao T Kawakubo K Yao T Okada M Fujishima M Endoscopic features in amyloidosis of the small intestine clinical and morphologic differences between chemical types of amyloid protein Gastrointest Endosc

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

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

RNAs circRNAs are a novel and unique class of noncoding RNAs that are backspliced from premRNAs It hasbeen confirmed that circRNAs are involved in various malignant behaviors of hepatocellular carcinoma HCCHowever the role of circRNA in the regulation of ferroptosis and the underlying mechanism remain unknown HerecIARS hsa_circ_0008367 was found to be the most highly expressed circRNA after sorafenib SF treatment in HCCcells Small interfering RNA against cIARS sicIARS significantly suppressed the cellular sensitivity to SF or Erastinthrough inactivating ferroptosis which may be partially attributed to the inhibition of autophagy and ferritinophagyPrediction analysis and mechanistic identification revealed that cIARS physically interacted with RNA binding proteinRBP ALKBH5 which was a negative regulator of autophagic flux in HCC The dissociation of BCL2BECN1 complexmediated by ALKBH5 silencing was effectively blocked by sicIARS Furthermore the inhibition of ferroptotic eventsautophagic flux and ferritinophagy resulted from sicIARS were significantly rescued by ALKBH5 downregulationOverall cIARS may be an important circRNA positively regulating SFinduced ferroptosis through suppressing theALKBH5mediated autophagy inhibitionIntroductionHCC is a highly refractory and prevalent cancerworldwide with new cases and deathsevery year1 Rapid advances in diagnosis and treatmenthad improved patient outcomes However the survivalgains are stagespecific2 Liver resection transplantationablation ortransarterial chemoembolization benefitspatients in early or intermediate stages3 while treatmentfor latestage HCC has remained challenging The standard firstline systemic drug against advanced HCC issorafenib SF BAY Nexavar which is currentlyCorrespondence Xiaoqing Wei weixqrm163com orJie Li heplijie163com1Department of Hepatobiliary Surgery The First Affiliated Hospital ofShandong First Medical University Jinan Shandong China2Department of Hepatobiliary Surgery Shandong Provincial QianfoshanHospital Shandong University Jinan Shandong ChinaFull list of author information is available at the end of the These authors contributed equally Zhiqian Liu Qi WangEdited by Inna Lavrik The Authors known as a ferroptosis inducer4 exerting only limitedeffects on overall survival and time to tumor progression56 Therefore it is imperative to explore novel andeffective therapeutic target to improve the cellular sensitivity to SFˆ’ was targeted SubsequentlyIn recent years ferroptosis has been confirmed to be anessential mechanism in SF treatment7 It is a kind of finelycontrolled cell death featured with irondependent accumulation of lipid hydroperoxides8 When HCC cells wereexposed to SF the cystineglutamate antiporter systemsystem xcthe cystineuptake was blocked and the biosynthesis of glutathioneGSH was inhibited resulting in accumulation of lipidperoxidation products and eventually inducing ferroptosis Meanwhile SF also induces autophagic flux in cellsWhen autophagy flux is initially induced MTORC1 dissociates from the ULK12ATG13 complex leading todephosphorylation of the complex9 Then phagophorenucleation occursthe ATG14BECN1recruiting 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 0cLiu Cell Death Discovery Page of PIK3C3PIK3R4 complex10 Nexttwo ubiquitinlikeconjugation systems are involved in phagophore elongation11 When de novo autophagosome formation iscomplete they are delivered to the lysosome initiatinglysosomal degradationActually autophagy is a targetable pathway regulatingcellular sensitivity to ferroptosis12 The crosstalk betweenautophagy and ferroptosis has attracted more and moreattentions in the recent years Autophagy inhibition bybafilomycin A1 or chloroquine or lack of key autophagyassociated gene for example ATG5 ATG7 will partiallysuppressed ferroptotic eventsthrough ferritinophagyinactivation reducing the turnover of ironbinding ferritinand the iron release13“CircRNAs primarily originate from backsplicing eventsof premRNAs The covalently bonded loops are highlystable and abundant16 The deregulation of circRNAs isclosely related to various human diseases including cancer17“ In HCC several studies have revealed that circRNAs are involved in multiple malignant behaviors20However the functions and mechanisms of circRNAs inSF treatment are still not comprehensively understood Inthe current research we found a novel circRNA derivedfrom the IARS gene ID from circBase hsa_circ_0008367named cIARS cIARS was proven to be a promoter offerroptosis in HCC cells after SF treatment which waspartially attributed to the activation of autophagy andferritinophagy The underlying molecular mechanism ofcIARSmediated ferroptosis was also clarified in this studyResultsCircular transcript cIARS hsa_circ_0008367 is significantlyupregulated in SFtreated HCC cellsRNAseq was performed in three pairs of SFtreated anduntreated HCC celllines HepG2 SMMC7721 andHuh7 to screen differentially expressed circRNAs Cellswere treated with μM of SF for h uniquecircRNAs were found Among these circRNAs had already been reported in circBase21 and theother circRNAs were newly discoveredAmong the annotated circRNAs were ofextremely low abundance with fragments per kilobasemillion FPKM values in cellular samples and werethus excluded from this study The other circRNAscomprised two groups presented no significantchanges in expression levels after SF treatment and theother were differentially expressed circRNAs foldchange ‰¥ pvalue The volcano plots demonstrated that there were upregulated and downregulated transcripts Fig 1a The results of hierarchicalclustering are displayed in a heatmap Fig 1b generatedwith Heatmap Illustrator22 and suggest that there weretwo different clusters of transcripts The most highlyexpressed circRNA was cIARS hsa_circ_0008367 andOfficial journal of the Cell Death Differentiation Associationwas marked with black arrow in the volcano plotsAccording to circBase cIARS is an exonic circRNA nt in length originating from the exon and exon of the IARS gene on chr9 “ Fig1c qPCR showed that the relative expression levels ofcIARS were significantly higher in SFtreated cell linesthan in untreated ones Fig 1d Both convergent anddivergent primers of cIARS were applied for amplificationThe band of cIARS was only observed in cDNA samplenot the genomic DNA Fig 1e Sanger sequencing further validated that the sequence around the junction siteabout bp around the site was consistent with theresult of RNAseq and CircInteractome database23 Fig1f In addition cIARS was much more resistant to RNaseR Uμg which degrades linear but not circularthan IARS and GAPDH Fig 1g Whentranscriptsactinomycin D ActD a transcription inhibitor wasadded to HCC cells for the indicated time periods cIARSwas much more stable than its linear counterpart Fig1h These evidences suggested cIARS to be a highlyabundant and stable circular transcript in HCC cellscIARS is found to be a significant regulator of SFinducedferroptosisTo clarify the biological role of cIARS we first knockdown cIARS expression with a junction sitespecificsiRNA vector sicIARS The effects of the sicIARSwas shown in Fig 2a CCK8 assay showed that SFinduced growth inhibition was evidently weakened in sicIARS transfected cells Erastininduced growth inhibitionwas also attenuated by sicIARS Fig 2b c To determinethe underlying mechanism sicIARSintroduced HCCcells were treated with various cell death inhibitorsFerrostatin124 a specific ferroptosissignificantly undermined the therapeutic effects of either SFor Erastin in both sicIARS and NC transfected cellsHowever ZVADFMK an apoptosisinhibitor andNecrosulfonamide a necroptosis inhibitor exerted nosignificant influence on SF or Erastininduced growthinhibition Fig 2c Simultaneously malondialdehydeMDA and the level of Fe2 were significantly reducedwhile intracellular GSH obviously increased in the cIARSsilencing cells following SF or Erastin administration Fig2d These evidences suggested cIARS to be a positiveregulator of ferroptosis in HCC cellsinhibitorcIARS positively regulates SFinduced autophagy andferritinophagycIARS was also found to be an autophagy regulatorWestern blot WB assay showed that cIARS knockdownsignificantly decreased LC3 lipidation and increased p62accumulation Fig 3a Either autophagosomes or autolysosomes were observed via microscopic examinationafter AdmCherryGFPLC3 adenovirustransfection 0cLiu Cell Death Discovery Page of Fig See legend on next pageThis experiment is applied for concurrent observation ofautophagosome and autolysosome The signal of greenfluorescent protein will be quenched during fusion ofautophagosome and lysosome Thus the red signal ofmCherry indicates autolysosome and the merge of greenand red signals yellow puncta indicates autophagosomesicIARS significantly decreased the amount ofredautolysosome and yellow autophagosome puncta percell demonstrating an inhibition of autophagy fluxFig 3b TEM visuallyautophagicsuggested theOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of see figure on previous pageFig Circular transcript cIARS hsa_circ_0008367 is significantly upregulated in SFtreated HCC cells RNAseq was performed in three pairsof SFtreated μM h and untreated HCC cell lines HepG2 SMMC7721 and Huh7 a The differentially expressed circRNAs were visualized withvolcano plots the ˆ’log10 pvalue and the log2 fold change are plotted on the y and x axes respectively The dashed lines signify the filteringcriteria p fold change ‰¥ Upregulated circRNAs are shown in red and downregulated circRNAs are shown in green The most highlyexpressed circRNA was marked by a black arrow b Hierarchical cluster analysis showing the differentially expressed circRNAs The six columnsrepresent the six different cellular samples Each row indicates a circular transcript and the colors represent the abundance of the transcriptsc Schematic diagram of the backsplicing transcript generated from linear IARS Exon and exon are colored red and green respectively dRelative expression of cIARS after SF treatment for h in the HCC cell lines e Divergent and convergent primers for cIARS were applied to amplifyboth cDNA and gDNA Agarose gel electrophoresis visualized the products f The sequence around the junction site was confirmed through Sangersequencing The empty triangle indicates the junction site g RNase R exoribonuclease Uμg °C min was applied to remove the lineartranscripts from cellular extracts leaving circRNAs behind qPCR was applied to assess the resistance of RNAs to RNase R h RNA decay assayevaluating the stability of cIARS and IARS by qPCR after ActD μgml administration The error bars represent the standard deviation SD of at leastthree independent experiments p compartments sicIARS decreased the number of doublemembraned vacuoles to a relatively low level Fig 3cThese results showed that cIARS is a positive autophagyregulator in SFtreated HCC cells Furthermoretheprotein levels of FTH1 and NCOA4 the substrate andcargo receptor of ferritinophagy were determined by WBassay sicIARS resulted in remarkable accumulation ofboth FTH1 and NCOA4 Fig 3d This finding indicatedthatthe ferroptotic events in SFtreated HCC cellspositively regulated by cIARS may be partially associatedwith autophagy and ferritinophagycIARS specifically interacted with RBP ALKBH5 AlkBHomolog RNA demethylaseRecent studies have revealed that circRNA“RBP interaction has important roles in diverse biological processesAccording to CircInteractome database we found six RBPsbearing at least two binding sites matching to cIARSincluding FMRP Fragile X Mental Retardation SFRS1Serine And Arginine Rich Splicing Factor ALKBH5HuR ELAV like RNA binding protein IGF2BP1 InsulinLike Growth Factor MRNA Binding Protein andLIN28A Lin28 Homolog A Fig 4a RBP immunoprecipitation RIP assay in both HepG2 and Huh7 cell linesdemonstrated that the relative levels of cIARS in ALKBH5enriched samples were much higher than in the other fiveRBPs Fig 4b RNA pulldown and RNA EMSA were performed to determine the interaction using an antisenseprobe spanning the junction site of cIARS The presence ofspecific bands demonstrated the physical binding of cIARSand ALKBH5 Fig 4c d Interestingly SF administrationhad no influence on the protein levels of ALKBH5 in bothHepG2 and Huh7 cells Fig 4e but remarkably increasedthe cIARS“ALKBH5 interaction Fig 4f which may be dueto SFinduced expression of cIARScIARS repressed the role of ALKBH5 in the regulation ofautophagyALKBH5 had been previously proven to be an autoprole inin cancer2526 Howeverhagy inhibitoritsOfficial journal of the Cell Death Differentiation AssociationSFtreated HCC cells remains unclear WB assay showedthat siRNA against ALKBH5 siALKBH5 significantlypromoted the transformation of LC3B I to II and degraded p62 Fig 5a This result suggested ALKBH5 to be anegative regulator of autophagy in HCC cells qPCR andWB showed that sicIARS failed to influence ALKBH5mRNA and protein levels Fig 5b c similarly siALKBH5 also had no impact on the relative expressionof cIARS Fig 5d A previous study revealed that siALKBH5 promoted the dissociation of BECN1 and BCL a key step during phagophore nucleation In SFtreated HCC cells we gained similar results via IP assayMore importantly this process can be effectively blockedby cIARS silencing Fig 5e These results demonstratedthat cIARS repressed the biological role of ALKBH5 inautophagycIARS regulates ferroptosis through ALKBH5mediatedautophagyTo explore whether cIARSregulated ferroptosis viaALKBH5 we performed several phenotyperescueexperiments First siALKBH5 successfully rescued theeffects of sicIARS in autophagic flux and ferritinophagyFig 6a b Second ALKBH5 knockdown effectively reintensified the SF cytotoxicity which was remarkablyimpaired by sicIARS Fig 6c Third sicIARSmediateddecrease of MDA Fe2 and increase of GSH can berescued by siALKBH5 Fig 6d“fTaken together a novel circRNA in HCC was revealedin our research We partially clarified its role andmechanism in ferroptosisDiscussionAs a novel class of noncoding RNA circular transcriptshave attracted widespread attention However circRNAregulated ferroptosis in human diseases has not beenwidely investigated A small portion of studies focused onthe circRNAmediated autophagy For instance Chenet al27 reported that circHIPK3 depletion significantlyvia miR1243pSTAT3PRKAAinduced autophagy 0cLiu Cell Death Discovery Page of Fig cIARS is found to be a significant regulator of SFinduced ferroptosis a The expression levels of cIARS after transfection of sicIARS or NCb The evaluation of growth inhibition induced by SF in sicIARS or NC transfected cells at the indicated concentrations for h c The evaluation ofcytotoxicity of SF μM h and Erastin μM h with or without several inhibitors of cell death including ferrostatin1 μM ZVADFMK μM or necrosulfonamide μM d The assessment of MDA Fe2 and GSH during SF treatment μM h p AMPKa axis and there was an antagonistic regulation onautophagy between circHIPK3 and linear HIPK3 Du WWet al28 showed that the oncogenic circDnmt1stimulatedautophagy flux in breast carcinoma via interaction withboth p53 and AUF1 These findings demonstrated thepotential of circRNAsin autophagy regulation andprompted us to explore the role of circRNA in ferroptosiswhich had been identified as an autophagic cell death12research we delineated a mechanism ofcircRNAmediated ferroptosis during SF treatment inHCC cells circRNA cIARS hsa_circ_0008367 wasIn ourOfficial journal of the Cell Death Differentiation Associationscreened from RNAseq analysis Phenotypically cIARSpositively regulated ferroptosis which may be partiallydependent on autophagy and ferritinophagy Mechanistically cIARS physically interacted with RBP ALKBH5and negatively regulated its role in autophagyitis essentialTo comprehensively investigate the complicated cirto deeply evaluate itscRNA networkbinding partners In this study cIARS is found to be aninteractor of RBP ALKBH5 which had previously beenreported to be a N6methyladenosine m6A eraser Itsrole in autophagy regulation is completely different in 0cLiu Cell Death Discovery Page of Fig cIARS positively regulates SFinduced autophagy and ferritinophagy a The expression levels of LC3B and p62 in sicIARSintroduced cellswith or without SF treatment b Microscopic observation of autophagy flux after transfection of AdmCherryGFPLC3 for h mCherryGFPLC3 wasvisualized by fluorescence microscopy The green fluorescent protein is acid sensitive and will be quenched in the acidic condition of lysosome while themCherry is stable at low pH Thus the red puncta represent autolysosomes and the yellow puncta generated from the merge of both green and red signalsrepresent autophagosomes The autophagosomal and autolysosomal abundance was measured by the number of puncta lower density of yellow and redpuncta suggested lower level of autophagic flux c Visualization of autophagic compartments via TEM The red arrowheads indicate doublemembranedvacuoles d The assessment of the protein levels of FTH1 and NCOA4 in sicIARS or NC transfected cells with or without SF treatment μM hlower m6A leveldifferent tissues and diseases252629“ In lung cancer25ALKBH5 upregulation stabilized UBE2C an autophagyinhibitor with maintenance ofInovarian cancer26 ALKBH5 inhibited autophagy throughactivating PI3KAktmTOR signaling pathway stabilizedBCL2 mRNA and promoted the interaction betweenBCL2 and BECN1 Herein cIARS was proven to be apivotal regulator of autophagy ferroptosis and ferritinophagy depending on negatively regulating the biologicalinHCC cellsrole of ALKBH5 an autophagy inhibitibco Carlsbad CA USA and penicillin Umlstreptomycin µgml solution in the medical researchcenter of Shandong Provincial Qianfoshan HospitalShandong First Medical University All of the cell linesmentioned in this research were cultured within tenpassages The transfection experiments were performedaided by Lipofectamine Life Technologies Carlsbad CA USA The RNA oligonucleotides in this workwere designed and constructed by GenePharma Shanghai China including siRNAs against cIARS or ALKBH5and the corresponding negative controls NC and NC™Sequences were shown as follows ²“² sicIARS GACUUU GAG GAG AUC AGA CAC siALKBH5 GGAUAU GCU GCU GAU GAA ATT NCNC² UUC UCCGAA CGU GUC ACG UIn this study the cIARS“ALKBH5 axis was demonstrated to be a key mechanism regulating ferroptosisduring SF treatment Further studies are still needed atthe clinical and mechanistic levelsMaterials and methodsCell culture and transfection assayUnder humidified conditions with CO2 at °C theHCC cell lines HepG2 SMMC7721 and Huh7 boughtfrom the National Infrastructure of Cell Line Resourcewere cultured in Dulbecco™s Modified Eagle™s mediumHyClone Logan UT USA with fetal bovine serumCell Counting Kit8 CCK8 assayThe growth inhibition rate of HepG2 and Huh7 cellswas assessed by the CCK8 Dojindo Laboratories Japanassay The blank or transfected cells × cells perwell were seeded into 96well plates with three replicatewells After the treatment with SF or Erastin or variouscell death inhibitorsFerrostatin1 ZVADFMK orOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Fig cIARS specifically interacts with ALKBH5 a Prediction analysis of the interaction between cIARS and RBPs through CircInteractome b RIPassay evaluating the physical interaction between cIARS and the six candidate RBPs in HCC cells c d RNA EMSA and RNA pulldown evaluating thephysical interaction between cIARS and ALKBH5 e The protein levels of ALKBH5 in HCC cells in the absence or presence of SF treatment f Theassessment of the physical binding of cIARS and ALKBH5 by RIP assay in the absence or presence of SF treatment p Necrosulfonamidefor h a µl volume CCK8reagent was added to each well Then measuring theabsorbance at nm after incubation with the CCK8solution at °C for hRNA extraction and analysisExpression profiles of genomewide circRNAs in threepairs of HCC cell lines before and after SF treatmentwere explored on an Illumina HiSeq platform byNovogene Beijing China The cDNA from divergentprimers was subjected to Sanger sequencing by Zhonghong Boyuan Biological Technology Jiangxi ChinaAgarose gel electrophoresis was used to detect the qPCRamplification of cDNA and genomic DNA after applyingwith the divergent and convergent primers of cIARSTherelative fold changes in expression were calculated withthe formula ˆ’ΔΔCt The sequences of all the primers werelisted as follows ²“² cIARS F AGC GAT GAC TTTGAG GAG ATC A R CCC AGT AGC ACA GGT CATTG IARS F CAT ATC CAG TTT CTC CAT CGG A RTGG ATT TTC CAG GAG CAA TAC T ALKBH5 FGCA AGG TGA AGA GCG GCA TCC R GTC CACCGT GTG CTC GTT GTA C U6 F CTC GCT TCGGCA GCA CAT A R ATT TGC GTG TCA TCC TTGCG GAPDH F CAG AAC ATC ATC CCT GCC TCTAC R ATG AAG TCA GAG GAG ACC ACC TGRibonuclease R RNase R assayRNase R assay R0301 Uµl Geneseed GuangzhouChina was used for the identification of circRNAAccording to the manufacturer™s guidance µl ×Reaction Buffer and U RNase Rµg RNA were mixedto the total RNA and then added RNaseFree Water toform a µl reaction solution system After digestion withRNase R for min at °C the enzyme then was inactivated at °C for min and then directly performOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Fig cIARS repressed the role of ALKBH5 in the regulation of autophagy a The expression levels of LC3B and p62 in siALKBH5 or NC²introduced cells with or without SF administration b c The relative expression of ALKBH5 mRNA and protein in sicIARS or NC introduced HCC cellsd The relative expression of cIARS in siALKBH5 or NC² transfected cells e The assessment of the role of sicIARS in ALKBH5mediated interactionbetween BECN1 and BCL2Official journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of see figure on previous pageFig cIARS regulates ferroptosis through ALKBH5 a b The evaluation of the role of siALKBH5 in sicIARSregulated autophagy and ferritinophagyThe WB assay demonstrated that the effect of sicIARS on LC3 lipidation and p62 accumulation can be significantly reversed by siALKBH5 in SF μM h treated HCC cells a the effect of sicIARS on the accumulation of FTH1 and NCOA4 can be evidently rescued by siALKBH5 in either HepG2 or Huh7cells treated by SF μM h b c Identification of the role of ALKBH5 in cIARSregulated growth inhibition by CCK8 The results demonstrated thatsiALKBH5 significantly reversed sicIARSmediated growth inhibition in SF administered HCC cells d“f The rescue experiments evaluating the effects ofsiALKBH5 in the sicIARSmediated ferroptotic events The relative levels of MDA and iron were obviously decreased and the level of GSH increased insicIARS introduced HCC cells these impacts can be effectively reversed by ALKBH5 knockdown The black whiskers indicated the difference between theœsicIARS and œNC groups while the red ones indicated the difference between the œsicIARSNC™ and œsicIARSsiALKBH5 groups p reverse transcription reaction The qPCR assay was usedto determine the relative expression of cIARS IARS andGAPDH compared to the mock groupActinomycin D ActD assayActD assay HY17559 MedChem Express New JerseyUSA was used to detect the stability of RNA μgmlActD reagent was used to treat HepG2 and Huh7 cellsAfter or h of administration the total RNAwas extracted respectively to determine the relativeexpression of cIARS or IARS by qPCR assayWestern blot analysisRadioimmunoprecipitation assay buffer RIPA was appliedto lyse cells Total proteins were then harvested and quantified with bicinchoninic acid assays Beyotime ShanghaiChina The target proteins were separated through SDSPAGE and transferred to polyvinylidene fluoridemembranes Merck Millipore Burlington MA USA Themembranes were blocked with nonfat milk incubated withprimary antibodies and then incubated with secondaryantibody diluted at a ratio of Jackson ImmunoResearch West Grove PA USA The primary antibodieswere antiLC3B Cell Signaling Technology BeverlyMA USA antip62 Cell Signaling TechnologyantiHuR ab28660 Abcam Cambridge MA USA antiSFRS1 ab133689 antiFMRP ab17722 antiALKBH5ab195377antiLIN28Aab46020 antiFTH1 ab65080 antiNCOA4 ab86707antiBCL2 ab32124 antiBECN1 ab62557 and antiGAPDH ab9485 The protein signals were visualized withenhanced chemiluminescence detection reagentsECLMillipore Burlington MA USA and quantified with ImageLab software BioRad Hercules CA USAantiIGF2BP1ab82968based on the 3rd edition of the Guidelines for the Interpretation of Assays for Monitoring Autophagy32ImmunoprecipitationandImmunoprecipitation KitRIP and IP were performed to confirm the RNA“proteinand protein“protein interactions using RIP Kit Milliporeab206996according to the manufacturer™s guidance RIP related detailshad been described in our previous studies3334 The IP assayconsists of four steps including antibody binding beadspreparation bead capture and elution The volume of theantibody binding system was made up to µl with lysisbuffer containing the protease inhibitor cocktail and gentlymixed for h the protein AG sepharose µlreactionwas washed twice with wash buffer centrifuged at × gfor min and aspirated the supernatant between washesRNA pulldown assayPierce„¢ Magnetic RNAProteinPullDown KitThermo was applied to evaluate RNA“protein interaction using biotinlabeled junctionspecific probe and itsnegative control designed and synthesized by ViageneBiotech Jiangsu China Detailed procedure strictly followed the manufacturer™s guide cIARSprobe ²TGTAAA TTA GAG GAG TGT CTG ATC TCC TCA AAGTC3² Negative control ²GCA GCC TGA TCA CGACTG ACT TTA GTG TTT GCA TT3²RNA EMSAThe LightShift Chemiluminescent RNA EMSA KitThermoFisher Scientific was applied to evaluate theinteraction between cIARS and ALKBH5 according to themanufacturer™s guidance The details were described inour as previous research34Observation of autophagy fluxLipid peroxidation assayAdmCherryGFPLC3 adenovirus Servicebio Technology Wuhan China was transfected into HCC cellsAfter h incubation cells were observed and photographed using a fluorescence microscope OlympusFSX100 Tokyo Japan Autophagic compartments werefinely observed through transmission electron microscopyTEM HT7700 HITACHI Tokyo Japan The identification of autophagic vacuoles in TEM images was mainlyThe relative level of MDA was evaluated through Lipidab118970 according to the manuPeroxidation Kitfacturer™s guidanceIron assayThe relative level of intracellular iron was determinedby an Iron Assay Kit ab83366 according to the manufacturer™s instructionsOfficial journal of the Cell Death Differentiation Association 0cLiu Cell Death Discovery Page of Glutathione assayThe relative level of intracellular GSH was assessedthrough a GSH Colorimetric Detection Kit CS0260SigmaAldrich St Louis USA according to the manufacturer™s instructionsStatistical analysisThe statistical analyses in this work were carried outusing Prism The results from at least three independenttests are shown as the mean value ± standard deviationSD The mean values of two groups were compared viaunpaired Student ttests œpvalue  was defined assignificant p p AcknowledgementsThis work was supported by the National Natural Science Foundation of China[Grant No ] Shandong Natural Science Foundation [Grant NoZR201808200282] and Medical and Pharmacological TechnologyDevelopment plan Shandong [Grant Nos 2017WS188 and 2017WS284]Author details1Department of Hepatobiliary Surgery The First Affiliated Hospital ofShandong First Medical University Jinan Shandong China2Department of Hepatobiliary Surgery Shandong Provincial QianfoshanHospital Shandong University Jinan Shandong China 3Department ofReproduction Medicine Jinan Maternal and Child Health Care HospitalAffiliated to Shandong First Medical University Jinan Shandong ChinaAuthor contributionsStudy design ZL XW and JL data collection QW ZX and XW dataanalysis ZL QW and XW cellular experiments QW ZL XW and ZXmanuscript preparation ZL QW ZX and JL figures and manuscriptproofing ZL QW JL and XW project administration XW and JLConflict of interestThe authors declare that they have no conflict of interestPublisher™s noteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsReceived May Revised July Accepted July References Bray F Global cancer statistics GLOBOCAN estimates of incidenceand mortality worldwide for cancers in countries CA Cancer J Clin “ Siegel R L Miller K D Jemal A Cancer statistics CA Cancer J Clin “ Forner A Reig M Bruix J Hepatocellular carcinoma Lancet “ Li J Ferroptosis past present and future Cell Death Dis Llovet J M Sorafenib in advanced hepatocellular carcinoma N Engl JMed “ Cheng A L Efficacy and safety of sorafenib in patients in the AsiaPacificregion with advanced hepatocellular carcinoma a phase III randomiseddoubleblind placebocontrolled trial Lancet Oncol “ Official journal of the Cell Death Differentiation Association Dixon SJ Pharmacologicalinhibition of cystineglutamateexchange induces endoplasmic reticulum stress and ferroptosis Elife e02523 Stockwell B R Ferroptosis a regulated cell death nexus linking metabolism redox biology and disease Cell “ Jung C H ULKAtg13FIP200 complexes mediate mTOR signaling to theautophagy machinery Mol Biol Cell “ Itakura E Mizushima N Characterization of autophagosome formation siteby a hierarchical analysis of mammalian Atg proteins Autophagy “ Geng J Klionsky D J The Atg8 and Atg12 ubiquitinlike conjugation systems in macroautophagy ˜Protein modifications beyond the usual suspects™review series EMBO Rep “ Zhou B Ferroptosis is a type of autophagydependent cell death SeminCancer Biol httpsdoi101016jsemcancer201903002 Torii S An essential role for functional lysosomes in ferroptosis of cancercells Biochem J “ Gao M Ferroptosis is an autophagic cell death process Cell Res “ Hou W Autophagy promotes ferroptosis by degradation of ferritinAutophagy “ Memczak S Circular RNAs are a large class of animal RNAs with regulatory potency Nature “ Bi W CircRNA circRNA_102171 promotes papillary thyroid cancer progression through modulating CTNNBIP1dependent activation of betacateninpathway J Exp Clin Cancer Res Su Y circRIP2 accelerates bladder cancer progression via miR1305Tgfbeta2smad3 pathway Mol Cancer Chen J Circular RNA profile identifies circPVT1 as a proliferative factorand prognostic marker in gastric cancer Cancer Lett “ Wang M Yu F Li P Circular RNAs characteristics function and clinicalsignificance in hepatocellular carcinoma Cancers httpsdoi103390cancers10080258 Glazar P Papavasileiou P Rajewsky N circBase a database for circular RNAsRNA “ Deng W Wang Y Liu Z Cheng H Xue Y HemI a toolkit for illustratingheatmaps PLoS ONE e111988 Dudekula D B CircInteractome A web tool for exploring circular RNAsand their interacting proteins and microRNAs RNA Biol “ Dixon S J Ferroptosis an irondependent form of nonapoptotic celldeath Cell “ Guo J Deregulation of UBE2Cmediated autophagy repression aggravates NSCLC progression Oncogenesis Zhu H A

Thyroid_Cancer

Neurogenesis From Neural CrestCells Molecular Mechanisms in theFormation of Cranial Nerves andGangliaKarla MndezMaldonado12  Guillermo A VegaLpez34  Manuel J Aybar34 andIv¡n Velasco15 Instituto de Fisiolog­a Celular “ Neurociencias Universidad Nacional Autnoma de Mxico Ciudad de Mxico Mexico Departamento de Fisiolog­a y Farmacolog­a Facultad de Medicina Veterinaria y Zootecnia Universidad Nacional Autnomade Mxico Ciudad de Mxico Mexico Instituto Superior de Investigaciones Biolgicas INSIBIO CONICETUNT SanMiguel de Tucum¡n Argentina Instituto de Biolog­a œDr Francisco D Barbieri Facultad de Bioqu­mica Qu­mica yFarmacia Universidad Nacional de Tucum¡n San Miguel de Tucum¡n Argentina Laboratorio de Reprogramacin CelularInstituto Nacional de Neurolog­a y Neurocirug­a œManuel Velasco Su¡rez Ciudad de Mxico MexicoThe neural crest NC is a transient multipotent cell population that originates in thedorsal neural tube Cells of the NC are highly migratory as they travel considerabledistances through the body to reach their final sites Derivatives ofthe NC areneurons and glia of the peripheral nervous system PNS and the enteric nervoussystem as well as nonneural cells Different signaling pathways triggered by BoneMorphogenetic Proteins BMPs Fibroblast Growth Factors FGFs Wnt proteins Notchligands retinoic acid RA and Receptor Tyrosine Kinases RTKs participate in theprocesses of induction specification cell migration and neural differentiation of the NCA specific set of signaling pathways and transcription factors are initially expressed inthe neural plate border and then in the NC cell precursors to the formation of cranialnerves The molecular mechanisms of control during embryonic development havebeen gradually elucidated pointing to an important role of transcriptional regulatorswhen neural differentiation occurs However some of these proteins have an importantparticipation in malformations of the cranial portion and their mutation results in aberrantneurogenesis This review aims to give an overview of the role of cell signaling and of thefunction of transcription factors involved in the specification of ganglia precursors andneurogenesis to form the NCderived cranial nerves during anogenesisKeywords cranial nerve peripheral nervous system hindbrain cell signaling transcriptional regulatory networktrigeminal nerve facial nerve vagus nerveAbbreviations BMP bone morphogenetic proteins CN cranial nerve CNS central nervous system DRG dorsal rootganglia FP floor plate hESCs human embryonic stem cells Msx Muscle segmentrelated homeobox NC neural crestNCCs neural crest cells NT neural tube PA pharyngeal arches Pax Paired box PNS peripheral nervous system rrhombomere RA retinoic acid RTK receptor tyrosine kinase Shh Sonic hedgehog Sox Srybox VSMC vascular smoothmuscle cells Wnt Wingless and Int1 WRPW TrpArgProTrp Zic Zinc finger protein of cerebellumEdited byMichael PiperThe University of QueenslandAustraliaReviewed byRoberto MayorUniversity College LondonUnited KingdomRebecca McLennanStowers Institute for MedicalResearch United StatesCorrespondenceManuel J AybarmanuelaybarfbqfunteduarIv¡n Velascoivelascoifcunammx These authors have contributedequally to this workSpecialty sectionThis was submitted toStem Cell Researcha section of the journalFrontiers in Cell and DevelopmentalBiologyReceived April Accepted June Published August CitationMndezMaldonado KVegaLpez GA Aybar MJ andVelasco I Neurogenesis FromNeural Crest Cells MolecularMechanisms in the Formationof Cranial Nerves and GangliaFront Cell Dev Biol 103389fcell202000635Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alINTRODUCTIONDuring the embryonic development of vertebrates one of themain events after the gastrulation process is neurulation whichallows the formation of the neural tube NT The neuralectoderm generates not only the central nervous system CNSbut also another set of cells between the NT and the nonneural ectoderm located in the most dorsal part of the NT calledthe neural crest NC Hall SimµesCosta This versatile and plastic cell population was first described byWilhelm His years ago Hall The NC is one of themost important features that separate vertebrates from otherchordate anisms It arises at the posterior and lateral bordersof the neural and nonneural ectoderm the neural plate borderFigure Cerrizuela the embryo NCCs are divided into cranialNC cells NCCs are multipotent and give rise to several celltypes depending on the site of origin along the anteroposterioraxis oftrunkincluding cardiac vagal and sacral A Minouxand Rijli SimµesCosta and Bronner VegaLopez Cranial nerves CN transmit sensory and motorinformation between the brain and tissues of the head andcervical region The CN are formed from the contribution oftwo specialized embryonic cell populations cranial NC andectodermal placodesOrigin of the Neural CrestNCCs which are multipotent delaminate from their origin andmigrate throughout the body to diï¬erentiate into several celltypes including cells of the peripheral nervous system PNSmelanocytes cranial cartilage and bone neuroendocrine cellsand several other phenotypes B In humans at least cell types have been defined as NC derivatives Vickaryousand Hall Proper NC migration relies on environmentalcues such as EphEphrins Smith Semaphorin3F Gammill Versican Szab thechemokine Stromal cellderived factor Theveneau or Robo2 Shiau The migration patternsof NCCs have been clearly described for model anisms likebirds frogs and mice In all vertebrates cranial NCCs emergefrom the forebrain midbrain and hindbrain regions Coulyand Le Douarin Serbedzija Depending ontheir axial origin cranial NCCs will either migrate through thefacial mesenchyme and into the frontonasal process or willpopulate the branchial arches Noden Lumsden Serbedzija The sensory module of the PNSin the cranial region is composed of an array of paired gangliaadjacent to the hindbrain that transduce the perception of touchpain temperature position and special sensory informationfrom the periphery to the CNS Cranial NCCs migrate to formsensory ganglia such as the trigeminal V the facial VII theglossopharyngeal IX the vagus X CN and also to form themotor ganglia for the oculomotor III and accesory XI CNTable and Figures acomplex and multistep processinitially directed by cell signaling molecules including BoneMorphogenetic Proteins BMPs Wnts Wingless and Int1NC formation isNeural CrestDerived Cranial NervesFibroblast Growth Factor FGF and retinoic acid RA Thesesignals reveal the tissue interactions into the ectodermal cellpopulations the neural plate the nonneural ectoderm and theunderlying mesoderm in a highly coordinated manner VegaLopez It has been proposed that NC specificationoccurs during gastrulation as a consequence of the action oftwo successive gradients of secreted signals A combinationof intermediate levels of activity of BMP and Wnt signalingacting on the ectoderm to induce and specify NC precursorsat the neural plate border and a subsequent requirement ofboth signals is needed for maintenance of specification duringneurulation Aybar and Mayor Steventon Inchick embryos it was shown that NCCs are specified as earlyas the blastula stage Prasad It was demonstratedthat during gastrulation Pax7 expression is restricted to cellslocated in a region in the medial epiblast which are NCfated andcontribute to the neural folds and later to migrating NCCs Basch The inhibition of Pax7 function in chicks inhibitedthe expression of key NC markers such as Snai2 OMIM Sox9 Srybox OMIM Sox10 OMIM andHNK1 beta13glucuronyltransferase like OMIM Basch and BronnerFraser This evidence suggests thatthe neural plateprospective ectoderm interaction at the neuralplate border might not be a requisite for NC specification orinduction and that neural plate border formation and NCinduction might be separable eventsThe variousresearch works carried outto study theorigin of NCCs have identified genes anized into a generegulatory network that participate in and control the inductionspecification and diï¬erentiation of NC SimµesCosta An example of this are the transcription factors involvedin induction such as FoxD3 Forkhead Box D3 OMIM Snai2 and Sox9 SaukaSpengler and BronnerFraser Garc­aCastro and coworkers identified a novel preneuralborder state characterized by early Wntβcatenin signalingtargets that displayed diï¬erent responses to BMP and FGFsignaling from the neural border genes in human cells Leung These preborder genes Gbx2 Gastrulation brainhomeobox OMIM SP5 OMIM Zic3 OMIM and Zeb2 OMIM had their induction andpeak of expression before the classical neural plate borderspecifier genes such Msx12 Muscle segmentrelated homeobox OMIM Pax37 OMIM and Zic1 OMIM Such specifier genes together withsignaling molecules direct the expression of NCspecific geneslike AP2 OMIM FoxD3 Snai2 Sox9 and Sox10Specifiers regulate NC eï¬ector genes involved in migrationSox9 Sox10 Cad7 and diï¬erentiation [Col1a Collagen typeI alpha OMIM Ngn1 Neurogenin OMIM MitfMicrophthalmiaassociated transcription factor OMIM] in human NC development Betters The NC population migrates to diï¬erent regions of the mouseembryo from the NT after the epithelialmesenchymal transitionmaintaining its multipotential character until completingdiï¬erentiation in its final destination Baggiolini To study the ontogeny of the NC diï¬erent model anismsboth in vivo and in vitro have been used Several proteinsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Neural crest origin regions in human and mouse embryos and some of its cranial derivatives A The topleft part of the scheme shows the origin of theneural crest cells green that migrate through the embryo On the topright side the level of axial origin see axial color key of different regions of the neural crest isrepresented in developing mouse or equivalent human embryos the migration of neural crest is represented in green inside the embryos and the direction ofmigration is marked with black arrows Depending on their axial level of origin and migratory pathways neural crest cells adopt different fates and contribute tovarious tissues and ans B The main cranial derivatives labeled in green are shown Abbreviations d days E mouse embryonic stage NCCs Neural CrestCells s somite St human stage VSMC vascular smooth muscle cellsincluding transcription factors as well as epigenetic modifiersthat take part in the specification and diï¬erentiation of the NChave been described The study of transcription factors and ofthe signaling pathways in which they participate is importantto understand the diï¬erentiation programs and how thesemultipotent cells are committed to a specific destination On theother hand transcriptome analysis during the development of theNC from specification to migration Meulemans and BronnerFraser and a more recent study covering the migrationto the diï¬erentiation of the NC show the importance of theinteraction between the diï¬erent transcription factors and thesignaling pathways at every stage of NC development SimµesCosta However these authors acknowledge thatit is difficult to have a complete global map since only a fewtranscriptional regulators have been characterized and little isknown about the function of the products of the eï¬ector genesacting on NC migration Betancur SimµesCosta andBronner VegaLopez NC and cranial placodes are thought to appear togetherduring the evolution of vertebrates to give rise to specific sensorystructures of the head Northcutt and Gans Northcutt The components of the sensory nervous system of the headare derived from the NC and from an embryonic cell populationdeveloping in close proximity the cranial sensory placodes theolfactory lens otic trigeminal epibranchial and paratympanicplacodes A series of events induce develop and anizethese cell precursors which through reciprocalinteractionswith NCCs build the functional sensory system in vertebratesSteventon Singh and Groves Migrating NCCsarrive first at the site of ganglia development ie the trigeminalFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cFrontiersinCellandDeveopmentallliBoogywwwfrontiersniAugustlVoumeAlrticeTABLE Contributions of neural crest cells and placodes to ganglia and cranial nervesCranial nerveGanglion and typeOrigin of neuronsReferencesCNI “ Olfactory Ensheating gliaof Olfactory nervesCNIII “ Oculomotor mCiliary visceral efferentCNV “ Trigeminal mixTrigeminal general afferentCNVII “ Facial mixSuperior general and special afferentCNVIII “ Vestibulocochlear sCNIX “ Glossopharyngeal mixCNX “ Vagus mix Superiorlaryngeal branch and recurrentlaryngeal branchCNXI “ Accessory mInferior geniculate general and special afferentSphenopalatine visceral efferentSubmandibular visceral efferentAcoustic cochlear special afferent and Vestibularspecial afferentSuperior general and special afferentInferior petrosal general and special afferentOtic visceral efferentSuperior general afferentInferior nodose general and special afferentVagal parasympathetic visceral efferentNo ganglionTelencephalonolfactory placode NCCs at forebrainNCCs at forebrainmidbrain junction caudal diencephalonand the anterior mesencephalonNCCs at forebrainmidbrain junction from r2 into 1st PAtrigeminal placodeHindbrain NCCs from r4 into 2nd PA 1st epibranchialplacode1st epibranchial placode geniculateHindbrain NCCs 2nd PAHindbrain NCCs 2nd PAOtic placode and hindbrain from r4 NCCsHindbrain NCCs from r6 into 3rd PA2nd epibranchial placode petrosalHindbrain NCCs from r6 into 3rd PAHindbrain NCCs from r7r8 to 4th and 6th PAHindbrain NCCs 4th and 6th PA 3rd nodose and 4thepibranchial placodesHindbrain NCCs 4th and 6th PAHindbrain from r7r8 to PA NCCs 4th PABoyd Muller O™Rahilly andM¼ller Barraud Noden Couly Wahl Lee d™AmicoMartel and Noden Forbes and Welt D™amicoMartel and Noden D™amicoMartel and Noden Lumsden Barlow and Northcutt Begbie andGraham Barlow Krimm Sandell Narayanan and Narayanan D™amicoMarteland Noden O™Rahilly and M¼ller Barlowand Northcutt Narayanan and Narayanan D™amicoMarteland Noden Muller and O™Rahilly O™Rahilly and M¼ller Abbreviations CN Cranial Nerve m purely motor nerve mix mixed nerve sensory and motor NC neural crest PA pharyngeal branchial arch r rhombomere s purely sensory nerve There is no known ganglionof the accessory nerve The cranial part of the accessory nerve sends occasional branches to the superior ganglion of the vagus nerveMndezMadonadoletalNeuralCrestDerivedCranailNerves\x0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Contribution of neural crest cells to the formation of cranial nerves I III V VII VIII IX X and XI These selected cranial nerves are formed by thecontribution of cranial placodes and neural crest cells indicated in green Neural crestderived Schwann cells produce peripheral myelination of cranial nerves III“XIIThe sensory nerves are the olfactory I the optic II and the vestibulocochlear VIII The motor nerves are the oculomotor III the trochlear IV the abducens VIand the accessory XI The remaining are mixed nervesganglion but the diï¬erentiation of these cells is delayed untilthe migration and diï¬erentiation of the corresponding placodalcells in chicks Covell and Noden Placodal specificationand development as well as its contribution to the assembly ofplacodal derivatives is a complex and wideranging topic thatis beyond the scope of this review We will focus on discussingthe main signaling pathways and relevant transcription factorsinvolved in the specification of cranial NCCs precursors theirdiï¬erentiation to form CNs and ganglia that are exclusively NCderived and the alterations caused by the mutations of certaingenes that are important for the neurogenesis of NC derivativesSIGNALING PATHWAYS INVOLVED INCRANIAL NEURAL CRESTDEVELOPMENTareseveralsignaling pathwaysThereand transcriptionfactors that are known to regulate NC and CN formationduring development We discuss some important pathwaysinvolved in cranial NCCs induction and specification in closerelationship with the cranial ganglia and nerves derived from theNC Figure BMPsBone morphogenetic proteins are proteins that control severalimportant steps in the formation and diï¬erentiation of the CNSof vertebrates These proteins act in diï¬erent regions of theCNS to regulate fate proliferation and diï¬erentiation Aftergastrulation the presence of BMPs and the activation of thissignaling pathway are essential for the diï¬erentiation of thenonneural ectoderm whereas the inhibition of this pathway isrequired for the proper formation of the neural plate It has beenproposed that the later activation of BMPs receptors participatesin the induction of the NC through a very fine regulation wherethe presence of BMPs at a specific time will give rise to the NCin mouse and human Embryonic Stem Cells ESCs Figure 3BMizuseki Leung Seminal studies in Xenopus have shown that there is an activitygradient of BMPs controlled by their antagonists and that anintermediate level is needed to induce the formation of the NCLaBonne and BronnerFraser Marchant Barth Tribulo Thus the BMP antagonistsChordin OMIM and Noggin OMIM areexpressed in a spatiotemporal manner thatinfluences theformation of the NC In mouse at embryonic day E Nogginis expressed in the neural folds and in the dorsal region afterthe closure of the NT The expression of Chordin is low atthe level of the neural plate and in the paraxial mesodermThese antagonists participate in the induction of NC as wellas in delamination but also protect from apoptosis induced byBMP during migration and diï¬erentiation of NCCs Importantlyit was observed that the decrease in the expression of theseBMP antagonists alters the PNS derived from the NC andcraniofacial skeletal elements Noggin knockout mice presentedall cranial nerves but the vagus X and glossopharyngeal IX aredisanized and fused Doubleknockout mice of Noggin andChordin lack CN and only a structure similar to the trigeminalganglion V is present Anderson In the chickembryo the activity of BMP signaling during the formation ofNC precursors is modulated by CKIPSmurf factors throughthe regulation of Smad degradation resulting in intermediateFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesFIGURE Gene regulatory network involved in neural crest contribution to the formation of cranial nerves The cranial ganglia and cranial nerves are formed inprecise positions along the dorsoventral and anteroposterior axes of the midbrainhindbrain region A The drawing represents a human embryo at stage days somites equivalent to mouse day E9510 somites and chick stage h somites The cell signaling pathways that providedevelopmental cues to neural crest precursors are colorcoded when these factors diffuse the target regions are indicated with arrows with the same color In panelB an idealized scheme of the hindbrain shows the cell signaling gradients and the genes that establish the dorsoventral pattern C The illustration of the human days stage and chick stage hindbrain rendered flat to eliminate cerebral flexures The levels of origin of the neural crest cells NCCs and placodeswhich contribute to the formation on cranial nerves are indicated on the left NCCs from the corresponding rhombomeres also populate other embryo structures in asegmental fashion and generate different craniofacial derivatives The positions of the cranial ganglia and the otic vesicles are indicated on the right side thecontribution of NCCs is indicated in green The segmental nested expression of HOX genes is colorcoded On the right signaling pathways and the expression oftranscription factors involved in cranial nerve CN formation are indicated Adapted from Lumsden and Keynes Noden Yamamoto and Schwarting BallyCuif and Wassef Takahashi and Osumi and M¼ller and O™Rahilly Abbreviations CN cranial nerve FP floor plate Mmesencephalon NCCs neural crest cells OV otic vesicle r rhombomere PA pharyngeal archeslevels of BMP activity required for proper NC formationPiacentino and Bronner In contrast placode progenitorshave diï¬erential BMP signaling requirements as they can bespecified under low or no BMP signaling Thiery A study of human ESCs hESC showed that if BMPs areblocked with Noggin for h on days or of thediï¬erentiation protocol there is a dramatic decrease in theinduction of human NCCs However if the inhibition is madeon day the inhibition is partial so the participation of BMPsat the beginning of the induction of the NC is very importantwhile the inhibition of this pathway promoted the expressionof neural genes such as SOX1 OMIM HES5 OMIMFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial Nerves and SOX2 OMIM Leung Thisprotocol produced sensory peripheral neurons and it will beof interest to investigate if such neurons can contribute to thesensory CN after grafting them in experimental animals aswell as the eï¬ect of modulating BMPs on peripheral neurondiï¬erentiation Interestingly BMP antagonism upregulates theseneural stem cell markers but several reports indicated that Sox1Hes5 and Sox2 are involved in the suppression of neuronaldiï¬erentiation by maintaining neural stem and progenitorcells in an undiï¬erentiated state in mammalian cells Kan BaniYaghoub The generation ofneurons from stem cells depends on the decrease of Sox13expression caused by proneural proteins However if Sox13target genes were repressed independently of proneural activityneural progenitor cells diï¬erentiated prematurely and someneuronal features emerged These results demonstrate a dualrole of proneural proteins in the acquisition of a definitiveneuronal fate and indicate that the proneural proteindirectedrepression of Sox13 expression is a required and irreversiblestep in the commitment to neuronal diï¬erentiation in severalspecies including mammals Guillemot Farah Bylund BMP4 OMIM and Smad proteins have beeninvolved in an interesting mechanism called retrograde signalingin trigeminal ganglia from rats Ji and Jaï¬rey Thismechanism elicits a specific transcriptionalresponse thatcontributes to the specification of diï¬erent subpopulations ofsensory neurons in the trigeminal ganglia CN V As axonsfrom the neurons oftrigeminal ganglia grow and extendinto their three main peripheral axonal branches ophthalmicmaxillary and mandibular that innervate the correspondingregions of the face they encounter BMP4 which results in aretrograde signal that leads to transport back transcription factorsSMAD1 and from axons to the somata where nuclearaccumulation of the phosphorylated and transcriptionally activeSmad forms contributes to neuronal specification and gangliapatterning Nohe Ji and Jaï¬rey BDNF BrainDerived Neurotrophic Factor OMIM signaling wasalso found to regulate axonal levels of SMAD1 and inconcert with BMP4 for patterning of the trigeminal gangliaJi and Jaï¬rey Hippo PathwayGenetic studies have demonstrated that Hippo signaling is crucialin an size regulation controlling cell number by modulatingcell proliferation and apoptosis processes Huang Hippo is a criticalfactor for proliferation and epithelialmesenchymal transition during embryonic development andcancer In the neural tube of the mouse chicken and frogYAP YesAssociated Protein OMIM is expressed inthe ventricular zone progenitor cells and colocalizes with theneural progenitor cell marker Sox2 Milewski Cao It has been observed that the ectopic expressionof one ofthis pathwayTAZ Transcriptional Coactivator With PDZBinding MotifOMIM in mammalian cells stimulates cell proliferationthe transcriptional regulators ofreduces the inhibition by contact and promotes the epithelialmesenchymal transition Lei A relationship between this signaling pathway and the classicalNC genes such as interaction with Pax3 is through TAZ andthe phosphoprotein YAP65 These proteins participate as coactivators of Pax3 It has been suggested using transgenicmice that Tead2 TEA Domain Family Member OMIM is an endogenous activator of Pax3 in NCCs Milewski Through expression assays Pax3 and Yap65were colocalized in the nucleus of NC progenitors in thedorsal region offorSchwann cell proliferation and diï¬erentiation in a stagedependent manner Nuclear TAZYAP complexes activate cellcycle regulators to promote Schwann cell proliferation whiledirecting diï¬erentiation regulators in cooperation with Sox10 formyelination in rodents Deng the NT HippoTAZYAP are criticalNeurofibromatosis Nf2 OMIM is a tumorsuppressor that inhibits YAP during dorsal root ganglia DRGdevelopment Merlin encoded by the NF2 tumorsuppressivegene was identified through genetic studies in mouse embryosand proved to be an important upstream regulator of theHippoYap pathway Neurofibromatosis is an inherited diseasecharacterized by the development of bilateral Schwann celltumors originated from CN VIII Mouse with specific Schwanncellinactivated Nf2 alleles developed schwannomas and SChyperplasia McClatchey Giovannini Merlin has also been shown to act as a suppressor ofmouse neural progenitor proliferation by inhibiting TAZYAPpathway activity Lavado The mechanism bywhich Merlin regulates YAP activity might involve p21 Proteinactivated kinase PAK1 OMIM activation whichinduces phosphorylation of Merlin thus abrogating its scaï¬oldfunction for YAP and LATS12 OMIM andthereby attenuates YAP phosphorylation by LATS12 in mousecells Sabra it has been suggested that nuclearexport signals of Merlin mediate YAP nuclear export in epithelialmammalian cells Furukawa Hindley and coworkers investigated the role of HippoYAPsignaling in NC development and neural diï¬erentiation Theyshowed thatthe activity of YAP promotes an early NCphenotype accompanied by premature migratory behavior andthat HippoYAP interacts with RA signaling in hESCs Hindley A recent study demonstrates that YAP is necessaryfor the migration of a premigratory pool of NCCs sincethey incorporated YAP signaling into a BMPWntdependentmolecular network responsible for the migration of trunklevelNC in avians Kumar Notch SignalingNotch is a family of conserved receptors whose activation isinduced by specific ligands Delta1 OMIM Delta OMIM Delta4 OMIM Jagged1 OMIM and Jagged2 OMIM through interactionwith four possible receptors Notch14 Perdigoto and Bardin Once the Notch receptors are activated through thecellcell interaction proteolytic cuts are carried out resultingin the release ofthe Notch Intracellular Domain NICDFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cMndezMaldonado et alNeural CrestDerived Cranial NervesMumm NICD translocate to the nucleus andforms a transcriptional complex together with the DNA bindingprotein CBF1 C promoter binding factor OMIM This complex recognizes the specific sequence CTGTGGGAAin itstarget genesfor example Hes1 OMIM Kageyama Notch1 receptor is present during development oftherhomboencephalon at E95 in mice showing strong expressionwithin the hindbrainincluding the trigeminal geniculatepetrosum and nodose placodes which give rise to CN VVII IX and X respectively and is also expressed in the oticand olfactory vesicle Reaume A study wherehuman induced pluripotent stem cells were induced toward NCdiï¬erentiation showed that when Notch signaling is blockedusing a Îsecretase inhibitor DAPT or shRNA for JAGGED the genes specifying NC [DLX5 Distalless homeobox OMIM PAX3 SNAI2 SOX10 and TWIST1 OMIM] are downregulated However the ectopic expression ofNICD1 increased its expression demonstrating that Notch alsoparticipates significantly in NC induction Noisa Mead and Yutzey evaluated the function of Notch signaling inmurine NCderived cell lineages in vivo They demonstratedthat cellautonomous Notch has an essential role in properNCCs migration proliferation and diï¬erentiation with criticalimplications in craniofacial cardiac and neurogenic developmentMead and Yutzey Sonic HedgehogSonic Hedgehog Shh signaling is involved in the correctdevelopment of NC and therefore in the generation of its cellularderivatives Figure 3B Shh is a member of the family ofthe secreted Hedgehog proteins Sonic Shh OMIM Indian Ihh OMIM and Desert Hedgehog Dhh OMIM Shh regulation during NC diï¬erentiation is crucialduring head and face morphogenesis Mutant mice and humanslacking Shh present holoprosencephaly and cyclopia due to thelack of separation of the forebrain lobes Chiang It is suggested that Shh inhibition maintains Pax3 expressionso the lack of Shhmediated regulation for Pax3 inductionpromotes the constitutive induction of NC generating theaforementioned phenotypes A subset of Fox genes regulated byShh signaling is important during lip morphogenesis in miceEither Shh addition or Foxf2 OMIM overexpressionwas shown to be sufficient to induce cranial NCCs proliferationEverson On the other hand enhanced Shh signaling in mousemediated by lossoffunction Ptch1WigWig of the Shh receptorPatched1 Ptch1 OMIM suppressed canonical Wntsignaling in the CN region This critically aï¬ected the survivaland migration of cranial NCCs and the development ofplacodes as well as the integration between NC and placodesKurosaka Ptch1WigWig mutants exhibited severelydisanized trigeminal CNV and facial nerves CNVII thatdid not develop properly and failed to project to their appropriatetargettissues Kurosaka High levels of Shhsignaling have been correlated with Moebius Syndrome whichis characterized by cranial nerve defects including trigeminalabducens CNVI and facial alterations concurrent with othercraniofacial defects Verzijl VegaLopez NCCs migration is particularly sensitive to Shh levels since inmice lacking Shh these cells continue their migration beyondthe normal position and fuse medially condensing into a singlemidline ganglion Fedtsova Mutation in the mouseHedgehog acyltransferase Hhat OMIM gene producedhypoplasia and aberrant fusion of cranial ganglia CN V VII IXand X and aï¬ected NC and placode gene markers expressionsuggesting that a regionalized action of the Hedgehog signalingis required for proper cranial ganglia and nerve developmentand patterning Dennis In vitro analyses showedthat Shh increased the number of cranial NC progenitors fromquail embryos yielding neural and mesenchymal lineages Shhcan decrease the neuralrestricted precursors without aï¬ectingsurvival or proliferation These data also suggestthemesenchymalneural precursor was able to yield both the PNSand superficial skeleton Calloni thatReceptor Tyrosine Kinase RTK FamilyHumans have known RTKs which fall into subfamiliesA few years ago a systematic work summarized the contributionof the mouse model to the understanding of the role of a subsetof RTKs in regulating the activity of NCCs in developmentFantauzzo and Soriano With respect to its downstreamsignaling RTKs induce the activation of various pathwaysincluding PLCÎ PI3K MAPK JNK Shc Erk and the JAKSTATpathways In this section we discuss insights pointing tomechanisms of action of some RTK families in relation tothe development of the cranial NC that have emerged fromrecent evidenceEph ReceptorsEphrin ligands and Eph erythropoietinproducing humanhepatocellular carcinoma receptors comprise an increasinglywell studied family of signaling molecules Ephrins bind totwo families of transmembrane tyrosine kinase receptors EphAand EphB While Atype Ephrins preferentially bind to EphAreceptors Btype Ephrins do so to EphB receptors In Xenopusthe streams of NCCs going to the second branchial arch expressEphrinB2 whereas cells reaching the third arch express EphB1disruption of EphEphrin signaling results in aberrant migrationof NCCs causing mixing of the streams in the branchial pouchesSmith Eph receptor functions are best characterizedin the mouse nervous system where they are involved inneuronal development and axon guidance Wilkinson Xuand Henkemeyer migration and proliferation Conover H

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

This study aimed to investigate serum matrix metalloproteinase MMP2 and MMP9levels in patients with papillary thyroid carcinoma PTCMethods Fortyone patients with PTC undergoing ultrasoundguided radiofrequency ablationRFA and controls were included Serum MMP2 and MMP9 levels were determined byenzymelinked immunosorbent assay before and after surgery Potential affecting factors wereevaluated by logistic regression analysisResults Serum MMP2 and MMP9 levels were significantly higher in PTC patients comparedwith controls and decreased significantly after surgery According to receiver operating characteristic curve analysis diagnostic values for preoperative serum MMP2 and MMP9 levels were and There was no contrastagent perfusion in the ablation zone in of lesionsand enhancement within or at the lesion edge in The volume reduction at months™followup was Age microcalcification irregular shape and lesion diameter and numberwere influencing factors for PTC Age and lesion diameter and number were independent riskfactors while calcification and morphology were protective factorsConclusion Serum MMP2 and MMP9 levels have important clinical values for the diagnosis andtreatment of PTC by RFA Preoperative serum MMP2 and MMP9 levels combined with otheraffecting factors contribute to disease prognosisDepartment of Ultrasound Beilun People™s Hospital ofNingbo Beilun Branch of the First Affiliated Hospital ofZhejiang University Ningbo Zhejiang ChinaThese authors contributed equally to this studyCorresponding authorQian Ding Department of Ultrasound Beilun People™sHospital of Ningbo Beilun Branch of the First AffiliatedHospital of Zhejiang University No East LushanRoad Ningbo Zhejiang ChinaEmail qianding02sohucomCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cJournal of International Medical ResearchKeywordsPapillary thyroid carcinoma radiofrequency ablation matrix metalloproteinase2 matrix metalloproteinase9 disease prognosis serum levelDate received June accepted March IntroductionPapillary thyroid carcinoma PTCis acommon thyroid malignancy accountingfor about of systemic malignancies1PTC is currently characterized by a highincidence and low mortality2clinicalRecentimprovementsin highfrequencyultrasonic diagnostic technologies and theapplication of ultrasoundguided puncturetechniques have led to an apparent increasein the incidence of PTC year by year3“Regardingthyroidtumors the palpation detection rate forthyroid microtumors in the general population is about compared with as highas to for highfrequency ultrasound6 which has thus greatly improveddisease diagnosisdiagnosistheofSurgical resection is a routine treatmentfor thyroid tumors but the recurrence rateis usually high and the consequent reduction in thyroid function can seriously affectthe patient™s quality of life7 Associatedwith the increasing detection rate of thyroidtumors and the pursuit of minimally invasive treatments radiofrequency ablationRFA has been gradually applied in theclinic RFA uses local hyperthermia tocause tissue necrosis The thermal effectsdo not depend on the tissue type andmost lesions can be completely eliminatedby RFA89 RFA has thus become a novellocal treatment for tumorsThe clinical diagnosis of benign andmalignant thyroid tumors currently dependson the clinical manifestations and pathological examinations Howeverthe clinicalmanifestations are mostly derived frominvolvessubjective empirical analysis while a finalpathological diagnosisinvasiveprocedures with lesssatisfactory specificity It is therefore necessary to identifyappropriatepredictivetumor markersdiagnosticandforimplicationsThe collagenases matrix metalloproteinase MMP2 and MMP9 can degradetype IV collagen in the basement membranewith importanttumorangiogenesis and tumor cell invasion andmetastasis10 MMP2 and MMP9 expression levels were found to be upregulatedin thyroid cancer tissue11 however thesestudies mostly examined pathological tissuesafterinvasive surgery and lessinvasivemeasures such as serum levels of MMP2and MMP9 have been lesswell consideredIn this study we detected serum levels ofMMP2 and MMP9 in patients with PTCbefore and after ultrasoundguided RFAWe also determined the therapeutic effectsof RFA during the followup period andinvestigated the relevant prognostic factorsMaterials and methodsStudy subjectsPatients who underwent ultrasoundguidedRFA in our hospital from May toOctober were included in this studyThe inclusion criteria were as follows patients diagnosed by preoperative fineneedle aspiration cytology no historyof neck surgery and patients requiringminimally invasive treatment for aestheticreasons and because of neck oppression 0cPan et alwith anxiety The exclusion criteria were asfollows benign lesions confirmed by fineneedle aspiration cytology history ofneck surgery and severe coagulopathyPeripheral venous blood samples wereobtained from the included patients beforeand at and months after the operation and serum levels of MMP2 andMMP9 were determined Additional subjects with confirmed benign thyroid noduleswithout RFA were included as a controlgroup Prior written informed consent wasobtained from all patients and the studywas approved by the ethics review boardof our hospitalPreoperative preparationcalcificationThe number size nature echo boundarymorphologysurroundinghalo and nodular blood flow distributionof the tumors were assessed before the operation After skin disinfection local anesthesia was performed with lidocainesolution A total of mL Sonovue Bracco Milan Italy was injected via the elbowvein and the bloodsupply characteristicswere then evaluated by contrastenhancedultrasound CEUS of the ablationtargetedlesions using a Mylab90 ultrasonic devicewith 10MHz probe Esaote ShenzhenGuangdong ChinaAccording to the location of the thyroidnodules the thyroid and carotid space thyroid and tracheal space thyroid and esophageal space and posterior thyroid spacerecurrent laryngeal nerve were separatedusing a saline and lidocaine mixture basedon the intraoperative conditions to form aliquid separation zone to protectthesestructures from thermal damageAblation treatmentUnder ultrasound guidancethe tip ofthe RFA needle rated power Woutput frequency kHz was accuratelypenetrated into the nodule and RFA wasperformed using an OlympusCelon PowerRFA System Germany in mobile mode12following the fromdeeptoshallow principle The lesions were subjected to multipointed and multifaceted ablation untilthe thyroid tissue layer with the noduleswas completely covered by the strong echogenerated by heat accumulation The wholeprocess was carried out under continuousultrasound monitoring A highecho areawas produced in the ablation zone duringthe ablation treatment The position of theelectrode needle was gradually adjustedaccording to the lesion size After ensuringthat there was no residual enhancement inthe ablation zone the ablation needle wasremoved and the ablation was completedAfter the operation an ice compress wasapplied for h to avoid skin burnsSerum MMP determinationFor all subjects mL venous blood wascollected from the elbow vein under fastingconditions before and after the operationrespectively Blood samples in the controlgroup were collected after ultrasound contrast examination The blood samples wereplaced at room temperature for minutesand then subjected to centrifugation at 02 g for minutes The serum washarvested and serum levels of MMP3 andMMP9 were determined using enzymelinked immunosorbentELISAkitsBoster Bioengineering WuhanHubei ChinaassayFollowup and efficacy evaluationImmediately after the operation the ablation range was evaluated by CEUS If residualtissues were detected ablation wasrepeated immediately Ultrasound detectionwas performed at and monthsafter surgery to determine the nodule sizesand volumes The volumereduction rate 0cwas calculated according to the followingformula volume reduction rate¼ preoperative volume followup volumepreoperative volume 02 Echo and bloodflowchanges in the ablation zone were alsoobserved and analyzed The efficacy wasdetermined based on the criteria for RFAfor treating tumors13 disappearance ofnodules indicated by complete disappearance of blood flow confirmed by ultrasonography indicated complete cure noduleby 15 indicatedvolumemarked effect and nodule volume reducedby to indicated improvementreducedClinicopathological featuresInformation on ultrasoundbased clinicopathologicalincluding numbersize and calcification of the lesion wereobtainedfeaturesversion Statistical analysisData were expressed as mean 06 standarddeviation Statistical analysis was performed using IBM SPSS StatisticsforWindowsIBM CorpArmonk NY USA Comparisons betweengroups were performed using v2testsPotentially related factors were analyzedby univariate or multivariate logistic regression The prognostic predictive effects ofserum MMP2 and MMP9 levels wereevaluated by receiver operating characteristic ROC curve analysis P was considered statistically significantResultsPatientsJournal of International Medical Research men mean age 06 years range“ yearsSerum MMP2 and MMP9 levels beforeand after treatmentThe characteristics of the ultrasound images inthe included subjects are shown in Table Serum levels of MMP2 and MMP9 weremeasured before and after treatment Serumlevels of MMP2 and MMP9 were significantly higher in patients with PTC comparedwith the control subjects P Serumlevels of MMP2 and MMP9 had declinedat month after the operation comparedwith before surgery but the difference wasnot significant However serum levels ofMMP2 and MMP9 had declined significantly in the PTC patients at and monthsall P Table These results suggestthat changes in serum MMP2 and MMP9levels between before and after surgery mayhave significant implications for the therapyof PTCROC curve analysis of preoperative serumMMP2 and MMP9 levelsPreoperative serum MMP2 and MMP9levels were used as potential diagnostic indicators In the patients with PTC the predictive probability from theregressionmodel was used as the diagnostic resultsand the gold standard classification criteriawere used as the pathological results ROCcurves were obtained accordingly The areaundercurve AUC values for serum MMP and MMP9 levels were and respectively Figure These results suggestthat serum levels of MMP2 and MMP9could contribute to the disease diagnosisFortyone patients with PTC lesionswere enrolledincluding women and men mean age 06 years range to þ65 years The control group included patients with confirmed benignthyroid nodules including women andEvaluation of RFA efficacyWe also evaluated the efficacy of RFA CEUSof the lesions before ablation showed hypoenhancement in nodules isoenhancementin nodules and slight hyperenhancement 0cPan et alTable Characteristics of thyroid ultrasound imagesPTC patientsNormal controlLesion numberOneTwoMultipleLesion size 14 cm cmCalcificationMicrocalcificationCoarse calcificationMorphologyRegularIrregularAge years 15 yearsnP compared with the control groupnTable Serum matrix metalloproteinase2 and levels in controls and inpatients with papillary thyroid carcinoma before and after treatmentControlsPTC patientsBefore surgery month after surgery months after surgery months after surgery months after surgeryMMP2 06 06 06 06 06 06 P“MMP9P“ 06 06 06 06 06 06 MMP matrix metalloproteinase PTC papillary thyroid carcinomain nodules Ultrasound examination afterablation showed no contrastagent perfusionin the ablation zone in lesions and enhancement of different degrees at theedge or inside the lesion in the other lesions and the ablation area wasgradually reduced with prolonged ablationFigure There was no significant changein ablation volume in any patients at month after surgery compared with beforesurgery However the volume was reducedby at months offollowupcompared with before surgery P Table These results showed that RFAtreatmentthetumor volume in patients with PTCcould effectively reduceInfluence of relevant factors on diseaseprognosisTheclinical data of patients beforeRFA were retrospectively analyzed by 0cJournal of International Medical Researchirregularlogistic regression to identify factors thatmay affectthe disease prognosis Agemicrocalcificationshape anddiameter and number of lesions were significant influencing factors for PTC P The hazard ratios HRsfor age andlesion diameter and number were indicating that these represented independentrisk factors In contrast the HRs for microcalcification and irregular shape were negativeindicating that a greater degree ofcalcification and regular shape were associated with lower risks of developing the disease and were thus protective factorsTable Figure ROC analyses of serum MMP2 andMMP9 levels MMP matrix metalloproteinaseFigure Efficacy evaluation of radiofrequency ablation a Twodimensional 2D ultrasound showingobvious bloodflow signals around the tumor and fewer signals within the tumor b Preoperative contrastenhanced ultrasound showing no obvious enhancement in the lesion with lowperfusion performance c Inthe 2D imaging localization the ablation needle was inserted into the tumor for ablation d The tumor wascompletely ablated with no bloodflow signal at year after ablation 0cPan et alDiscussionitis difficultPTC is a type of thyroid tumor with a highincidence14 which has been increasingrapidly worldwide1516 Mostthyroidtumor cases are currently diagnosed by hiscytological detectiontopathological orHoweverto distinguishbetween benign and malignant papillaryhyperplastic nodules and it is therefore difficult to diagnose PTC There is also a lackof effective and specific prognostic molecular markers for PTC17 The relationshipbetween MMPs and tumors is a currenthotspot of modern cancer research MMPsplay important roles in pathophysiologicalprocesses such as the dynamic extracellularmatrix balance as well as in tissue remodeling and repair10 Tumor cells may inducethe matrix to secrete MMPs via a series ofsignalingprovidingpathwaysthusTable Volume reductions after radiofrequencyablation of papillary thyroid carcinomasReductionrate Lesionvolumecm3 06 06 06 06 months after surgery 06 P P compared with before surgeryBefore surgery month after surgery months after surgery months after surgery“favorable conditions for tumor cell invasionand metastasisalternatingthe extensive surgicalSurgical resection is a traditional methodthyroid nodules18for the treatment ofHowevertraumaunsightly neck scars and risks of laryngealnerve injuries postoperative recurrenceand multiple operations mean that increasing numbers of patients are opting for minimally invasive ablation methods RFA is athermal ablation therapy that uses highfrequencyelectromagneticwaves generated by the radiofrequencyinstrument inserted into the tumor tissueto accumulate heat by rapid friction of positive and negative ions within the cells causing local coagulation in the tumor tissuewhich isthen removed by the body™ssystem19 Reduction ratesimmuneforbenign thyroid nodules of to after month of ablation and to after months of ablation have beenreported20 In this study ultrasound performed immediately after ablation of lesions showed no perfusion of contrastagents in lesions and enhancementto varying degrees at the edge or inside thelesion in of lesions Although therewere no significant changes in lesion volumeat month after the operation the lesionvolumes were reduced by to at months after surgery Considering thatthe ablation effects might be associatedwith the heat and the ablation needle aTable Logistic regression analysis of relevant risk factors for disease prognosisAgeMicrocalcificationIrregular morphologyLesion diameterLesion numberB HRHR hazard ratio CI confidence interval95CILower limitUpper limitP 0cJournal of International Medical Researchfine needle is good for mobile conformalablationthe highfrequency alternatingelectromagnetic wave only circulates in theeffective region between the two needle tipsthus allowing accurate control of the ablation zone The ablation safety zone aroundthe PTC was relatively small in the currentstudy and the nodulereduction rate afterablation was thus relatively greaterWe analyzed the serum levels of MMP2and MMP9 in PTC patients by ELISAPreoperative serum levels of both enzymeswere significantly higher in patients withPTC compared with patients with benigninthyroid nodules Regarding changesserum MMP2 and MMP9 levelstheAUC values based on the ROC curveswere and for MMP2 andMMP9 respectively suggesting satisfactory clinical diagnostic and prognostic valuesIn the present study serum levels MMP2and MMP9 were lower in the first monthafter surgery compared with before surgerybut the difference was not significant Thismight be because before ablationthetumor parenchyma induced the tumorstroma to produce larger amounts ofMMP2 and MMP9 which were releasedinto the blood These results suggest thatfailure to completely ablate the tumor ortumor recurrence may result in the secretionof high levels of MMP2 and MMP9 intothe blood However serum levels of MMP and MMP9 were significantly decreasedat and months after surgery compared with before surgery suggesting thatserum MMP2 and MMP9 were secretedby the tumor The lesions disappearedafter PTC ablation thus reducing the secretion of MMP2 and MMP9 and therebyreducing the degradation and destructionof type IV collagen protecting the basement membrane of normaltissues andinhibiting the growth and metastasis oftumorresults alsoshowed that age microcalcification irregular shape and lesion diameter and numbercells Thecurrentwere risk factors for PTC Logistic regression analysis showed that age 14 yearswas an important risk factor for PTC inline with the findings of Yu et al21Microcalcification is caused by the deposition of calcium salts at the tip of the nippleor the secretion of calcium salts by thetumor itself and has been considered tobe the most specific sign of PTC In thisstudy the incidence of microcalcificationwas higher in PTC patients compared withthe control group and logistic regressionidentified it as an independent risk sign forthyroid PTC Our results also identifiedirregular morphology two nodules and anodule diameter 14 cm as danger signs forPTClargely consistent with previousfindings22This study had some limitations It was asinglecenter study with a relatively smallnumber of cases Moreover the relevantthyroid hormone analysis and other serumindicators could not be followed up for alonger periodtreating PTC FurthermoreIn summary the results of this studyshowed that RFA could shrink or eliminatethyroid lesions thus representing a minimally invasive safe and effective methodforserumlevels of MMP2 and MMP9 before RFAcould provide a valuable reference for thediagnosis of PTC These serological indexes combined with relevant risk factors mayalso help to predict the prognosis of PTCafter ablationAcknowledgementsThis work wasProvincial HealthCommission Project WJ2017F102supported by the HubeiPlanningand FamilyDeclaration of conflicting interestThe authors declare that there is no conflict ofinterest 0cPan et alFundingThis research received no specific grant from anyfunding agency in the public commercial ornotforprofit sectorsorcid000000026050ORCID iDQian DingReferences Zhang YB Zhang B Yan DG et al[Central compartment reoperation for recurrentpersistent differentiated thyroid cancer]Zhonghua Er Bi Yan Hou Tou Jing Wai KeZa Zhi “ [in Chinese] Pellegriti G Frasca F Regalbuto C et alWorldwide increasing incidence of thyroidcancer update on epidemiology and risk factors J Cancer Epidemiol DOI Brito JP Gionfriddo MR Al Nofal A et alThe accuracy of thyroid nodule ultrasoundto predict thyroid cancer systematic reviewand metaanalysis J Clin Endocrinol Metab “ DOI 101210jc2013 Zhao P Zheng D Dong X et al Clinicaldiagnosis and treatment of thyroid microcarcinoma a report of cases Chinese JGene Surg “ Anil G Hegde A and Chong FH Thyroidnodules risk stratification for malignancywith ultrasound and guided biopsy CancerImaging “ DOI Levine RA Current guidelines for the management of thyroid nodules Endocr Pract “ DOI 104158ep12071co Zhang Y Zhang MB Luo YK et al Effectof chronic lymphocytic thyroiditis on theefficacy and safety of ultrasoundguidedradiofrequency ablation for papillary thyroid microcarcinoma Cancer Med “ Liu Y Wang W Wang Y et al Ultrasoundguided percutaneous microwave ablation inthe treatment of recurrent thyroid nodulesJ Clin Otolaryngol Head Neck Surg “ Weslley Rosario P Franco Mourao GRegina Calsolari M et al Role of adjuvanttherapy with radioactive iodine in patientswith elevated serum thyroglobulin afterneck reoperation due to recurrent papillarythyroid cancer a monoinstitutional comparative study Endocrine “ Zhang WJ Song B and Yang T MMP2MMP9 TIMP1 and TIMP2 in theperipheral blood of patients with differentiated thyroid carcinoma Cancer Manag Res “Wu R Luo Y Tang J et al Ultrasoundguidedradiofrequency ablation for papillary thyroidmicrocarcinomaa retrospective analysis of patients Int J Hyperthermia “ Zhao CK Hu HX Lu F et al Factors associated with initial incomplete ablation forbenign thyroid nodules after radiofrequencyablation First results of CEUS evaluationClin Hemorheol Microcirc “ Faggiano A Ramundo V Assanti AP et alThyroid nodules treated with percutaneousradiofrequency thermal ablation a comparative study J Clin Endocrinol Metab “ DOI 101210jc20122251 Tomayko EJ Cachia AJ Chung HR et alResveratrol supplementation reduces aorticatherosclerosis and calcification and attenuates loss of aerobic capacity in a mousemodel of uremia J Med Food “ DOI 101089jmf20120219 Xhaard C Rubino C Clero E et alMenstrual and reproductive factors in therisk of differentiated thyroid carcinoma inyoung women in France a populationbased casecontrol study Am J Epidemiol “ DOI 101093ajekwu220 Tafani M De Santis E Coppola L et alBridging hypoxia inflammation and estrogen receptors in thyroid cancer progressionBiomed Pharmacother “ DOI101016jbiopha201310013 Bumber B Marjanovic Kavanagh MJacovcevic A et al Role of matrix metalloproteinases and their inhibitors in the development of cervical metastases in papillary 0cJournal of International Medical Researchthyroid cancer Clin Otolaryngol “ofAssociation Gharib H Papini E Paschke R et alAmericanClinicalEndocrinologists Associazione MediciEndocrinologiand European ThyroidAssociation medical guidelines for clinicalpractice for the diagnosis and managementof thyroid nodules executive summary ofrecommendations J Endocrinol Invest “ Baek JH LeeJH Valcavi Ret alThermal ablation for benign thyroid nodules radiofrequency and laser Korean JRadiol “ DOI kjr2011125525 Vuong NL Dinh LQ Radiofrequency ablation for benign thyroid nodules 1yearfollowup in patients World J Surg “ Lang BHH Woo YC and Chiu KWIdentifying predictive factors for efficacy inhigh intensity focused ultrasound HIFUablation of benign thyroid nodules a retrospective analysis Int J Hyperthermia “ Buryk MA Simons JP Picarsic J et al Canmalignant thyroid nodules be distinguishedfrom benign thyroid nodules in children andadolescents by clinical characteristics Areview of pediatric patients with thyroidnodules Thyroid “ 0c'

Thyroid_Cancer

Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedObjective To investigate whether preventive administration of a proton pump inhibitor PPI can reduce the occurrence anddevelopment of traumatic granuloma TG following type IVVI cordectomy Methods We retrospectively analyzed the statusof postoperative granulomas in patients who underwent type IVVI cordectomy due to glottic cancer and determinedwhether postoperative administration of a PPI had any impact on granuloma formation and development Results Thepercentage and number of patients with granuloma in the PPI treatment group experimental group at the 1st 2nd 3rd and6th month following surgery were and respectively The percentageand number of patients with granuloma in the noPPI group control group were and respectively The granuloma percentage of the PPI treatment group was lower than that of thecontrol group at all postoperative time points assessed The diï¬erences were not statistically significant at the 1st monthp but were statistically significant at the 2nd and 3rd months after surgery p p ConclusionPreventive use of a PPI in patients after type IVVI cordectomy can shorten the TG recovery duration and may reduce theseverity of TG but it cannot prevent TG from occurring Our results should be confirmed by prospective randomized controlledtrials with large sample sizes IntroductionLaryngeal squamous cell carcinoma LSCC is a commonhead and neck cancer It had an incidence of approximately in China between and [] and new cases were reported worldwide in [] Itis estimated that there will be new cases worldwidein [] Approximately twothirds of LSCCs originatein the glottic area The presence of hoarseness in patientswith earlystage glottic cancer GC prompts the patients toseek medical treatment Anatomically the larynx is surrounded by cartilage and has sparse lymphatic tissue As aresult patients with GC are mostly diagnosed at an earlystage which is clinically defined as T12N0M0 []In recent years surgery has been gradually abandoned in the treatment of earlystage GC and has beenreplaced with transoral microsurgery TM or radiotherapyTM has the advantages of being minimally invasive and having a high laryngeal preservation rate and high costeï¬ectiveness Transoral laser microsurgery TLM is the mostcommon surgical method used for GC although a few studies in the literature have adopted transoral coblation microsurgery TCM Although TM methods diï¬erthermalinstruments are often needed for the surgery [“]Traumatic granuloma TG is a common complication ofTM especially after type IVV cordectomy Mild TG mayonly manifest as hoarseness foreign body sensation and frequent throat clearing The severe granuloma may cause or becomplicated by perichondritis which leads to severe symptoms such as dyspnea Additionally this granulation isbelieved to be an important factor in the formation of glotticweb and larynx stenosis [“] Reflux is an important 0cBioMed Research Internationalfactor that aï¬ects granuloma formation Proton pump inhibitors PPI are often used empirically in the treatment ofpatients with postoperative granulomas [ ]We found in our previous clinical practice that patientswith a wide range of resections such as type IVVI cordectomies have a higher risk of postoperative granuloma Thiscan sometimes be very severe even requiring temporary tracheotomy to alleviate dyspnea which greatly and adverselyimpacts patients™ quality of life PPIs have been empiricallyused for the treatment of patients with postoperative granuloma and evidence of its eï¬ectiveness has been published[] However there has not been any research on the prevention of postoperative granuloma formation with PPIsTherefore we initiated PPI treatment for patients who hadundergone type IVVI cordectomies and compared theresults with those of patients who had also undergone thistype of surgery but were not treated with PPIs to evaluatethe eï¬ect of PPI treatment for preventing postoperative granuloma formation in this patient population Materials and Methods Patients This was a retrospective study Patient inclusioncriteria i patients with vocal cord cancer who agreed toundergo type IV V and VI cordectomies ii patients whodid not undergo preoperative and postoperative radiotherapy Exclusion criteria i patients who were complicatedwith diabetes and were not treated routinely ii patientswho used PPIs regularly iii patients who continued tosmoke and drink after surgeryA total of patients between January and December were recruited as the control group who did not use PPIsimmediately after surgery and did not take PPIs persistentlyA total of patients between January and June were recruited as the experimental group PPI treatment group who took PPIs immediately after the surgeryand routinely Since this was a retrospective study only thepatients in the experimental group underwent preoperativereflux symptom index RSI and reflux finding score RFSassessments Patients with an RSI score points andoran RFS ‰¥ points were considered to have laryngopharyngeal reflux disease LPRD [ ]All patients signed an informed consent form prior to thesurgery All clinical experiments conformed to the guidelinesissued by the committee on clinical research of Peking UnionMedical College Hospital PUMCH Ethics Committeeapproval was obtained at PUMCH and all patients providedspecific written informed consent Surgical Procedure According to the European Laryngological Society classification endoscopic cordectomies includethe following types type IV total cordectomy type Vaextended cordectomy encompassing the contralateral cordtype Vb extended cordectomy encompassing the arytenoidtype Vc extended cordectomy encompassing the ventricularband type Vd extended cordectomies encompassing thesubglottis and type VI extended cordectomies encompassing the anterior commissure [ ] During the surgerythe larynx was fully exposed with a selfretaining laryngo°scope Karl Storz Tuttlingen Germany and the tumor°was resected en bloc under direct visualization of a orlaryngoscope Karl Storz Tuttlingen Germany using amodel coblator ArthroCare Corp Sunnyvale CA witha coblation level of and a coagulation level of Postoperative Treatment Procedure and FollowUp Thepatients in the experimental group were treated with intravenous omeprazole mg per day before the recovery of oralfeeding cases recovered oral feeding on the first day aftersurgery and cases recovered oral feeding on the third dayThen they were given mg oral omeprazole twice daily minutes before breakfast and dinner for consecutiveweeks The patients in the control group were not treatedwith PPIs but if severe granulomas formed during followup and required intervention they were also given PPIs routinely patients in the control group developedgranulomas But only patients developed severe granulomas on the 2nd 3rd and 3rd month after surgery respectively and began to be given PPIs for weeks the same asthe experimental group while the other cases in the control group were not given PPIs throughoutthe wholefollowup period All patients were treated with cefuroximeat mg twice daily for week to prevent infection Afterthe surgery the patients were required to quit smoking anddrinking and engage in reasonable vocal useThe followup procedure included regular checkups at and months after surgery If a granuloma was detectedduring the 3month checkup the patients were followedmonthly until the granuloma disappeared Postoperativegranuloma was defined as relatively smooth tissue in the surgery region that protruded from the surrounding area thatmay be attached to a pseudomembrane The granulomaand the surrounding area did not have obvious vascularhyperplasia The proportion of patients with postoperativegranuloma was used as an observation indicator Additionally the number of unscheduled visits and the rate of reoperationincluding tracheotomy and granulectomy wererecorded as indicators of granuloma severityDuring followup if the granuloma was found to severelyimpact vocalization andor breathing or if patients were suspected of having a recurrent tumor the granuloma was surgically resected and the specimen was sent for examination Statistical Analysis Data were statistically analyzed withSPSS software SPSS Chicago IL Nonnormally distributed quantitative data are represented as median and interquartile range and were subjected to the Wilcoxon ranksumtest Normally distributed measurement data are representedas mean ± standard deviation and were subjected to theindependent sample t test Count data were subjected to thechisquared test Diï¬erences with p were consideredstatistically significant Results Baseline Characteristics The baseline data of the patientsin the PPI treatment group and the control group are shownin Table Among the patients in the PPI treatment 0cBioMed Research InternationalTable General information DiscussionVariableSex MFAge yearsTumor staging T1aT1bT2Surgery type IVVVIPPIn ± Controln ± p valuegroup patients were male and patient was female withan average age of ± years patients were at theT1 stage and patients were at the T2 stage patients underwent type IV surgery patients underwent type V surgeryand patients underwent type VI surgery Among the patients in the control group patients were male and patient was female with an average age of ± years patients were at the T1 stage and patients were at theT2 stage patients underwent type IV surgery patientsunderwent type V surgery and patients underwent typeVI surgery The two groups of patients did not diï¬er significantly in the baseline conditions Table Postoperative Granulation The numbers and percentageof patients with granuloma in the PPI treatment group atthe 1st 2nd 3rd and 6thmonth followup were and respectively The numbers and percentage of patients with granuloma in the control group at those time points were and respectively Although the percentage of granuloma in the PPItreatment group was lower than that of the control group ateach stage only the diï¬erences at the 2nd and 3rd monthsafter surgery were statistically significant p and p respectively Only one patient in the PPItreatment group required a second surgery due to persistentgranulation patients in the control group underwent a second surgery among whom patients had granuloma complicated with chondronecrosis and required totracheotomy due to dyspnea Figure However the diï¬erence between the two groups was not statistically significantp In the PPI treatment group only patients hadtwo unscheduled visits In the control group patients had unscheduled visits among them one patient had visitsdue to dyspnea The diï¬erence in the number of unscheduledvisits was not statistically significant p Table Eï¬ects of PPI Treatment in Patients with LPRD in theExperimental Group Among the patients in the experimental group were diagnosed with LPRD according to preoperative RSI and RFS scores and patients did not have LPRD The numbers of LPRDpatients with granuloma at the 1st 2nd 3rd and 6th monthsafter surgery were and respectively and the numbers of LPRD patients with granuloma at these time pointswere and While the data showed that the percentageof granuloma in the LPRD patients was higher than that inthe LPRD patients only the diï¬erence at the 3rd month aftersurgery was statistically significant p Table Transoral microsurgery TM for earlystage glottic cancerGC can achieve oncological therapeutic eï¬ects similar tothose of radiotherapy TM has the advantages of being minimally invasive and having a high laryngeal preservation rateand a low tracheotomy rate its disadvantage includes postoperative complications such as bleeding infection airwayburns and granuloma formation Therefore currently thetreatment selected for patients with earlystage GC is determined by the disease conditions as well as patient needs[ ] Postoperative traumatic granuloma TG is a common complication during the healing process after TLM surgery Severe TG may cause or be complicated by chondritis orchondronecrosis leading to severe complications includingdyspnea With the increase in the range of cordectomy theincidence of TG is also significantly increased Therefore itis necessary to investigate ways to reduce the incidence andseverity of postoperative TG [ ] Like TLMthecoblationassisted endoscopic cordectomy or TCM used inour study is also based on thermal damage whose working°C much lower than laser™s workingtemperature is °°temperature which is C1000C and as a result its healing process and the mechanism of TG formation are alsosimilar to those of TLM Current literature indicates thatreflux may be an important factor in the occurrence of postoperative TG [ “] Therefore we aimed to investigatewhether antiacid therapy could reduce TG through an analysis of the eï¬ects of PPI treatment in patients who underwenttype IVVI cordectomy which has rarely been reported°C70Our study showed that the incidences of TG after transoral surgery were as high as and in the twogroups which were much higher than the incidences of reported by Nerurkar and Shah and reported by Wang The reason for this discrepancy may be that the patients in our study all underwent typeIV or higher surgeries Enlarged wounds and damage to theperichondrium or cartilage may lead to increased TG andchondronecrosis Nerurkar and Shah reported that the incidence of TG in patients who have undergone type IV TLMwas and the study by Wang showed that the incidences of TG in patients who have undergone type IV andtype V TLM were and respectively [ ]Meanwhile the incidence of chondronecrosis was not rare especially on whom the surgeon had to expose thethyroid cartilage during tumor resection in TLM [] Thesestudies suggest that type IV or higher surgeries lead to a highlikelihood of TG occurrenceOur study showed that PPI treatment did not suppressthe formation of TG at the 1st month after surgery but thepercentage of TG in the PPI treatment group graduallybecame lower than that of the control group and the diï¬erences were statistically significant at the 2nd and 3rd monthsafter surgery At the 6th month granulomas disappeared inboth groups Our findings suggest that although PPI treatment cannot reduce the incidence of TG it can significantlyshorten the duration of granulomas a finding that is similarto the study by Wang [] Additionally we did notobserve any severe cases of TG that were complicated with 0cBioMed Research InternationalabcdefghiFigure a“f Shows a typical case in the control group who was a male patient for years old with glottic cancer T2N0M0 at the left sidefollowed by type V cordectomy a One month after surgery a granuloma was found in the left vocal cord under a fibrolaryngoscope b months after surgery the granuloma enlarged and the right vocal cord become edema obviously c months after surgery d monthsafter surgery e Computerized tomography CT scan taken before surgery f months after surgery chondronecrosis was found in the CTscan where the white arrow points out g“i Shows a typical case in the experimental group who was a male patient for years old withglottic cancer T2N0M0 at the right side followed by type V cordectomy g h and i were taken under a fibrolaryngoscope in month months and months after surgery respectively A granuloma could be found in g but disappeared in h and idyspnea in the PPI treatment group however TG in patients in the control group caused or was complicated bychondritis or chondronecrosis which led to severe dyspneaThere were only unscheduled visits among the PPI treatment group compared to unscheduled visits in the controlgroup Unfortunately the diï¬erences in the two indicators ofTG severity between the two groups were not statistically significant although we believe PPI treatment did alleviate the 0cBioMed Research InternationalTable Percentage and severity of granuloma in the two groups1st month2nd month3rd month6th monthResurgeryNumber of unscheduled visits asmedian and interquartile rangePPI treatment groupControl groupp valueNote aindicates that the diï¬erence is statistically significant0005a0037a Table Percentage of granuloma in patients with or without LPRDin the experimental group1st month 2nd month 3rd month 6th month”LPRD n LPRD n p valueNote aindicates that the diï¬erence is statistically significant0029aseverity of TG In this study postoperative PPI was used for weeks referring to the antiacid duration weeks in amedical routine of vocal process granulation and laryngopharyngeal reflux disease J R Lechien™s report in andChinse experts consensus on diagnosis and treatment of laryngopharyngeal and reflux disease in [] The resultsshowed that PPI could shorten the recovery time and potentially prevent severe complicationsOur study showed that the percentage of TG did not differ significantly between the LPRD and LPRD patientsp However it took longer for granulomas to disappear in LPRD patients than in LPRD patients and the difference in the number of patients with granulomas wasstatistically significant between the two groups at monthsafter surgery p The reason for this phenomenonmay be that during the early stage of recovery after vocalcord injury changes in the extracellular matrix are the mainmanifestation and injury impacts a lot while acid reflux hasa little eï¬ect Acid reflux may impact the repair process in themiddle and late stages [ ]This was a retrospective nonrandomized controlledstudy with a small sample size We only analyzed granulomaformation in the patients and did not assess their oncologicaloutcome We based the diagnosis of LPRD on RSI and RFSscores and did not perform dualprobe 24hour pH monitoring Therefore we were unable to determine whethernonacid reflux had an impact on the formation and development of postoperative granuloma ConclusionThe preventive use of PPI in patients who have undergonetype IVVI cordectomy cannot reduce the incidence of TGwhile it can shorten the TG recovery duration and may alsoreduce the severity of TG Our findings should be confirmedby prospective randomized controlled studies with largersample sizesData AvailabilityAccess to these anonymized data will be made available bythe corresponding author Dr Jian Wang upon reasonablerequestConflicts of InterestThe authors declare that they have no conflicts of interestfinancial or nonfinancial to discloseAuthors™ ContributionsXiaofeng Jin and Yanyan Niu contributed equally to thisstudyAcknowledgmentsThis study was funded by the Nature Science Foundation ofBeijing China Grant No References[] L B Du W M Mao W Q Chen œIncidence and mortality of larynx cancer in China during  ZhonghuaLiu Xing Bing Xue Za Zhi vol no pp “ [] F Bray J Ferlay I Soerjomataram R L Siegel L A Torreand A Jemal œGlobal cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA A Cancer Journal for Clinicians vol no pp “ [] B Gupta N W Johnson and N Kumar œGlobal epidemiology of head and neck cancers a continuing challenge Oncology vol no pp “ [] M F Vaculik C A MacKay S M Taylor J R B Trites R DHart and M H Rigby œSystematic review and metaanalysisof T1 glottic cancer outcomes comparing CO2 transoral lasermicrosurgery and radiotherapy Journal of Otolaryngology Head and Neck Surgery vol no p [] W Steiner œResults of curative laser microsurgery of laryngealcarcinomas American Journal of Otolaryngology vol no pp “ [] M S Strong œLaser excision of carcinoma of the larynxLaryngoscope vol no pp “ [] A S Carney M S Timms C N Marnane S KrishnanG Rees and S Mirza œRadiofrequency coblation for the resection of head and neck malignancies Otolaryngology and Headand Neck Surgery vol no pp “ [] M Lee M A Buchanan F Riï¬at and C E Palme œComplications after CO2 laser surgery for early glottic cancer an 0cBioMed Research Internationalinstitutional experience Head Neck vol no S1 pp E987“E990 [] B Liu L Cheng H Ming and C Zhong œTreatment of theearlystage glottic cancer using lowtemperature radiofrequency coblation Journal of Cancer Research and Therapeutics vol no pp “ [] Y Zhang B Wang G Sun G Zhang L Lu and G LiangœCarbon dioxide laser microsurgery versus lowtemperatureplasma radiofrequency ablation for T1a glottic cancer asingleblind randomized clinical trial BioMed Research International vol Article ID pages [] M Remacle H E Eckel A Antonelli œEndoscopic cordectomy A proposal for a classification by the Working Committee European Laryngological Society European Archivesof OtoRhinoLaryngology vol no pp “ [] L Wang S Sun S Wang D Liang and W Ji œClinical observation of traumatic granuloma after CO‚‚ laser cordectomy andlaryngopharyngeal reflux Zhonghua Er Bi Yan Hou Tou JingWai Ke Za Zhi vol no pp “ [] M Canis F Ihler A Martin C Matthias and W SteinerœTransoral laser microsurgery for T1a glottic cancer reviewof cases Head Neck vol no pp “ [] A Galli L Giordano D Sarandria D di Santo and M BussiœOncological and complication assessment of CO2 laserassisted endoscopic surgery for T1T2 glottic tumours clinicalexperience Acta Otorhinolaryngologica Italica vol no pp “ [] N K Nerurkar and R Shah œFactors responsible for the development of carbon granuloma post transoral laser cordectomy Lasers in Medical Science vol no pp “ [] P C Belafsky G N Postma and J A Koufman œThe validityand reliability of the reflux finding score RFS Laryngoscopevol no pp “ [] P C Belafsky G N Postma and J A Koufman œValidity andreliability of the reflux symptom index RSI Journal of Voicevol no pp “ [] M Remacle C van Haverbeke H Eckel œProposal forrevision of the European Laryngological Society classificationof endoscopic cordectomies European Archives of OtoRhinoLaryngology vol no pp “ [] J Yoo C Lacchetti J A Hammond R W Gilbert and Headand Neck Cancer Disease Site Group œRole of endolaryngealsurgery with or without laser versus radiotherapy in themanagement of early T1 glottic cancer a systematic reviewHead Neck vol no pp “ [] C M Chiesa Estomba F A Reinoso A O Velasquez J LFernandez J L Conde and C S Hidalgo œComplications inCO2 laser transoral microsurgery for larynx carcinomas IntArch Otorhinolaryngol vol no pp “ [] J R Lechien F Mouawad M R Barillari œTreatment oflaryngopharyngeal reflux disease a systematic review WorldJournal of Clinical Cases vol no pp “ [] M K Wani and G E Woodson œLaryngeal contact granuloma Laryngoscope vol no pp “ [] C Ling M Yamashita J Zhang D M Bless and N V Welham œReactive response of fibrocytes to vocal fold mucosalinjury in rat Wound Repair and Regeneration vol no pp “ 0c'

Thyroid_Cancer

Deacetylases inSkeletal Muscle Physiology andSystemic Energy HomeostasisImplications for Metabolic Diseasesand TherapyHaili Tian1  Sujuan Liu2  Jun Ren3 Jason Kai Wei Lee456 Ru Wang1 and Peijie Chen1 School of Kinesiology Shanghai University of Sport Shanghai China Department of Anatomy and Histology Schoolof Basic Medical Sciences Tianjin Medical University Tianjin China Department of Cardiology Shanghai Instituteof Cardiovascular Diseases Zhongshan Hospital Fudan University Shanghai China Department of Physiology Yong LooLin School of Medicine National University of Singapore Singapore Singapore Global Asia Institute National Universityof Singapore Singapore Singapore N1 Institute for Health National University of Singapore Singapore SingaporeSkeletal muscle is the largest metabolic an in the human body and is able torapidly adapt to drastic changes during exercise Histone acetyltransferases HATs andhistone deacetylases HDACs which target histone and nonhistone proteins are twomajor enzyme families that control the biological process of histone acetylation anddeacetylation Balance between these two enzymes serves as an essential elementfor gene expression and metabolic and physiological function Genetic KOTG murinemodels reveal that HDACs possess pivotal roles in maintaining skeletal muscles™metabolic homeostasis regulating skeletal muscles motor adaptation and exercisecapacity HDACs may be involved in mitochondrial remodelinginsulin sensitivityregulationturn onoff of metabolic fuel switching and orchestrating physiologicalhomeostasis of skeletal muscles from the process of myogenesis Moreover manymyogenic factors and metabolic factors are modulated by HDACs HDACs areconsidered as therapeutic targets in clinicaltreatment of cancer‚ammation and neurological and metabolicrelated diseases This review will focuson physiological function of HDACs in skeletal muscles and provide new ideas for thetreatment of metabolic diseasesresearch forKeywords histone deacetylases exercise capacity skeletal muscle metabolism muscle physiologyINTRODUCTIONSkeletal muscle is the largest metabolic an in the human body consuming about of theentire body daily expenditure of energy Durnin It produces various secrete factors andparticipates in the interplay among multiple tissues and ans Pedersen and Febbraio Mizgier Muscular contraction is one of the main physiological functions of skeletalmuscles and plays a role in maintaining an and systemic metabolic homeostasis Guo Proper exercises help build up body defense to combat various diseases including obesitytype diabetes Alzheimer disease osteoarthritis and so on Luan These importantEdited byBrian James MorrisThe University of Sydney AustraliaReviewed bySean L McGeeDeakin University AustraliaViviana MoresiSapienza University of Rome ItalyCorrespondenceRu WangwangrutysuseducnPeijie Chenchenpeijiesuseducn These authors have contributedequally to this workSpecialty sectionThis was submitted toIntegrative Physiologya section of the journalFrontiers in PhysiologyReceived May Accepted July Published August CitationTian H Liu S Ren J Lee JKWWang R and Chen P Roleof Histone Deacetylases in SkeletalMuscle Physiology and SystemicEnergy Homeostasis Implicationsfor Metabolic Diseases and TherapyFront Physiol 103389fphys202000949Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise Capacityfunctions require delicate regulations in muscle from thelevel of genome to signal transduction establishing muscleplasticity and responses to environmental stress AcetylCoAas a central metabolite of amino acidfatty acidglucose notonly serves as fuel for energy expenditure but also bridgesthe gap between environmental stress and anismal responseLempradl Liu This control occursthrough posttranslational modification on nucleosomes histonelysine residues and other signal transducing proteins whichin turn controls accessibility of DNA to regulatory factorsand protein activity Acetylation one of the most prevalentmodifications of proteinis believed to function as a keymodulator of chromatin structure and signal transduction andprovides an avenue to couple extracellular stimuli with genomeduring muscle metabolism process by regulating acetylation anddeacetylation Haberland histone deacetylasesHDACs as a family of proteindeacetylases have been demonstrated to moderate physiologicalhomeostasis and development by deacetylation Haberland Table In humans there are types ofHDACs which are classified into four categories based onhomologous proteins in yeast namely Class I Rpd3like proteinHDAC1 Class II Hda1like proteins are classifiedas Class IIa HDAC4 and Class IIb HDAC6 Class III Sir2plike nicotinamide adenine dinucleotideNADdependent SIRT1 and ClassIV HDAC11 containing homologous domain with bothRpd3 and Hda1 Seto and Yoshida HDACs modulategene expression and protein activity through deacetylatingproteins When histone lysine εamino acid in the nucleosomeis acetylated it can neutralize positive charge before looseningchromatin structuretranscriptionfactors to DNA and expression of downstream target genesBy contrast histone deacetylation compresseschromatinstructure thereby inhibiting transcriptional gene expressionShahbazian and Grunstein to promote binding ofBiochemical properties of HDACs have been comprehensivelyreviewed Shahbazian and Grunstein Howeverthephysiological functions of HDACs have yet to be well examined inskeletal muscles A series of researches shed light on the potentialof drugs targeting HDACs to improve muscle fitness and cardiacmuscle disease which needs further clarification of HDACsphysiological function to understand the mechanisms Bai Galmozzi Xie Zhang and Ren Gaur Ample work has revealed the role of HDACin muscle physiology such as skeletal muscles me olism and thusexercise capacity McGee and Hargreaves Table Class IHDACs can interact with myocyte enhancer factor MEF2MyoD regulating myogenesis and exercise capacity Mal Puri Ohkawa Gregoire HDAC3 participates in myocytes diï¬erentiation and is linked toskeletal muscles metabolic fuel switching Gregoire Hong Class II HDACs can be phosphorylated byHDAC kinases and are transduced from nucleus to cytoplasmin response to cellular stress mediating muscle fiber switch andaï¬ecting the pathway of insulin sensitivity such as Glut4 andAKT Haberland McGee and Hargreaves Class III HDACs target a crucial mitochondrial biogenesis factorperoxisome proliferatoractivated receptor gamma coactivator alpha PGC1α in skeletal muscles liver and fat tissue Whiteand Schenk Therefore we will further discuss howthese HDACs ‚uence respective downstream targets exercisecapacity and therapeutic eï¬ects in human diseasesCLASS I HDAC HDAC123Regulation of Myogenesis by Class IHDACsIn previous studies HDAC123 was shown to be associated withthe process of myogenesis or myocyte diï¬erentiation Mal Puri Ohkawa Gregoire HDAC1 tightly binds to MyoD and deacetylates specific sites toinhibit the expression of musclespecific genes such as MHC andMCK in myoblasts Mimicking muscle diï¬erentiation conditionby serum deprivation HDAC1 protein gradually decreasesduring myocyte diï¬erentiation and transfers to bind to the tumorsuppressor pRb accompanied by isolation of HDAC1MyoD andtranscriptional activation of musclespecific genes Puri A HDAC1 H141A mutant incapable of binding withMyoD loses its inhibitory property on musclespecific genes inmyoblast state Mal In the late stage of myocytediï¬erentiation activating factor gradually switches from MyoDto myogenin occurring with a decrease in the MEF2D inhibitoryregulator HDAC2 Simultaneous overexpression of myogeninand MEF2D can enhance the expression of the musclespecificgene MHC in the absence of MyoD Ohkawa MEF2D a key factor that controls myocyte diï¬erentiationcan only be eï¬ectively deacetylated by HDAC3 rather thanHDAC128 Gregoire In addition HDAC3 caninhibit autoacetylation of acetyltransferases p300 and p300CBPassociated factor PCAF Thus HDAC3 impedes MyoDMEF2PCAF to form a multicomplex disturbing MEF2dependentmyogenic transcription Gregoire Figure Redundant Roles of HDAC12 inMaintaining Sarcomere Homeostasis inMuscleLoss of function of HDAC12 inhibits autophagy flux in skeletalmuscles tissue accompanied by decreased LC3 III ratio andp62 accumulation under fasting condition Moresi Toxic autophagy intermediates accumulate in muscle fibersin which class I HDACs deficiency led to impaired exercisecapacity Moresi This oï¬ers convincing evidenceon the role of HDAC12 in stabilizing basic structure andfacilitating development in muscles Genetic models suggestthat Class I HDACs control the physiological homeostasis ofskeletal muscles A germline deletion shows that wholebodyknockout of either HDAC1 or HDAC2 leads to mortalityin mice prior to the perinatal period Montgomery However a tissuespecific single deletion of HDAC1or HDAC2 in myocardium does not cause any phenotypeMontgomery Double knockout HDAC12 in theFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityTABLE The targets of HADCs in skeletal muscles and their physiological functionsClassMembersDown targetsGenetic KOTG phenotypeReferencesIHDAC1MyoD†“ PTEN†“ FoxOHDAC2HDAC3HDAC8HDAC4HDAC5HDAC9HDAC7HDAC6MyoD†“ MEF2D†“ NFκBp65†“MEF2D PCAF†“ Ampd3†“RCAN1 MEF2ACpt1b†“MEF2†“ Glut4†“ Myosin HeavyChain PGC1α and Hsc70“Pax7†“MEF2†“ Glut4†“ Baf60c†“Atg7 Beclin1 LC3†“Dach2Myog Gdf5†“MEF2†“Pax7 MFN1†“ Fam65bdysferlin and MAFbxHDAC10SIRT1“PGC1α STAT3†“IIAIIBIII“MnSOD Hexokinase II PDHsubunit E1αMalonyl CoA decarboxylase†““NFκBMstnSIRT2SIRT3SIRT4SIRT5SIRT6SIRT7Functionally redundant HDAC12 DKO mice causesmitochondrial abnormalities and sarcomeredegenerationPuri Ohkawa Beharry Cho Zhu Bin mKO mice Enhanced amino acid\\lipid metabolism andendurance exercise Causes IR under basal conditionGregoire Han Yuan Hong “Functionally redundant HDAC45 DKO HDAC59 DKOHDAC459 TKO increases type I fiber percentage andMcKinsey 2000a Choi Niu Luo oxidation metabolismYuan Meng Niu Macpherson Zhang Dressel “KO mice Causes mitochondrial oxidative damagemitofusion defect Protect against muscle wastingBalasubramanian Lee Ratti “mKO mice Mediates CR induced insulin sensitivity hasno effect on glucose homeostasis or exercise capacityTG mice no effect on glucose homeostasis or exercisecapacityKO mice Exacerbates obesity and IR in HFDKO mice Exacerbates obesity and IR in HFDKO mice Increased exercise tolerance and protectagainst HFDinduced obesity“Rodgers Schenk Boyle Jing Lantier Laurent KO mice Abnormal hypoglycemia TG mice Protectagainst HFD mKO mice Impaired insulin sensitivity lossKanfi Xiao Cui Samant of muscle mass““HDAC11IVActivation inhibition†“ unknown KO wholebody deletion TG wholebody overexpression DKO double knockout TKO triple knockout mKO musclespecificknockout CR caloric restriction HFD high fat diet IR insulin resistance““““heart leads to severe cardiomyopathy in mice indicating theobligatory role for HDAC12 in specific tissues Montgomery In skeletal muscle double knockout of HDAC12by myogeninCre causes mitochondrial abnormalities andsarcomere degeneration disrupting fundamental structural unitsof myofibers Moresi Therefore these findingsdemonstrate that HDAC12 redundantly maintains sarcomerehomeostasis in skeletal muscleHDAC3 Functions as a Fuel Switch inMuscle Energy Metabolismthe enzymatic core of nuclearHistone deacetylases isreceptor corepressorNCoR and silencing mediator ofretinoic acid and thyroid hormone receptors SMRT corepressor correspondingly NCoR and SMRT corepressoractivate HDAC3 through its SANT domain enzyme activityKaragianni and Wong Mice suï¬er from insulin resistanceafter specifically knocking out HDAC3 in skeletal musclesmKO while their endurance exercise abilities are enhancedHong It seems a selfcontradictory phenomenonbecause former studies pointed out that increased enduranceexercise capacity enhances insulin sensitivity The study showsthat insulin signaling cascades including pAKTAKT pIRS1S1101 and pGSKGSK have no change in HDAC3 mKO musclewhile glucose uptake and insulin sensitivity are impaired inglucose tolerance test GTT and insulin tolerance test ITTHong which is called the œdissociation eï¬ect byresearchers Lipid tends to be used as energy fuel in HDAC mKOmice which inhibits glucose absorption but does not ‚uenceinsulin signaling sensitivity Hong Further workfound that the HDAC3 knockout upregulates the expressionof AMP deaminase AMPD3 the first ratelimiting enzymein purine metabolism in skeletal muscles Fortuin Hong AMPD3 can deaminate AMP to form IMPfacilitating aspartic acid to transmit into fumarate and malate theintermediate metabolites of tricarboxylic acid cycle TCA cycleHong Research studies found that exercise inducedglucose labeled 13C6 expressed a lower glycolysis flux rate inmuscles of HDAC3 mKO but a higher expression in the TCAFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Controls of myogenesis by Class I HDACs The inhibitory effects of HDACs on myogenic factors MEF2 and MyoD maintain a primary myoblast stateDuring myocyte differentiation and myogenic process the inhibition is lifted by other factors facilitating MEF2MyoD complex formation to promote myogenesis“Ac deacetylationcycle intermediates Hong Gong Thus ageneral increase in TCA cycle metabolites activates the oxidationin mitochondria In vitro radioactive aspartic acid isotope tracertest showed that inhibiting HDAC3 or overexpressing AMPD3can increase amino acid metabolism rate and enhance fatty acidmetabolism thereby downregulating glucose metabolism Hong Figure In liver knockout HDAC3 causes severeliver steatosis and can be rescued by wildtype or catalyticallyinactive mutants of HDAC3 indicating an enzymatic activityindependent function Sun However the musclefuel switching in HDAC3 mKO mice cannot be rescued by anenzymatic inactivity mutant HDAC3 Song Thisfinding demonstrates that HDAC3 controls the fuel utilizationin skeletal muscles and is dependent on its enzymatic activityThe relationship between exercise and glucose uptake is far morecomplex than one could expectCLASS II HDACs HDAC45796Phosphorylation and NucleusCytoplasmShuttle of Class II HDACsThe MEF2 family is a class of transcription factors that areclosely associated with myogenic basic helixloophelix domainsincluding MyoD Taylor and Hughes MEF2 may interactwith Class IIa histone deacetylase HDAC45 to inhibit thetranscription of MEF2dependent genes thereby impeding thediï¬erentiation from myoblast to myotube McKinsey et al2000a During exercise Ca2 flows out from sarcoplasmicreticulum and activate calciumcalmodulindependent proteinkinase CaMK which phosphorylates HDAC45 and mediatesHDAC45 shuttle from the nucleus to the cytoplasm thusreleasing the repressive eï¬ect of HDAC45 on MEF2dependentgenes McKinsey 2000b Yuan Further researchdemonstrates that the nucleoplasmic shuttle of HDAC4 relies on binding while the shuttle of HDAC5 depends on CaMKphosphorylation at serine and sites first and thenexporting from the nucleus by binding with McKinsey 2000b As expected HDAC7 in Class IIa also possessesthe ability to be exported from nucleus to cytoplasm Gao suggesting that there may be redundant functions of ClassIIa HDACs Except for Class IIa knocking out Class IIb HDAC6in embryonic stem cells ESCs can promote ESCs to diï¬erentiateinto myoblast cells and transplantation of ESCs with HDAC6knockdown can also help the regeneration of wound skeletalmuscles Lee type I fiberthe proportion ofRegulation of Class II HDACs onFiberType Switch MitochondriaRemodeling and Energy Stress inSkeletal MuscleEvidence showsinthatskeletal muscles has no change in animal knocked out aloneany member of Class IIa Potthoï¬ Howeverthe proportion of type I fiber is significantly increased inHDAC45 DKO HDAC59 DKO HDAC459 TKO miceas well as exercise capacity of skeletal muscles Potthoï¬ Altogether the finding indicated a functionalredundant mechanism in Class II HDACs family Previousstudy found that Class IIa HDACs are closely associated withthe MEF2 family and can repress their downstream targetgenes McKinsey 2000a Therefore similar to Class IIaHDACs DKO the proportion of type I fiber is decreased in theMEF2C and MEF2D knockout mouse models overexpressingMEF2C results in increasing type I fiber and exercise capacityHDAC45 not only regulates exercise capacity but also governsmotor adaptation Several studies had shown that exerciseFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class I HDACs regulates sarcomere structure and exercise capacity in mature muscle A schematic diagram shows Class I HDACs regulate exercisecapacity on different levels HDAC12 have redundant roles in modulating autophagy flux in muscle eliminating toxic factors in muscle HDAC3 serves as a fuelswitch and promotes muscle to use fatty acid to adapt endurance exercisepromotes phosphorylation of HDAC45 mediated by CaMK andadenosine monophosphateactivated protein kinase AMPKthen increasing a MEF2dependent transcription of Glut4 andmusclespecific genes which participates in the adaptationand plasticity of skeletal muscles McGee McGee and Hargreaves Besides exercise glucose can alsoactivate KATP channeldependent calcium signaling and CaMKwhich induce phosphorylationdependent nuclearcytoplasmtransportation of HDAC5 Meng The leavingHDAC5 releases its repressive eï¬ect on Baf60c and enhancinginsulinindependent AKT activation Meng Usingspecific inhibitor of class IIa HDAC activity myosin heavy chainPGC1α and heatshock cognate HSC70 are confirmed to beone of the substrates of HDAC4 in denervationinduced atrophymuscle in comparison with acetylated protein enrichment withuntreated group Luo Taken together Class IIaHDACs play critical roles in exercise capacity remodeling andnutrient sensing of skeletal muscles Figure For Class IIbit was revealed that glucose deprivationinduced mitochondrialfusion mediated by mitofusion1MFN1 is significantly disrupted in HDAC6 KO mice causingmitochondria degeneration which isreversed followingapplication of MFN1 acetylationresistant mutant Lee This study suggested that MFN1 deacetylation by HDAC6plays an important role in mitochondrial adaptive energyproduction and remodeling of skeletal musclesCLASS III HDACsSir2HOMOLOGSIRT1634NuclearLocated SIRT16Numerous studies have found that caloric restriction CRextends lifespan of mammals Caenorhabditis elegans and fruitflies while such eï¬ect is lost in SIR2 or NPT1 mutant C elegansstrains Imai Lin SIR2 encodes thesilencing protein Sir2p and NPT1 participates in the synthesis ofNAD NAD is an essential cofactor for Sir2pClass III HDACsto exert enzyme activity rather than Class I II and IV relayingon the zinc Imai Lin In mammalsSir2 has homologous proteins and these proteins are essentialto mitochondrial energy homeostasis antioxidant defense cellproliferation and DNA repair VargasOrtiz SIRT1 Mediates Energy Stress Adaptation in SkeletalMuscles reported that SIRT1 promotes theRodgers et altranscriptional activity of PGC1α by deacetylating the PGC1αat K13 site and mediates CRinduced gluconeogenesisrelatedgenes G6P PEPCK expression in liver Further research showedthat the NADNADH ratio and the enzymatic activity of Sir2 asensor of redox state were decreased in diï¬erentiating myocyteswhich relieved the inhibitory eï¬ect on MyoD and then promotedcell diï¬erentiation Resveratrol RSV a compound that maytarget SIRT1PGC1α can improve mitochondrial function andmetabolic homeostasis owning a potential to prolong lifespanRSV loses its activating eï¬ect on PGC1α in SIRT1 ˆ’ˆ’ MEFsLagouge Moreover RSVtreated mice at a dose of gkg increased their exercise capacity oxygen consumptionand improved insulin sensitivity against highfat induced obesityLagouge In addition RSV could enhance thedeacetylation of PGC1α in brown fat tissue BAT and skeletalmuscles which promotes mitochondrial production oxygenconsumption and thus improves metabolic syndrome Lagouge The function of SIRT1 in regulating skeletal musclesmetabolic homeostasis and exercise capacity has attracted greatattention from researchers Researchers further generated skeletalmusclespecific SIRT1mKO mice and found that mKO miceFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Control of muscle exercise adaption and mitochondrial function by Class II HDACs A Schematic of the Class IIa HDACs nucleoplasm shuttle Understress condition CaMK and AMPK could be activated and phosphorylate Class IIa HDACs Phosphorylated HDACs then bind with and shuttle to cytoplasmrelieving inhibitory effects on MEF2 and Baf60c B Class IIb HDAC6 can deacetylate MFN1 and facilitates its mitofusion function P phosphorylationlost the eï¬ect of CRinduced increasing insulin sensitivity andwere unable to deacetylate and inactivate STAT3 resulting inupregulating the phosphatidylinositol3kinase PI3K inhibitoryregulator p55αp50α expression Schenk Althoughknockout or overexpression of SIRT1 in skeletal muscles hasno eï¬ect on endurance capacity or glucose homeostasis in miceGurd Philp White Svensson some studies have shown that AMPK regulatesenergy metabolism of skeletal muscles partially mediated bySIRT1 and SIRT1 is significantly increased after enduranceexercise Suwa Canto Overexpressionof Sirt1 in skeletal muscles by adenoassociated virus AAV1promotes the expression of oxidationrelated genes includingPpargc1a Tfam Cpt1b and Pdk4 while it has no eï¬ect on insulinsensitivity of body Vila But overexpressing Sirt1in liver by AAV8 can protect from fatty liver induced by highcarbohydrate food HCD Vila Taken togetherthe aforementioned studies suggest that SIRT1 possesses limitedregulation capacity of skeletal muscles motility and SIRT1may modulate mitochondrial homeostasis and mediate skeletalmuscles adaptation under certain physiological conditions suchas CR aging and regeneration Gomes Ryall Figure In addition there may be more beneficial eï¬ectsof SIRT1 on metabolism in the liver or fat tissue Lagouge Vila Stefanowicz SIRT6 Improves Muscle Fitness andExercise CapacitySIRT6 is another Sir2like deacetylase located in the nucleusWhole body deletion of SIRT6 causes mice death around weeks owning to hypoglycemia Xiao Neverthelessthe skeletal musclespecific knockout of SIRT6 causes insulinresistance and impairs glucose homeostasis of mKO mice Cui Because the activity of AMPK is reduced inSIRT6mKO mice the glycolipids absorption and utilizationof skeletal muscles are impaired and the exercise capacity ofthe mice was also attenuated Cui Furthermoreresearchers constructed the overexpressing SIRT6 Sirt6BACmice and found that their body weight and fat content werenormal but the Sirt6BAC mice could protect from HCDinducedhyperglycemia via increasing pAKTAKT induced by insulinstimulation and glucose absorption Anderson Vitroexperiments further demonstrated that the ability of glucoseuptake was enhanced mainly in skeletal muscles not in brainiWAT eWAT tissues of Sirt6BAC mice Anderson These results shows the potential eï¬ects of SIRT6 targeted inskeletal muscles on improving exercise performance and treatingmetabolic diseases Figure MitochondriaLocated SIRT34SIRT34 Maintains Mitochondria Function in SkeletalMusclesSIRT34 are mitochondrialocalized deacetylases and responsiblefor regulating the deacetylation of proteins in mitochondria andmaintaining mitochondrial homeostasis After feeding highfatdiet HFD accelerated obesity attenuated insulin sensitivityworsened fatty liver and increased acetylation of proteins inmitochondria were observed in SIRT3 KO mice Hirschey Multitissue proteomics and physiological examinationreveals that SIRT3 is responsible for mitochondrial acetylatedFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class III HDACs promote a stressinduced adaptation in muscle and regulate mitochondrial function Class III HDACs are NADdependentdeacetylases and may function as a redox sensor in muscle cell SIRT1 could activate PGC1alpha by directly deacetylasing K13 site and regulate insulin sensitivityby targeting STAT3 Mitochondrialocated Class III HDACs control mitochondrial function by deacetylating certain mitochondrial proteins modulating muscle fuelutilization and exercise capacityproteome regulation and metabolic fuel switching in brainheart kidneyliver and skeletal muscles DittenhaferReed Protein enrichment analysis discovers that itsregulatory proteins were mainly concentrated in lipid metabolismDittenhaferReed Knocking out SIRT3 leadsto reduction in the levels of deacetylation of manganesesuperoxide dismutase MnSOD mitochondrial complex II andpyruvate dehydrogenase PDH subunit E1α a downregulationof hexokinase II HK II binding with mitochondria resultingin impaired glucose and lipid metabolism of skeletal musclesLantier Interestingly SIRT3 liverspecific knockouthepˆ’ˆ’ and skeletal musclespecific knockoutskmˆ’ˆ’mice did not aï¬ect glucose homeostasis under chow or HFDconditions FernandezMarcos The above resultsindicate that SIRT3 has an important role in metabolic regulationbut in specific physiological processes such as redox stateexercise and aging and its regulatory mechanism is yet definedin skeletal muscles Kong Robin Williams A recent study found that SIRT4 knockoutcan resist HFDinduced obesity and increase endurance exercisein mice by repressing malonyl CoA decarboxylase which wasa key enzyme controlling fatty acid betaoxidation and wasreported to regulate muscle fuel switching between carbohydratesand fatty acids Koves Laurent Moreover knockdown of SIRT4 by adenoviral shRNA canincrease the mRNA and protein content of SIRT1 therebyenhancing the expression of fatty acid oxidation genes andmitochondrial oxidation capacity in hepatocytes and myotubesNasrin Table THERAPEUTIC TARGETS OF HDACsAND THEIR POTENTIAL FORMETABOLIC DISEASESA comparative analysis used HDAC paninhibitor SAHA aclass I HDAC selective inhibitor MS275 and a class IIHDAC selective inhibitor MC1568 to treat C2C12 separatelyIt was found that only MS275 could significantly stimulatemitochondria biogenesis and oxygen consumption Galmozzi In obese diabetic mice it was found that specificallyinhibiting Class I rather than Class II HDACs improvedGTT and insulin sensitivityincreased oxidative metabolismof skeletal muscles and adipose tissue and reduced bodyweight Galmozzi Similar to the eï¬ect of MS275knockdown HDAC3 in C2C12 could also increase Pgc1αGlut4 Tfam Idh3α transcription suggesting that HDAC3 andits target genes played an important role in the above eventsGalmozzi For Class II HDACs studies have found that scriptaid a ClassIIa HDAC inhibitor has similar eï¬ects to exercise Six weeksof scriptaid administration significantly improved enduranceperformance and significantly increased the wholebody energyexpenditure and the expression of lipid oxidation related genesPdk4 Cpt1b Pgc1α Pparδ etc of C57BL6 mice Gaur One possible explanation of above observation is thepotential target of scriptaid inhibition of titin a structure proteinof sarcomere deacetylation and downstream genes of Class IIaHDACs Gaur Huang Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityIn mice treated with SPT1720 another activator of SIRT1overtly enhanced endurance exercise ability was noted Moreoverthese mice were protected from HFDinduced obesity and insulinresistance owing to an upregulation of the oxidative metabolismin skeletal muscles liver and BAT tissues Pacholec In order to further explore the principle of SIRT1 activation theresearchers purified SIRT1 in vitro and added SRT1720 and itsanalogs SRT2183 SRT1460 and RSV and they found that theenzymatic activity of SIRT1 was not enhanced suggesting thatthese drugs may indirectly activate SIRT1 Pacholec The enzymatic activity of SIRT1 largely depends on the contentof cofactor NAD This implied that the enhancement of SIRT1activity may be achieved through indirect upregulation of NADStudy has discovered that knocking out poly ADPribosepolymerase1 PARP1 which is a NAD consuming enzymecould increase NAD content and enhance the activity of SIRT1in BAT and skeletal muscles Bai PARP1 inhibitorscan upregulate the proteins of mitochondrial respiratory chaincomplexes in mice enhance the aerobic oxidation capacity ofmitochondria and improve mitochondrial defects in the primarymyotubes of obese humans Bai Pirinen et alResveratrol was initially reported as an SIRT1 activatorthat improves mitochondrial function and exercise capacity inmice and resists HFDinduced obesity Lagouge However subsequent studies have discovered that administeringthe same dose of RSV to rats or mice does not increasemitochondrial protein content Higashida Bycontrast overexpression of SIRT1 in the triceps muscle of ratsdecreases the mitochondrial protein content Higashida In a doubleblind human trial healthy and obesemen were supplied for 30day RSV in which the data showedthat RSV can improve systolic blood pressure and homeostasismodel assessment HOMA indexindicating glucose metabolismability simulating the eï¬ect of CR Timmers However some researchers found that the overexpression ofSIRT1 alone cannot mimic the CR eï¬ect in transgenic mice andthe transcriptomic changes in various tissues were quite diï¬erentor even opposite Boutant Such an opposite situationmay explain that the genetic model and compound stimulationare not completely consistent RSV as a potential metabolicsyndrome treatment drug still needs largescale populationsample verificationCONCLUSIONThe very first mammalian histone deacetylase HDAC1 wascloned and isolated by Taunton and sabout HDACs have been published in the last years CurrentlyREFERENCESAnderson J G Ramadori G Ioris R M Galie M Berglund E D CoateK C Enhanced insulin sensitivity in skeletal muscle and liver byphysiological overexpression of SIRT6 Mol Metab “ 101016jmolmet201509003we know at least HDAC proteins They are responsible foreradicating epigenetic modifications establishing an epigeneticoï¬ chromatin state and regulating heritable gene expressionYang and Seto Haberland In these processeseach HDACs may play a diï¬erent role Table What kind ofgeneprotein is the specific downstream target of these HDACsIs its enzyme activity related to intracellular localization andthe formation of multiprotein complex It is still one of thekey and difficult issues in this field Based on previous researchexperience some techniques may be used to further experimentsas follows HDACs interacting proteinscomplex coIP Proteinacetylation western Histone acetylation target geneChIP High through put proteomicsacetylome with specificHDACs inhibitor etctherapeuticare potentialHistone deacetylasestargetsand clinical drugs such as SAHA Vorinostat and FK228romidepsin have been used for antitumor treatment Bolden However they have two disadvantages greattoxic and side eï¬ects and difficulty in specific inhibition ofHDACs activity With the development of computer simulationtechnology and structural biology it is believed that more specificHDACs inhibitorsactivators can be constructed And researchesshould attach importance to HDACs regulatory factors likePARP1 when direct targeting on HDACs fails to show its eï¬ectsFurther development of their inhibitors with more specificityand trials for the treatment of metabolic diseases may have greatpotential as well

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

"Follicular dendritic cell sarcoma FDCS is a rare mesenchymal tumor that mostly occurs in systemiclymph nodes FDCS in the uterine cervix has not yet been reportedCase presentation A 49yearold woman was referred to our department with a cervical tumor which washistologically suspected to be undifferentiated carcinoma She underwent hysterectomy salpingooophorectomyand pelvic lymphadenectomy after neoadjuvant chemotherapy with paclitaxel and carboplatin The resectedspecimen contained high numbers of spindle cells and was immunohistochemically confirmed to be FDCS Thetumor was completely resected and recurrence was not detected at a 16month followupConclusion FDCS is an extremely rare malignant tumor in the uterine cervix and an accurate diagnosis andcomplete resection are essential for a good prognosisKeywords Follicular dendritic cell sarcoma Cervical cancerBackgroundFollicular dendritic cell sarcoma FDCS is a rare mesenchymal tumor that was initially reported in []Three types of tumors are derived from dendritic cellsFDCS derived from follicular dendritic cells that presentantigens to B lymphocytes in lymph follicles [] interdigitating dendritic cell sarcoma derived from the Tcellzones of lymphoid ans such as the paracortex anddeep cortex of the lymph nodes and fibroblastic reticular cell sarcoma derived from a reticular network oflymphoid ans [] An accurate diagnosis is challenging without an appropriate series ofimmunohistochemistry therefore some tumors may be diagnosed asundifferentiated carcinoma The accuracy of a diagnosisinfluences the prognosis of patients because of the lowresponse rate to established chemotherapy and radiotherapy [“] Correspondence myoshihara1209mednagoyauacjp2Department of Obstetrics and Gynecology Nagoya University GraduateSchool of Medicine Tsurumacho Showaku Nagoya Aichi JapanFull list of author information is available at the end of the Although previous studies described FDCS in the cervical lymph nodes liver stomach and tonsils FDCS inthe uterine cervix has not yet been reported We hereinpresent a 49yearold woman diagnosed with FDCS inthe uterine cervix which was successfully treated bycomplete surgery without postoperative adjuvant therapybased on a precise pathological diagnosis This is thefirst case of FDCS arising from the uterine cervix andwe described the time course of this patient from herinitial admission to diagnosis and treatment We alsoemphasized the importance of considering FDCS as adifferential diagnosis for cervical tumors because an accurate diagnosis and complete resection are essential fora good prognosisCase presentationA 49yearold woman gravida para with abnormalgenital bleeding was referred to our department with acervical mass She had a previous history of uterine myomectomy and cervical polypectomy which were both benign diseases Pelvic magneticimagingrevealed a — mm solid tumor developing from theresonance 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 0cNakamura BMC Women's Health Page of findingsrevealed aIntraoperativeand carboplatin AUC5 mgbody Massive genitalbleeding occurred months after the admission therefore the patient underwent extended total hysterectomybilateral salpingooophorectomy and pelvic lymphadenectomysmallamount of bloody ascites and the resected specimen hada necrotic mass at the posterior side of the cervix Fig Tumor cells exhibited the same histopathological characteristics as those in the previous biopsy Fig 3a Additionalimmunohistochemistry revealed cells that werepositive for both CD68 and FDC Fig 3b which wasconsistent with the diagnosis of FDCS The tumor wascompletely resected and lymphovascular invasion wasnot detected There was also no evidence of lymph nodemetastasis She did not receive adjuvant therapy and herCA19“ level decreased to within almost normal limitsTable Tumor recurrence was not detected at the month followup after admission The consent of the patient for publication was recorded according to the Ethics Committee of Nagoya University and the principlesof the Declaration of HelsinkiDiscussionThis is the first case of FDCS in the uterine cervix Afterreported surgery and chemotherapy we reached a finaldiagnosis Our experience demonstrates the difficultiesassociated with accurately diagnosing FDCS due to alack of familiarity with the pathologies of rare tumorsincluding sarcoma Although FDCS in the gynecologicalsystem is markedly rarer than other carcinomas it needsto be considered as a differential diagnosis because of itspotentiallyappropriatetreatmentprognosiswithoutfatalFDCS has been reported in approximately casesworldwide with an age range of to years and amalefemale ratio of The primary lesion of FDCS isFig Surgical specimen of the tumor The tumor with a necroticlesion arose from the posterior side of the cervix arrowheadsFig Magnetic resonance imaging of the patient at the initialpresentation A solid tumor developing from the posterior of theuterine cervix was confirmed arrowheadsposterior of the uterine cervix Fig and — and — mm spaceoccupying lesions were also detectedaround the rectus which were suspected to be lymphnode metastases Chestabdominal computed tomography CT showed no enlarged lymph nodes or distantmetastasis except for the tumors already detected Positron emission tomographyCT revealed the accumulation offluorodeoxyglucose maximum standardizeduptake value at the cervical tumor only Serumtumor marker levels were as follows carcinoembryonicantigen ngml cancer antigen125 UmLcancer antigen199 Uml squamous cell carcinomaantigen ngml soluble interleukin2 receptor Uml The other results of the blood examination including renal function liver enzymes and electrolyteswere within normal limitsBiopsy of the cervical tumor was performed and ahistopathological examination indicated that the tumorhad atypically spindle ovoid and polygonal cells with aneosinophilic hyalinerich cytoplasm with whorled andcordlike forms Nuclei were oval to round and had mildatypia Inflammatory cells were scattered to various degrees around tumor cells The results of immunohistochemical staining were as follows cytokeratin AE1AE3positive epithelial membrane antigen EMA negativeCAM52 negative cytokeratinMNF116 positive S100negative vimentin positive actin negative desminnegative cluster of differentiation CD negative CD8negative CD10 negative CD5 negative CD20 negativeCD79a negative PgR PgR inhibin negative thyroid transcription factor1 negative EpsteinBarr virusencoded RNA in situ hybridization negativeSince the tumor was suspected to be undifferentiatedcarcinoma of the uterine cervix the patient receivedchemotherapy with four cycles of paclitaxel mgm2 0cNakamura BMC Women's Health Page of Fig The histopathology of the cervical tumor The tumor had atypically spindle ovoid and polygonal cells with an eosinophilic hyalinerichcytoplasm with whorled and cordlike forms Nuclei were oval to round and had mild atypia a Tumor cells were positive for FDC bmostly in the lymph nodes in the neck axilla and mediastinum The remainder of FDCS originate from extralymphatic lesions such as the liver mesenterium stomach smallintestine pharynx tonsils retroperitoneumand ovary [“] however FDCS in the uterine cervixhas not yet been reported in the English literatureDue to the wide range of primary lesions there is noknown specific initial symptom of FDCS some patientsmay exhibit symptoms associated with an increase intumor volume such as lymph node swelling [] Sincethere are no specific findings in blood examinations orimaging studies difficulties are associated with makingan accurate diagnosis prior to the initiation of treatmentThe diagnosis of FDCS is pathologically confirmed usingbiopsy or resected specimens however due to the rarityof this tumor it is important to conduct appropriate immunohistochemistry in consideration of FDCS from thefindings of hematoxylin and eosin staining The tumorcells of FDCS are spindle to epithelioid in shape with thecoexistence of multinucleated cells Single nuclear inflammatory cells infiltrate and form bundles flowers andswirls structures Some giant cells and ReedSternbergcells are also detected in these tumors To reach a definitive diagnosis of FDCS FDC markers such as CD21CD35 KiM4p and CNA42 are used to distinguish itfrom other tumors that may have a mesenchymal structure such as undifferentiated carcinoma meningiomaand paraganglioma [ ] Alternatively FDCS may befound within lesions of Castleman™s disease particularlyits hyalinevascular type [] It has been reported thatFDCS occurred after the excision of a lesion of hyalinevascular type Castleman™s disease and occupied most ofthe lesions It is also important to distinguish FDCSfrom inflammatory pseudotumorlike FDCS associatedwith EpsteinBarr virus This tumor is often found in theabdominal ans particularly in the liver and spleenand has the characteristics of positive FDCS markersand the detection of EBV by in situ hybridization It progresses more slowly than FDCS and longterm survivalhas been reported even after recurrence []The treatment for cervical cancer principally involvessurgeryradiation and chemotherapy New surgicaltechniques such as laparoscopic radical hysterectomyand sentinel lymph node biopsy have also become available [ ] On the other hand a basic therapeuticstrategy for FDCS is prioritized to guarantee completesurgical tumor resection because of the low responserate to established chemotherapy and radiotherapy [“] Saygin examined patients with FDCS andreported 2year survival rates of and forearlylocally advanced and distant metastasis diseaserespectively Patients who underwent complete tumorresection had a better prognosis than those with unresectable localized tumors Furthermore no prognosticdifference was observed between the surgery group andpostoperative radiotherapy groups A large tumor sizelarger than cm and lymphoplasmacytic cell invasionhave been identified as poor prognostic factors [] InTable Level of tumor markers in each followup periodTumor marker Normal rangeCA125 UmL AdmissionCA199 UmL CEA UmL SCC ngmL At surgery m after admissionAt final followup m after admissionsIL2R UmL Abbreviation CA cancer antigen CEA carcinoembryonic antigen SCC squamous cell carcinoma antigen sIL2R soluble interleukin2 receptor 0cNakamura BMC Women's Health Page of the present case as the tumor remained localized andwas completely resected the patient could follow favorable clinical course without any tumor recurrenceConclusionFDC sarcoma is a rare tumor that may develop in theuterine cervix FDCS needs to be considered when confirming a mesenchymal cervical tumor and appropriateimmunohistochemistry needs to be performed for bothan accurate diagnosis and selection of a therapeuticstrategyAbbreviationsFDC Follicular dendritic cell sarcoma FDCS Follicular dendritic cellCT Computed tomography CD Cluster of differentiationAcknowledgmentsThe authors thank Dr Sakata and Dr Hirata for supporting the managementof the case with patience and knowledgeAuthors™ contributionsTN and MY developed the study concept interpreted the data and wrotethe manuscript ST and HK participated in designing the study and plannedthe investigations FK drafted the study design and supervised all of thework All authors have approved the final manuscriptShia J Chen W Tang LH Carlson DL Qin J Guillem JG Extranodalfollicular dendritic cell sarcoma clinical pathologic and histogeneticcharacteristics of an underrecognized disease entity Virchows Arch “Ruco LP Gearing AJ Pigott R Pomponi D Burgio VL Cafolla A et alExpression of ICAM1 VCAM1 and ELAM1 in angiofollicular lymph nodehyperplasia Castleman's disease evidence for dysplasia of folliculardendritic reticulum cells Histopathology “ Maeda K Matsuda M Suzuki H Saitoh HA Immunohistochemicalrecognition of human follicular dendritic cells FDCs in routinely processedparaffin sections J Histochem Cytochem “Arber D Kamel O van de Rijn M Davis RE Medeiros LJ Jaffe ES et alFrequent presence of the EpsteinBarr virus in inflammatory PseudotumorHum Pathol “ Casarin J Bogani G Papadia A Ditto A Pinelli C Garzon S et alPreoperative Conization and risk of recurrence in patients undergoinglaparoscopic radical hysterectomy for early stage cervical cancer amulticenter study J Minim Invasive Gynecol “ Rossetti D Vitale SG Tropea A Biondi A Lagan  AS New procedures for theidentification of sentinel lymph node shaping the horizon of futureManagement in Early Stage Uterine Cervical Cancer Updat Surg “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsFundingNo funding was obtained for this studyAvailability of data and materialsNot applicableEthics approval and consent to participateNot requiredConsent for publicationWritten consent to publish this information was obtained from studyparticipantsCompeting interestsAll authors declare that there are no competing interestsAuthor details1Department of Obstetrics and Gynecology Anjo Kosei Hospital Anjo AichiJapan 2Department of Obstetrics and Gynecology Nagoya UniversityGraduate School of Medicine Tsurumacho Showaku Nagoya AichiJapanReceived April Accepted August References Monda L Warnke R Rosai J A primary lymph node malignancy withfeatures suggestive of dendritic reticulum cell differentiation A report of cases Am J Pathol “Tew JG Kosco MH Burton GF Szakal AK Follicular dendritic cells asaccessory cells Immunol Rev “Saygin C Uzunaslan D Ozguroglu M Senocak M Tuzuner N Dendritic cellsarcoma a pooled analysis including cases with presentation of ourcase series Crit Rev Oncol Hematol “Pileri SA Grogan TM Harris NL Banks P Campo E Chan JK Tumoursof histiocytes and accessory dendritic cells an immunohistochemicalapproach to classification from the international lymphoma study groupbased on cases Histopathology “Chan JK Fletcher CD Nayler SJ Cooper K Follicular dendritic cell sarcomaClinicopathologic analysis of cases suggesting a malignant potentialhigher than currently recognized Cancer “ 0c"

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

Hydrophobicity drives receptormediateduptake of heatprocessed proteins by THP1macrophages and dendritic cells but notcytokine responsesYing Deng12 Coen Govers1 Malgorzata Teodorowicz3 Ieva Liobyte12 Ilaria DeSimone13 Kasper Hettinga4 Harry J WichersID12 Food and Biobased Research Wageningen University and Research Wageningen The Netherlands Laboratory of Food Chemistry Wageningen University and Research Wageningen The Netherlands Cell Biology and Immunology Wageningen University and Research Wageningen The Netherlands Food Quality and Design Wageningen University and Research Wageningen The Netherlands harrywicherswurnlAbstractAlthough an impact of processing on immunogenicity of food proteins has clearly been demonstrated the underlying mechanisms are still unclear We applied different processingmethods wet heating ˚C and low or hightemperature ˚C or ˚C respectivelydryheating in absence or presence of reducing sugars to lactoglobulin BLG lysozymeand thyroglobulin which represent dietary proteins with different pI or molecular weightUptake of the soluble fraction of the samples was tested in two types of genetically homogeneous antigenpresenting cells macrophages and dendritic cells derived from THP1monocytes This revealed a strong correlation between the uptake of the different proteinsamples by macrophages and dendritic cells and confirmed the key role of hydrophobicityover aggregation in determining the uptake Several uptake routes were shown to contribute to the uptake of BLG by macrophages However cytokine responses following exposureof macrophages to BLG samples were not related to the levels of uptake Together ourresults demonstrate that heattreatmentinduced increased hydrophobicity is the prime driving factor in uptake but not in cytokine production by THP1 macrophagesIntroductionDietary proteins play an important physiological role not only in providing amino acids andenergy but also for the maturation and regulation of the immune system Proteins are knownto be involved in the regulation of chronic inflammation and be the cause of allergies [] Forexample a higher intake of animal protein was found to enhance the proinflammatoryresponse of macrophages in mice [] Most of the proteins that humans ingest have beenthrough a heating process as part of the food processing as a result of which structural modifications such as glycation with reducing sugar andor aggregation of proteins may occura1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Deng Y Govers C Teodorowicz MLiobyte I De Simone I Hettinga K Hydrophobicity drives receptormediated uptake ofheatprocessed proteins by THP1 macrophagesand dendritic cells but not cytokine responses e0236212 101371journalpone0236212Editor Yi Cao Xiangtan University CHINAReceived June Accepted August Published August Copyright Deng 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 paper and its Supporting InformationfilesFunding This work was financial supported by theChina Scholarship Council No awarded to YDCompeting interests The authors have declaredthat no competing interests existPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseDifferent processing methods would alter the immunogenicity of food protein in differentmanner For example the antigenicity of prawn allergen was found to be significantlyincreased after frying but decreased after boiling acid treatment or highpressure processing[] The allergenicity of milk protein was reported to decrease after glycation []In an earlier study on lactoglobulin BLG the major whey protein of milk [] weobserved that heat processing under different conditions altered the physicochemical properties and its uptake by THP1 macrophages In particular heating in solution at ˚C for days introduced changes in hydrophobicity and molecular weight which were shown to be thekey determinants for uptake This event may be relevant for a possible subsequent immuneresponse [ ] and has been linked to heattreatmentinduced changes in protein™s pr sityto induce an allergic reaction [“] Despite the new findings in that study there are obviouslimitations relate to the use of a single protein cell phenotype and immunological readoutHere we extend previous findings by investigating other proteins lysozyme and thyroglobulin another cell phenotype THP1 dendritic cells and different immunologicalresponses to yield novel insight Lysozyme and thyroglobulin were subjected to the same heattreatments as used for BLG Lysozyme from chicken egg white is a single chain polypeptidewith four disulphide bridges and is a rather inflexible protein with stable structure and properties [] It has a molecular weight of kDa which is similar to kDa of BLG and an isoelectric point pI of [] which is clearly different from BLG pI [] resulting in apositive net charge of this protein at the physiological pH Moreover it is also known as anallergen as exemplified by the high incidence of sensitization and allergenicity to this protein[] On the other hand bovine thyroglobulin which originates from follicular cells of the thyroid gland is a protein that is considered as less allergenic It has a molecular weight of kDa as a monomer making it more than times larger than the mass of the other two proteins under investigation It has a complex quaternary structure composed of four subunitsthat are disulphide bonded [ ] Its pI is close to that of BLG conferring it a similarcharge as BLG at the pH of the exposure medium We analysed uptake of heattreated proteinsby macrophages and dendritic cells and correlated this to physicochemical characteristics Furthermore we investigated the mechanism and receptors that are involved in uptake and identified downstream immunological responses by quantifying cytokine responseTaken together our findings provide insights into the role of protein size and pI towardsphysicochemical changes upon foodprocessing related heattreatments This appears to yieldgeneralisable correlations between a protein™s physicochemical characteristics and its uptakeby antigenpresenting cells APCs Finally we identified routes of uptake and downstreamimmunological responses which will help to clarify the interaction of protein ps withmacrophages and the potential immunoregulatory effect The results of this study could helpto better understand how the immunogenicity of food proteins can be modulated throughprocessingMaterials and methodsChemicalsAll chemicals were purchased from Sigma Aldrich St Louis Missouri USA unless otherwisestatedSample preparation and fluorescent labellingLactoglobulin BLG from cow™s milk lysozyme from chicken egg white and thyroglobulinfrom bovine thyroid were dissolved in sodium phosphate buffer mM pH same belowto a protein concentration of mgmL Besides this solution without saccharides DglucosePLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseglu Dlactose lac and galactooligosaccharide GOS Royal FrieslandCampina Wageningen the Netherlands were added to reach a final molar ratio of total free amino groups tosaccharide reducing ends Identical heating methods being hightemperature dryheating Hwetheating W and lowtemperature dryheating L sample preparation and naming wereapplied to the proteins as indicated in a previous study [] Fluorescein isothiocyanate isomer IFITC labelling was performed as described previously []THP1 cell culture and differentiationThe human monocytic leukaemia cell line THP1 ATCC Manassas Virginia USA was differentiated into cells characteristic for resting macrophages M0 as described elsewhere []at a concentration of — cellsmL using ngmL phorbol12myristate13acetate PMAfor a hours stimulation followed by washing two times with medium and another hoursof rest Using a cell concentration of — cellsmL and incubation with ngmL of IL4and ngmL of PMA for days as described [] immature dendritic cells were generatedfrom THP1 monocytesPhysicochemical analysisThe analysis of solubility protein mass remaining in solution loss of amino group OPAmethod AGE formation fluorescent measurement exposure of hydrophobicity regionANS method surface charge zetapotential measurement secondary structure circulardichroism measurement and aggregation size exclusion chromatography were performedusing the protocols as described previously []Uptake and blocking of different uptake routesUptake experiments were performed as described previously [] No significant difference ofabsolute uptake value has been found for native BLG thyroglobulin or lysozyme in macrophages or dendritic cells S1 Fig As there is no bias for the basic uptake capability for the different proteins all the uptake results were presented relative to their corresponding nativeprotein to be able to compare among different proteins To determine which route of uptakewas involved in a protein™s uptake by THP1derived cells the cells were preincubated for minutes with DMSO control or different inhibitors μgmL nystatin caveolaedependentuptake inhibitor μgmL chlorpromazine clathrindependent uptake inhibitor μgmLcytochalasin B microphagocytosis inhibitor or all inhibitors combined dissolved in DMSOFollowing incubation the cells were washed with PBS and incubated for hours with FITClabelled protein samples Subsequently the cells were harvested and measured using flowcytometry as described previously [] For all uptake experiments μl trypan blue was addedper μl of cell suspension to quench extracellular FITC signal before flow cytometry analysis Uptake was calculated as either the fold change in mean fluorescence intensity MFI relative to control after correcting for FITC labelling efficiency control or as percentage tocontrol control Soluble Receptor for Advanced Glycation End Products sRAGE CD36and galectin3 inhibition ELISAThe experiment was performed according to the method of Liu [] As a positive control soy protein Bulk Powder Colchester UK was mixed with Dglucose in an equal wwratio in mM PBS buffer pH to a protein concentration of mgmL and heated for minutes at ˚C The positive control was diluted in mM of sodium carbonate buffer pHPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine response to μgml and used for plate coating by adding μL per well to a Nunc MaxiSorp„¢ flatbottom plate Thermo Fisher Waltham Massachusetts USA and incubating overnight at ˚C Then the plate was washed times with PBS buffer pH containing vv Tween After blocking with bovine serum albumin BSA in PBS for hour at room temperature the wells were washed times and incubated with μL protein samples μgmL at ˚C for hour The samples were preheated at ˚C for minutes with recombinanthuman sRAGE CD36 or galectin3 at a concentration of or μgmL RD SystemsMinneapolis Minnesota United States in PBS buffer with BSA and Tween20For detection the plate was washed times and incubated with μL per well of monoclonalmouse IgG2B human sRAGE or galectin3 antibody RD Systems Minneapolis MinnesotaUnited States at a concentration of μgml for minutes shaking at room temperatureAfter washing times the plate was incubated for minutes while shaking at room temperature with μL per well polyclonal goat antimouse HRPconjugated DAKO Glostrup Denmark at a concentration μgmL For galectin3 an additional incubation step with μgml Streptavidin SDT Baesweiler Germany μL per well for minutes was followed For CD36 only incubation with goat antihuman IgGHRP SouthernBiotech Birmingham Alabama United States at a concentration of μgml for minutes shaking atroom temperature was used for the detection step For all receptor inhibition ELISAs the platewas then washed times and μL per well TMB substrate was added and incubated for minutes before stopping the reaction by adding uL of HCl per well The absorbancemeasured at nm by an Infinite1 PRO NanoQuant Tecan Ma¨nnedorf Switzerlandwas subtracted from the measured absorbance at nm Each sample was measured in triplicate and values were averaged Absorbance of buffer was used as a control and was subtractedfrom every sample The absorbance of the control solution without any inhibition was codedby Abscontrol The percentage of inhibition for samples was equal to AbscontrolAbssampleAbscontrol�Cytokine production measurementTHP1 macrophages were incubated with μgmL of protein sample or medium as controlfor hours after which the supernatant was collected The cytokine production was measured using the ELISA Deluxe Set Human IL8CCL20IL1 Biolegend San Diego California United States following the manufacturer™s protocolStatisticsThe statistical analysis was performed using Prism software GraphPad Software San DiegoCalifornia United States with p considered to be significant The correlation analysiswas done by calculating the Pearson correlation coefficient r and the twotailed p value ThePCA plot was generated with the dataset of variables after being mean centred and weightedby 1standard deviation using Unscrambler software CAMO Oslo NorwayResultsWetheating significantly increased uptake of thyroglobulin by THP1derived macrophages whereas heattreatment did not increase uptake oflysozymeThyroglobulin and lysozyme were wetheated W hightemperature dryheated H or lowtemperature dryheated L in the absence or presence of saccharides with different lengthsmonosaccharide glucose disaccharide lactose or oligosaccharide GOS Soluble proteinPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responsefrom each sample was concentration calibrated fluorescently labelled and incubated with resting macrophages that were derived from THP1 monocytes Hightemperature dryheating ofthyroglobulin in the presence of glucose and lactose and lysozyme in the presence of glucoseled to complete insolubility S2A and S4A Figs and therefore these samples were excludedfrom further analysis Prior to the experiment the lipopolysaccharide LPS contaminationwas measured S1 Table and found to be below the threshold level that significantly affecteduptake capacity of THP1 macrophages ie ngmL as reported in an earlier study []For uptake of thyroglobulin samples by macrophages Fig 1A the type of heat treatmentappeared to play a more determining role than the presence or absence of saccharides Notably all wetheated samples regardless of the presence of saccharides showed significantlyincreased uptake compared to untreated protein Although not significantly differing fromuntreated protein high and lowtemperature dryheating increased the uptake of thyroglobulin as well On the contrary no significant differences in uptake of lysozyme after differenttreatments and saccharide exposures was noticeable Fig 1BUptake of heattreated protein samples by THP1derived immaturedendritic cells is qualitatively similar to that of THP1derivedmacrophagesThe uptake of processed thyroglobulin and lysozyme as well as BLG which was previouslyreported [] was only tested for macrophages To expand the understanding of the proteins™uptake beyond only macrophages THP1derived immature dendritic cells iDC were alsotested with identical methodology as for macrophages The uptake of wetheated thyroglobulinin the absence or presence of saccharides by these iDC was significantly increased compared tothe native protein Fig 2A For lysozyme the relative uptake remained the same regardless ofthe treatment Fig 2B The uptake of wetheated BLG in the absence or presence of saccharidesby iDCs was also increased compared to the native form although only reaching significanceFig Uptake of thyroglobulin by macrophages was significantly increased upon wetheating of the sample Thyroglobulin THY A and lysozymeLYS B were untreated or heated W H or L in the absence or presence of saccharides glu lac or GOS THP1derived macrophages Mϕ wereincubated with FITClabelled soluble fraction of protein samples for hours after which uptake was determined using flow cytometry The resultsrepresent mean values ± SD of n measurement of independent experiments based on independent sample sets Statistical differences comparedto native proteins were calculated with Dunnett™s Test �p ��p 101371journalpone0236212g001PLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Uptake of thyroglobulin lysozyme and BLG samples by THP1derived iDC was strongly and significantly correlated with uptake bymacrophages AC Thyroglobulin THY lysozyme LYS and BLG were untreated or heated W H or L in the absence or presence of saccharidesglu lac or GOS THP1derived iDC were incubated with FITClabelled soluble fraction of protein samples for hours after which uptake wasdetermined using flow cytometry The results represent the mean values ± SD of n measurement of independent experiments based on independent sample sets Statistical differences compared to native proteins were calculated with Dunnett™s Test ��p ���p D Therelative uptake values of all protein samples by iDC was plotted against macrophages Mϕ and the correlation analysis was done by calculating thePearson correlation coefficient r and twotailed p value101371journalpone0236212g002when heattreated in the presence of glucose Fig 2C When compared to the results obtainedwith macrophages the increase in uptake upon protein wetheating for iDC was less pronounced BLGmacrophage data from previous study [] However the uptake pattern of allsamples correlated strongly r p between the two cell types Fig 2DIncreased uptake of thyroglobulin by THP1derived macrophages wascorrelated to hydrophobicity and aggregationDue to the strongly correlated responses of iDC and macrophages in uptake with a higher relative uptake by macrophages the latter was used for further testing To explain thePLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responsephysicochemical mechanism behind the differing uptake capability of macrophages for differently treated thyroglobulin and lysozyme a set of measurements was performed on the solublefraction after treatmentFor thyroglobulin dryheating at hightemperature significantly decreased its solubilityresulting in the absence of a soluble fraction when heating was performed in the presence ofglucose and lactose S2A Fig The soluble fraction of samples in the presence of GOS or without saccharide revealed a strong decrease of free amino groups and increases in AGErelatedfluorescence and formation of polymers in the presence of GOS S2B S2C and S3 Figs Wetheating did not result in loss of solubility but also led to formation of polymers and albeit lesspronounced as for hightemperature dryheating significant losses of amino groups andincreases in AGErelated fluorescence Moreover wetheating induced a significant increasein the exposure of hydrophobic regions as measured by ANSbinding S2D Fig Dryheatingat lowtemperature did not have much influence on the measured parameters for thyroglobulin except for a significant loss of amino groups and a slight increase in oligomer and polymerformation S2B and S3 Figs There was no significant change in the secondary structure forany of the treated thyroglobulin samples S2E and S2F FigApplying identical heating and glycation conditions as for thyroglobulin did not generateany soluble aggregates for lysozyme checked by size exclusion chromatography Hightemperature dryheating led to a significant decrease of solubility with complete insolubility forthe samples heated in the presence of glucose S4A Fig Among the soluble fraction of thehightemperature dryheated samples there was a significant decrease of free amino groupsincreases of AGErelated fluorescence and exposure of hydrophobic regions in the presence ofsaccharides S4B“S4D Fig Moreover results showed a significant loss of sheet structure forall samples except when heated in the presence of GOS S4F Fig Similarly lowtemperaturedryheating resulted in a significant loss of sheets in all lysozyme samples For wetheatedlysozyme only the exposure of hydrophobic regions in the presence of saccharides was significantly increased S4D FigTo identify the most important physicochemical properties of heatprocessed thyroglobulinthat are related to its uptake by macrophages we performed a principle component analysisPCA and a correlation analysis In the PCA plot Fig all lowtemperature dryheated samples clustered together with the untreated thyroglobulin indicating that the physicochemicalproperties of these samples were similar Uptake was strongly related with wetheating andpositively related to hydrophobicity ANS and polymer proportion and inversely to monomer and oligomer proportion These relations were further confirmed by correlation analysesAs shown in Fig there was a strong correlation between uptake and hydrophobicity and amoderate correlation between uptake and aggregation as indicated by the fraction of monomers polymers and oligomers Of note is that the samples were strongly clustering into twogroups at the extremes of the data scale for both the uptake and monomer or polymer contentcorrelation plots Fig 4B and 4C the correlation between uptake and oligomer content wasless profound Fig 4D p Contrastingly to thyroglobulin no clear correlation could befound between treatment and physicochemical properties for lysozyme in the PCA plotS5 FigEDCmediated increase in hydrophobicity but not crosslinking is themain factor driving uptake by macrophagesEDC links the carboxyl and amino group of amino acids and thereby offers the possibility tocrosslink proteins without heating This allows for a discrimination between aggregation andother heatingrelated effects like hydrophobicity changes with regard to their impact onPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Uptake of thyroglobulin by macrophages is related to wetheating hydrophobicity and aggregation Thyroglobulin THYwas untreated or heated W H or L in the absence or presence of saccharides glu lac or GOS and tested for solubility uptake ofsoluble fraction by THP1 macrophages uptake AGE formation AGE glycation OPA percentage of αhelix helix or sheetsheet aggregation monomer and smaller mono oligomers oligo polymers poly and exposure of hydrophobic regions ANSPCA scores of the samples were given in blue and the parameter loadings of the principal components were given in red The clusteringof uptake with wetheated samples the formation of hydrophobic regions and polymer proportion is indicated in the red circle101371journalpone0236212g003uptake As shown in Fig 5A and 5B the proportion of polymer and monomer significantlyincreased resp decreased in the EDC crosslinked BLG and thyroglobulin On the contraryonly limited aggregation of lysozyme was observed upon EDCtreatment EDC crosslinkedBLG and thyroglobulin did not demonstrate an increase in uptake Surprisingly EDCmediated crosslinking significantly increased the hydrophobicity of lysozyme Fig 5C and also itsuptake by macrophages Fig 5DUptake of heatedtreated BLG was mediated via several routesTo better understand the mechanism of uptake we set out to identify which routes of uptakewere utilized for the various proteins that differed in physicochemical characteristics andtherefore in their recognition motifs for cells Native and heattreated BLG in the presence ofglucose was used as a subset because these samples covered the whole range of levels of physicochemical modifications and macrophages were pretreated with inhibitors targeting phagocytosis caveolae or clathrinmediated uptake or a combination thereof Preincubation ofmacrophages with nystatin caveolae inhibitor and the combination of inhibitors prior toBLG samples exposure significantly inhibited the uptake Fig The usage of cytochalasinmicrophagocytosis inhibitor significantly decreased the uptake of both hightemperaturedryheated and wetheated BLG Fig 6B and 6C Moreover chlorpromazine significantlyPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Correlation analysis of uptake of heattreated thyroglobulin with physicochemical parameters Uptake of soluble fraction ofthyroglobulin THY samples by macrophages was correlated to hydrophobicity r07 and proportion of monomer polymer and oligomer07r05 The correlation analysis was done by calculating the Pearson correlation coefficient r and twotailed p value101371journalpone0236212g004reduced uptake of wetheated BLG indicating that clathrin was involved in this processFig 6CInhibition ELISA showed high binding of hightemperature dryheated andwetheated BLG by sRAGE CD36 and galectin3We investigated several receptors that might be involved in binding and subsequent uptake ofthe BLG samples that were heattreated in the presence of glucose Using an inhibition ELISAthe binding of the BLG samples to the soluble receptor for advanced glycation end productssRAGE CD36scavenger receptor and galectin3 was analysed Fig Native BLG did notshow any binding to sRAGE galectin3 or CD36 In contrast hightemperature dryheating ofBLG induced significant receptor binding for all three tested receptors Wetheating of BLGsimilarly resulted in binding to all three receptors albeit to a lesser extent and only significantly for sRAGE and galectin3 Finally lowtemperature dryheating did not induce anyreceptorbindingPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig EDCmediated hydrophobicity but not aggregation resulted in increased uptake of proteins by THP1 macrophagesThyroglobulin THY lysozyme LYS and BLG were nontreated or treated with EDC and subsequently analysed for the proportion ofpolymer A the proportion of monomerdimer B and protein hydrophobicity C EDCtreated and nontreated proteins were fluorescentlylabelled and their uptake by macrophage Mϕ was measured D The results represent the mean values ± SD of “ independent experimentswith independent sample sets Statistical differences were calculated with twotailed unpaired Ttest with Welch™s correction �p ��p ���p 101371journalpone0236212g005Cytokine responses of macrophages following exposure to BLG werereduced upon heattreatment of BLGReceptor binding and uptake of protein samples can lead to immune activity The immunological response could be estimated by the expression of secreted cytokines or chemokines Uponexposure to medium or native or heattreated BLG in the presence of glucose the response ofmacrophages was analysed by measuring the levels of secreted IL8 CCL20 and IL1 Asshown in Fig nonprocessed native BLG and lowtemperature dryheated BLG induced significant levels of IL8 CCL20 and IL1 secretion in macrophages when compared to mediumPLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Several routes appeared to be involved in the uptake of wetheated BLG BLG was untreated or heated W H or L in the presence of glucose gluand the soluble fraction was FITClabelled Macrophages were pretreated with DMSO control or inhibitors dissolved in DMSO of clathrindependentuptake chlorpromazine chlor caveolaedependent uptake nystatin nys microphagocytosis cytochalasin cyto or a combination of them combinedfor min before a 2hour incubation with protein samples Protein uptake was subsequently measured using flow cytometry The relative MFI wascalculated by dividing the MFI of a sample by the MFI of the corresponding control The results represent mean values ± SD of multiple independent cellexperiments N “ with independent sample sets The statistical differences were calculated with onetailed unpaired Ttest compared to control�p ��p ���p 101371journalpone0236212g006Hightemperature dryheating of BLG significantly reduced the secretion of all three analysedcytokines when compared to native BLG A similar reduction was observed in wetheated BLGfor IL8 and IL1 but less strongly and not significantly for CCL20DiscussionIn an earlier study we observed that hydrophobicity and the aggregation state of heatprocessed BLG were important parameters that were related to the protein uptake by THP1PLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseFig Heattreatment in the presence of glucose confers receptor binding capacity to BLG BLG was untreated orheated W H or L in the presence of glucose glu and the soluble fraction was used to inhibit the binding of receptorto a positive control in the inhibition ELISA Data shown are the mean values ± SD of n based on measurement of parallel prepared samples Statistically significant differences between native and processed BLG for each receptorwas determined using unpaired twotailed tTest �p ��p ���p 101371journalpone0236212g007macrophages [] Here we extended these findings by analysing whether similar relationswould apply for a protein with similar pI but higher molecular weight thyroglobulin aprotein with similar molecular weight but higher pI lysozyme EDCaggregated proteinsand another cell type by using THP1 dendritic cells The comparison between THP1derived macrophages and dendritic cells regarding uptake revealed similar patterns for thethree tested proteins and the different heattreatments Macrophages and DCs are both able tocapture exogenous ps and induce an immunogenic response playing an essential role inFig Cytokine and chemokine responses of macrophages upon exposure to native or heattreated BLG varies BLG was untreated or heated W Hor L in the presence of glucose and the soluble fraction was incubated with macrophages The cytokine secretion of IL8 A CCL20 B and IL1 Cwas analysed in the supernatant with ELISA The results represent mean values ± SD of “ independent experimental measurements The statisticaldifferences were calculated with Tukey™s Test with different letters a“c in the bars indicated significantly different p 101371journalpone0236212g008PLOS ONE 101371journalpone0236212 August PLOS ONE 0cUptake of heatprocessed proteins by THP1 macrophages and dendritic cells and subsequent cytokine responseimmunological defence [] We did observe however a quantitative difference in uptake asthe uptake values for wetheated thyroglobulin and BLG were relatively higher in macrophagesthan in DCs This suggests that macrophages are more efficient in their phagocytic role thanDCs which is in line with literature stating that uptake by macrophages is part of their corerole in eliminating harmful substances whereas uptake by DCs is a means to initiating anadaptive immune response [] Upon comparing the three tested proteins we found that thephysicochemical alterations upon heattreatment to thyroglobulin and BLG were similar butdifferent from lysozyme These proteins differ in size thyroglobulin kDa BLG kDa lysozyme kDa pI lysozyme BLG thyroglobulin anddenaturation temperature lysozyme ˚C BLG ˚C thyroglobulin ˚C [“]suggesting that both pI and heat stability may be important for heatinduced structural proteinchanges The high pI may have conferred a positive charge to lysozyme during wetheatingpH thereby hindering aggregation under this condition due to electrostatic repulsionThe strong internal coherence and stability of lysozyme™s structure may also have contributedto its stability during heat processing [] It is reported

Thyroid_Cancer

" The adopted strategy was the same as that used in prior years [] and is based on four exclusive queries that return four disjoint citation subsets The first query QPub_plain is based on a plaintext search in PubMed titles and s using keywords The second query QPub_indexed relies on the PubMed indexing scheme using MeSH terms and results are made exclusive of the previous set The third one QWoS_restricted is based on a plaintext search in WoS restricted to the two research areas œMedical Informatics and œHealth Care Sciences Services The fourth query QWoS_filtered is based on the same plaintext search used in WoS but filtered by nonrelevant research areas eg Archeology Dance Zoology etc and the two research areas of the previous query It is of note that the two WoS queries select only nonPubMedindexed papers that are supposed to be caught by the two PubMed queriesA first review of the four subsets of retrieved citations was performed by the two section editors to select candidate best papers Following the IMIA Yearbook protocol these candidate best papers were then individually reviewed and rated by both section editors the chief editor of the Decision Support section and external reviewers from the international Medical Informatics community Based on the reviewers™ ratings and comments the Yearbook editorial committee then selected the best papers of the year in the decision support domainIMIA Yearbook of Medical Informatics IMIA and Ge Thieme Verlag KG 0cReview Results The literature search has been performed on January A total of unique references were obtained distributed as follows for QPub_plain for QPub_indexed for QWoS_restricted and for QWoS_filtered yielding subtotals of references from PubMed and from WoS Compared to the previous year the global query retrieved more papers After a first individual screening independently performed by both section editors based on the title and of papers not rejected by both section editors were discussed by the two editors to achieve a final selection of candidate best papers After the external review of these s the editorial committee finally selected three of them as best papers for [“] Table They are discussed in the next section and summaries of their contents are available in the AppendixDiscussion and OutlookIn the first paper Hendriks [] propose an approach to the modeling of clinical practice guidelines which certainly builds on already existing approaches but which is systematically conducted in order to be scalable and used to represent complex guidelines They promote the formalism of clinical decision trees CDTs as they are both clinically interpretable by healthcare professionals and computerinterpretable thus suitable for implementation in datadriven CDSSs The disambiguation of textual guidelines is supported first by the formal unequivocal specification of data items used as decision criteria using international coding systems to enforce interoperability and second by the representation of guideline knowledge as CDTs The method is applied to the Dutch breast cancer guidelines Sixty CDTs were built involving a total of data items among which could not be linked to standard terminologies The authors report the ambiguity of certain criteria which could be subjective or had multiple definitions The resulting knowledge base was implemented in a decision support application where it can be interactively browsed or automatically executed By modeling guidelines in such a way this work is a step forward in the sharing of encoded knowledgeIn the second paper KamiÅ¡alić [] tackled the issues linked to the formalization of the medical processes used for managing chronic diseases and their execution in CDSSs They analyzed the decisionmaking dimensions of the therapeutic management of chronic diseases like those known to increase the cardiovascular risk and identified three basic levels therapy strategy dosage adaptation and intolerance management To handle these different aspects consistently they propose a formalism called extended Timed Transition Diagram eTTD With eTTDs they illustrate the multilevel and finegrained modeling required to capture the contents of arterial hypertension management guidelines This detailed demonstration on how procedural knowledge for hypertension management can be formalized to develop a CDSS could certainly be used in other medical domainsThe third paper by Khalifa [] presents a conceptual and practical framework to help assess confidence in predictive tools GRASP for Grade and Assess Predictive Tools is both a method to look for evidence from the published literature and an analysis grid It standardizes the assessment of the available literature associated to a predictive tool and the grading of its level of proof Three phases of evaluation are considered i before the implementation of the tool to assess both its internal and external validity ii during the implementation to assess its potential effect and usability and iii after the implementation to assess its effectiveness and safety In each phase the level of evidence can be assessed from the study design A qualitative summarizes the direction of evidence positive negative mixed This grid can be considered as similar to existing grids for instance the CONSORT statement for clinical trials However it gives a rigorous methodology for a critical appraisal of predictive tools and could be extended to all kind of CDSSs It might be a useful tool to extend the evidencebased culture in the field of medical informaticsBesides the three best papers selected for the Decision Support section of the edition of the IMIA Yearbook several other works retrieved from the literature review deserve to be cited Some of them deal with the personalization of decisions Laleci [] propose a scientific and technical approach to develop personalized care plans that comply with clinical practice guidelines for the management of complex polypathology situations Jafarpour [] propose a solution to dynamically manage the conflicts that can rise in this type of complex contexts Ben Souissi [] introduce the use of health information technology involving multiple criteria decision to support the choice between antibiotics alternatives Interestingly other works promote the creation and sharing of operational knowledge bases as exemplified by Hendriks [] Thus Huibers [] transform the textual STOPPSTART criteria into unambiguous definitions mapped to medical terminologies Canovas et al [] formalize EUCAST expert rules as an ontology and production rules to detect antimicrobial therapies at risk of failure M¼ller [] propose an diagnostic knowledge base that can compete with commercial ones Replacing humans is another topic of research and Spnig [] work on two aspects to virtualize a doctor the automatic acquisition of data through sensors and speech recognition and the automation of diagnostic reasoning Rozenblum et al[] propose a machine learning method to generate clinically valid alerts to detect errors in prescriptions Acceptability of CDSS is another key point Kannan [] propose a method for a CDSS design to best meet a precisely specified and assessable user purpose Design alerts may also avoid rejection of CDSSs by caregivers Fernandes [] created algorithms able to aggregate filter and reduce the notifications delivered to healthcare professionals Amrose et al [] tried to understand in real life the impact of alerts on users and to find the actions they triggered Finally it is always interesting to obtain varied evaluation results of controversial CDSSs In this respect Kim [] evaluated Watson for Oncology in thyroid carcinoma and reported a concordance rate with local practices considered as too low to adopt the tool As evidenced by the number and the variety of works around decision support research in the field is very active This year™s selection highlighted pragmatic works that promote the transparency and sharing of the IMIA Yearbook of Medical Informatics 2020Duclos 0cTable Best paper selection of s for the IMIA Yearbook of Medical Informatics in the section 'Decision Support' The s are listed in alphabetical order of the first author™s surname Section Decision Support\uf0a7 Hendriks MP Verbeek XAAM van Vegchel T van der Sangen MJC Strobbe LJA Merkus JWS Zonderland HM Smorenburg CH Jager A Siesling S Transformation of the National Breast Cancer Guideline Into DataDriven Clinical Decision Trees JCO Clin Cancer Inform \uf0a7\t KamiÅ¡alić\tA\tRia±o\tD\tKert\tS\tWelzer\tT\tNemec\tZlatolas\tL\tMultilevel\tmedical\tknowledge\tformalization\tto\tsupport\tmedical\tpractice for chronic diseases Data Knowledge Engineering “\uf0a7 Khalifa M Magrabi F Gallego B Developing a framework for evidencebased grading and assessment of predictive tools for clinical decision support BMC Med Inform Decis Mak knowledge bases used by decision support tools as well as the grading of their utility The ultimate goal is that users could trust such tools to then use themAcknowledgementWe would like to thank all the present and past editorial boards of the IMIA Yearbook especially Martina Hutter and Adrien Ugon for their support as well as the reviewers for their participation to the selection of the best papers for the Decision Support section We cannot end this synopsis without a meaningful thought for our colleague and friend Vassilis Koutkias who started this year again to tackle the tasks of a Decision Support section coeditor but passed away in last December and unfortunately could not finishReferences Jankovic I Chen JH Clinical Decision Support and Implications for the Clinician Burnout Crisis Yearb Med Inform Koutkias V Bouaud J Contributions on Clinical Decision Support from the Literature Yearb Med Inform Aug2811357 Hendriks MP Verbeek XAAM van Vegchel T van der Sangen MJC Strobbe LJA Merkus JWS Transformation of the National Breast Cancer Guideline Into DataDriven Clinical Decision Trees JCO Clin Cancer Inform KamiÅ¡alić A Ria±o D Kert S Welzer T Nemec Zlatolas L Multilevel medical knowledge formalization to support medical practice for chronic diseases Data Knowledge Engineering “ Khalifa M Magrabi F Gallego B Developing a framework for evidencebased grading and assessment of predictive tools for clinical decision support BMC Med Inform Decis Mak Laleci GB Yuksel M Sarigul B Arvanitis TN Lindman P Chen R A Collaborative Platform for Management of Chronic Diseases via GuidelineDriven Individualized Care Plans Comput Struct Biotechnol J “ Jafarpour B Raza Abidi S Van Woensel W Raza Abidi SS Executiontime integration of clinical practice guidelines to provide decision support for comorbid conditions Artif Intell Med Ben Souissi S Abed M El Hiki L Fortemps P Pirlot M PARS a system combining semantic technologies with multiple criteria decision aiding for supporting antibiotic prescriptions J Biomed Inform Huibers CJA Sallevelt BTGM de Groot DA Boer MJ van Campen JPCM Davids CJ Conversion of STOPPSTART version into coded algorithms for software implementation A multidisciplinary consensus procedure Int J Med Inform C¡novasSegura B Morales A Juarez JM Campos M Palacios F Impact of expert knowledge on the detection of patients at risk of antimicrobial therapy failure by clinical decision support systems J Biomed Inform M¼ller L Gangadharaiah R Klein SC Perry J Bernstein G Nurkse D An access medical knowledge base for community driven diagnostic decision support system development BMC Med Inform Decis Mak Spnig S EmbergerKlein A Sowa JP Canbay A Menrad K Heider D The virtual doctor An interactive clinicaldecisionsupport system based on deep learning for noninvasive prediction of diabetes Artif Intell Med Rozenblum R RodriguezMonguio R Volk LA Forsythe KJ Myers S McGurrin M Using a Machine Learning System to Identify and Prevent Medication Prescribing Errors A Clinical and Cost Analysis Evaluation Jt Comm J Qual Patient Saf Kannan V Basit MA Bajaj P Carrington AR Donahue IB Flahaven EL User stories as lightweight requirements for agile clinical decision support development J Am Med Inform Assoc Fernandes CO Miles S Lucena CJP Cowan D Artificial Intelligence Technologies for Coping with Alarm Fatigue in Hospital Environments Because of Sensory Overload Algorithm Development and Validation J Med Internet Res 20192111e15406 Amroze A Field TS Fouayzi H Sundaresan D Burns L Garber L et al Use of Electronic Health Record Access and Audit Logs to Identify Physician Actions Following Noninterruptive Alert ing Descriptive Study JMIR Med Inform 201971e12650 Kim M Kim BH Kim JM Kim EH Kim K Pak K Concordance in postsurgical radioactive iodine therapy recommendations between Watson for Oncology and clinical practice in patients with differentiated thyroid carcinoma Cancer Correspondence toPr Catherine DuclosLIMICS INSERM Facult© L©onard de Vinci rue Marcel Cachin Bobigny FranceEmail catherineduclosaphpfr IMIA Yearbook of Medical Informatics 2020Pragmatic Considerations on Clinical Decision Support from the Literature 0cAppendix Content Summaries of Best Papers for the Decision Support Section of the IMIA YearbookHendriks MP Verbeek XAAM van Vegchel T van der Sangen MJC Strobbe LJA Merkus JWS Zonderland HM Smorenburg CH Jager A Siesling STransformation of the National Breast Cancer Guideline into datadriven clinical decision treesJCO Clin Cancer Inform May3114Since clinical practice guidelines are still narrative and described in large textual documents the aim of this work was to model complex guidelines as datadriven clinical decision trees CDTs that could be still humaninterpretable while computerinterpretable for implementation in decision support systems The Dutch national breast cancer guidelines were translated into CDTs Data items which characterize the patient and the tumor and represent decisional criteria were encoded unambiguously using existing classifications and coding systems related to breast cancer when feasible In total CDTs were necessary to cover the whole guidelines driven by data items Of all data items could be coded using existing classification and coding systems All CDTs represented unique patient subpopulations Complex guidelines could be transformed as systematically constructed modular datadriven CDTs that are clinically interpretable and executable in a decision support applicationKamiÅ¡alić A Ria±o D Kert S Welzer T Nemec Zlatolas LMultilevel medical knowledge formalization to support medical practice for chronic diseasesData Knowledge Engineering “This research is focused on knowledge representation to support the medical processes involved in chronic diseases management which can be viewed as a procedural and sequential application of knowledge An intuitive easy and effective mechanism for medical knowledge formalization is proposed through a formalism called extended Timed Transition Diagram eTTD This formalism allows for the consistent representation of three basic levels of decision making that should be taken into account in the prescription and adaptation of longterm treatment therapy strategy dosage and intolerances The methodology can be manually applied to build eTTDs from clinical practice guidelines eTTDs implementation is demonstrated by modeling clinical practice guidelines for the therapeutic management of arterial hypertension The obtained models can be used as a baseline framework for the development of decision support systems involving medical proceduresKhalifa M Magrabi F Gallego BDeveloping a framework for evidencebased grading and assessment of predictive tools for clinical decision supportBMC Med Inform Decis Mak Oct Deciding to choose a clinical predictive tool in clinical practice should be guided by its correctly assessed effectiveness The objective of this work is to developp a conceptual and practical framework to Grade and Assess Predictive tools GRASP and provide clinicians with a standardised evidencebased system to support their search for and selection of efficient predictive tools The GRASP framework grades predictive tools based on published evidence across three dimensions phase of evaluation level of evidence and direction of evidence The final grade of the tool is based on the phase of evaluation that gets the hightest grade supported by the highest level of positive or mixed evidence that supports a positive This framework was successfully applied to five predictive tools GRASP report updates could be a way to maintain a data base that documents the evidence of predictive tools IMIA Yearbook of Medical Informatics 2020Duclos 0c"

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

"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

"variability around prevalence estimates of multimorbidity due to poorconsensus regarding its definition and measurement Medicationbased measures of morbidity may be valuableresources in the primarycare setting where access to medical data can be limited We compare the agreementbetween patient selfreported and medicationbased morbidity and examine potential patientlevel predictors ofdiscordance between these two measures of morbidity in an older ‰¥ years communitybased populationMethods A retrospective cohort study was performed using national pharmacy claims data linked to The IrishLongituDinal study on Ageing TILDA Morbidity was measured by patient selfreport TILDA and two medicationbased measures the RxRisk years and RxRiskV ‰¥ years which classify drug claims into chronic diseaseclasses The kappa statistic measured agreement between selfreported and medicationbased morbidity at theindividual patientlevel Multivariate logistic regression was used to examine patientlevel characteristics associatedwith discordance between measures of morbidityResults Two thousand nine hundred twentyfive patients were included years N and ‰¥ years N Hypertension and high cholesterol were the most prevalent selfreported morbidities inboth age cohorts Agreement was good or very good κ “ for diabetes osteoporosis and glaucoma andmoderate for high cholesterol asthma Parkinson™s and angina κ “ All other conditions had fair or pooragreement Age gender marital status education poordelayed recall depression and polypharmacy weresignificantly associated with discordance between morbidity measuresConclusions Most conditions achieved only moderate or fair agreement between selfreported and medicationbased morbidity In order to improve the accuracy in prevalence estimates of multimorbidity multiple measures ofmultimorbidity may be necessary Future research should update the current RxRisk algorithms inline with currenttreatment guidelines and reassess the feasibility of using these indices alone or in combination with othermethods to yield more accurate estimates of multimorbidityKeywords Agreement Selfreport Rxrisk RxriskV Morbidity Polypharmacy Older people Correspondence caitrionacahirrcsiie Clionadh Mannion and John Hughes are joint first authors2Division of Population Health Sciences Royal College of Surgeons in IrelandDublin IrelandFull 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 0cMannion BMC Geriatrics Page of Key pointsKey findings and implications Agreement between patient selfreported morbidityand medicationbased measures of morbidity RxRisk and RxRiskV was mainly moderate or fairDiabetes was the only condition for which the levelof agreement was found to be very good The results of our study indicate that neithermeasure of morbidity is completely reliable and wesuggest that researchers may require multiplemeasures selfreport and medicationbased measures of morbidity to fully capture accurate prevalence estimates of multimorbidity Our study identified several limitations of thecurrent versions of the RxRisk indices which require updating if medicationbased measures ofmorbidity are to be used to assess the epidemiologyof chronic conditions and multimorbiditytheofIndeedattentionBackgroundMultimorbidity is commonly defined as the presence oftwo or more chronic medical conditions and its prevalence has been shown to increase with age [] As theworld™s older population continues to grow multimorbidity has become an important public health issue caphealthcareturingresearchersprofessionals as well as policy makersforhealthcare systems to effectively adapt and manage thedelivery of healthcare to our growing older populationan accurate description of the epidemiology of chronicconditions is required However to date studies in theliterature reveal wide disparities in prevalence estimatesof multimorbidity ranging from to [ ] Thislarge variability is thought to be due to the lack of standards defining multimorbidity and validated methods forhow it should be measured [] A recent systematic review reported definitions of multimorbidity involving differenttheappropriateness of different measures of multimorbidityis also variable depending on both the outcome of interest as well as the type of data that is available []In additioncriteria[]Measures of multimorbidity include diagnosisbasedmeasures eg Charlson Index based on hospital diagnosis codes ICD codes [] medicationbased measureseg RxRisk and RxRiskV for those aged ‰¥ yearsbased on pharmacy data [] and patient selfreportDiagnosisbased measures of multimorbidity are themost common measures and are generally based on hospital or physician records [] Medicationbased measures of multimorbidity include the RxRisk and RxRiskV “ two algorithms which determine an individual™scurrent comorbidities based on their dispensed medication The RxRisk indexes only include morbidities forwhich a medicine could be prescribed and include categories of morbidities based on the World Health anisation WHO Anatomical Therapeutic Classification ATC system [“] The RxRisk and RxRiskVhave good reliability and criterion validity against ICD9diagnoses and have been shown to predict costs of caremortality and health care utilisation [] Previous studies have reported medicationbased measures of morbidity such as the Medicines Disease Burden Index MDBIand RxRiskV to be useful in epidemiological studieswhen adjusting for comorbidity [] However there arefew studies describing the use of these indices to directlymeasure chronic conditions Patient selfreport is also avalid method of identifying disease categories A study ofolder patients with multimorbidity reported good agreement between patient selfreport and general practitioner GP report for a wide range of diseases []A number of studies have compared the differentmeasures of multimorbidity with differing results [ ] A study of older primary care patients inIreland found that medicationbased measures ofmultimorbidity such as RxRiskV performed betterthan diagnosisbased measures of multimorbidity inpredicting emergency and ambulatory care sensitiveACS admissions [] Studies comparing patientselfreport and diagnosisbased measures of multimorbidity have reported a stronger association between selfreport measures of multimorbidity andqualitythandiagnosisbased measures [ ] However no previous research has compared selfreported morbidityin the primary care or community setting with theRxRisk measures of morbidity Comparison betweenselfreported morbidity data and pharmacy records isimportant in order to understand the relative meritsof each measure of morbidity and the potential formisclassification particularly in the community setting where access to medical or clinical data can belimitedfunctionaloutcomesandlifeofStudies have also indicated that agreement betweenselfreport measures and other measures of morbiditymight be influenced by patient recall bias [] Patientrecall has been reported to be influenced by age maritalstatus and education [] There is also some evidencethat cognition and memory influence patient recall []The impact of these factors needs to be explored furtherwhen assessing and comparing measures of morbidityThe aim of this study was to compare the agreementbetween patient selfreported morbidity and medicationbased morbidity RxRisk and RxRiskV and examine potential patientlevel predictors of discordance between theincludingdemographic cognitive and mental health factors in anolder community based populationtwo measures of morbidity 0cMannion BMC Geriatrics Page of MethodsThe STrengthening the Reporting of ObservationalStudies in Epidemiology STROBE guidelines were usedin the reporting of this study []Study populationThis was a retrospective cohort study using data froma national pharmacy claims database the Health Service ExecutivePrimary Care Reimbursement ServiceHSEPCRS General Medical Services GMS schemelinked to the first wave of The Irish LongituDinalstudy on Ageing TILDA TILDA is a nationally representative sample of community dwelling individualsaged ‰¥ years in Ireland The sampling framework isbased on the Irish Geodirectory a comprehensive anduptodate listing and mapping ofresidential addresses in Ireland compiled by the Ordinance SurveyOffice and participants aged ‰¥ years were randomlyselected using the RANSAM sampling procedureThis meant that each residential address in Irelandhad an equal probability of selection and thus ensured that the TILDA sample was representative ofthe Irish population aged ‰¥ years The first wave ofdata collection began in October through toFebruary N participants aged ‰¥ yearswhere participants completed a computeraided personal interview CAPI and a health assessment measuring their health economic and social circumstancesFurther information on TILDA™s study design andsampling framework is described in detail elsewhere[]The HSEPCRS GMS scheme is the largest pharmacy claims dataset in Ireland covering more than of the general Irish population [] It is meanstested and provides free health servicesincludingmedications to eligible persons in Ireland Qualification for the GMS scheme is on the basis of incomerelated meanstesting Automaticforthose aged ‰¥ years occurred between July andDecembercurrent study period meanstesting was introducedbut with a higher income threshold than the generalpopulation As of of men and ofwomen in the general population aged ‰¥ yearswere eligible [] The HSEPCRS GMS pharmacyclaims data were available for consenting TILDAparticipants aged ‰¥ years with GMS eligibility N entitlementhoweversinceJanuaryWithin the HSEPCRSGMS pharmacy claims dataprescriptions are coded using the WHO ATC classification system and prescriber information defineddaily doses strength quantity method and unit ofadministration of each drug dispensed are all available Pharmacy claims data was extracted for yearprior to each participant™s TILDA interview GMSpatientstypically receive their medications on amonthly basis []ifthey had any ofSelfreported morbidityAs part of the TILDA interview participants wereasked to reportthe followingdoctordiagnosed chronic diseases high blood pressure or hypertension high cholesterol angina congestive heart failure heart attack diabetes stroke orministroke abnormal heart rhythm arthritis osteoporosis cancer Parkinson™s disease emotional nervous or psychiatric problems alcohol or substanceabuse dementia serious memory impairment stomach ulcers glaucoma incontinence or chronic painParticipants were also asked to selfreport urinaryincontinence in the past months as well as painmoderate or severe and if they were taking medication for pain management If participants reportedthat they had arthritisthey were asked to clarifythe type of arthritis eg osteoarthritis rheumatoidarthritis some other kind of arthritis Similarlyifparticipants reported emotional nervous or psychiatric problems they were asked to clarify from a listof conditions eg anxiety depression emotionalproblems psychosis manic depressionfillsthatclassify prescription drugMedicationbased measures of morbidity “ Rxrisk andRxriskVThe RxRisk and RxRiskV indices were applied tothe HSEPCRS pharmacy claims data The RxRiskindex was applied to the population aged yearswhile the RxRiskV was applied to the populationaged ‰¥ years The RxRisk and RxRiskV are algorithmsintochronic disease classes for older populations basedon the WHO ATC classification system [“]Within the RxRiskV cardiac disease is separatedinto a number of categories anticoagulation antiplatelet agents arrhythmias congestive heart failureCHFhypertension hypertensionischaemic heartdisease IHDangina and ischaemic heart diseaseIHDhypertension [] For a medication to be eligible as a measure of morbidity per RxRisk and RxRiskV chronic disease classes a patient was required to have been dispensed two or more consecutive prescriptions of the medication in question eg˜donepezil™ was required to be dispensed on ‰¥ consecutive prescriptions to link this medication withthe RxRiskV condition ˜dementia™ This definitionhas previously been used by other pharmacoepidemiological studies [] 0cMannion BMC Geriatrics Page of Comparison of selfreported morbidity with Rxrisk andRxriskVEach selfreported condition in TILDA was matched tothe equivalent RxRisk and RxRiskV condition at theindividual patient level for those aged years and ‰¥ years respectively This was performed by consensusbetween two pharmacists FM CM For some selfreported conditions the ATC classes of medicationsspecific to these conditions “ eg antiwere notthrombotic agents B01AC04 “ B01AC30 were matchedto the selfreported condition of a heart attack and alsoto stroke There were four selfreported TILDA conditions which could not be matched to an RxRisk or RxRiskV condition but the prevalence was low Appendix in Tables and The RxRisk and RxRiskV alsoreported conditions which patients had not been askedabout during their TILDA interview Appendix in Tables and Patientlevel characteristics associated with discordancebetween the two measures of morbidityPatient characteristics were assessed to determine discordance patient recall bias between selfreported morbidity TILDA and the RxRisk years and RxRiskV ‰¥ years medicationbased measures of morbidity These characteristics were age gender maritalstatus education poor delayed recall depression andpolypharmacy Marital status was subcategorised intomarried never married separated or divorced Educationwas categorised into primarynone secondary or thirdhigher level education Delayed recall based on participants being presented with words during the interview and being later asked to recall as many as possiblewas defined as poor where or fewer words wererecalled Depression was defined as scoring or greateron the Centre for Epidemiologic Studies DepressionScale CESD [] Polypharmacy was defined as reporting regular use of five or more prescription medications[]Statistical methodsAgreement between selfreported morbidity TILDAand the RxRisk and RxRiskV measures of morbiditypharmacy claims was assessed using Cohen™s Kappastatistic as neither source was considered to be a goldstandard for reporting morbidity Interpretation of thevalue of Kappa was as follows poor fair “ moderate “ good “ and verygood “ []Multivariate logistic regression was used to examinethe association between the patientlevel characteristicsand discordance between the two measures of morbidityAdjusted odds ratios OR and confidence intervalsCIare presented Discordance was defined asparticipants reporting to have the condition in the absence of any dispensed medication for the condition perRxRisk years or per RxRiskV ‰¥ years andparticipants reporting to not have the condition butmedication was found to be dispensed for the conditionper RxRisk years or RxRiskV ‰¥ years Allsignificance tests were twotailed Statistical significancewas set at P after adjustment for a false discoveryrate of [] Analyses were performed using Stata SEVersion statistical package StataCorp College Station TXResultsStudy populationIn total patients were included in this cohortstudy patients were aged years and were aged ‰¥ years Characteristics ofthe study participants are presented in Table On average patients aged years had SD conditionsper the RxRisk and patients aged ‰¥ years had SD conditions per the RxRiskV The proportion ofpatients with thirdhigher level education was relatively years N low across both age ‰¥ years N Poor delayed recall years N ‰¥ years N years N ‰¥ years N were significantlymore prevalent in the older cohort compared to theyounger cohort p polypharmacygroupsandAgreement between selfreported morbidity andmedicationbased measures of morbidity Rxrisk and RxriskVTables and present a comparison between the number and percentage of patients™ selfreported morbiditiescompared to the RxRisk Table aged years andRxRiskV Table aged ‰¥ years measures of morbidity High blood pressure or hypertension yearsN ‰¥ years N and highcholesterol years N ‰¥ years N were the most prevalent selfreportedmorbidities in both age cohorts in the TILDA datasetHigh cholesterol was also found to be highly prevalentin the RxRisk N and RxRiskV N measures of morbidity Other prevalentRxRisk and RxRiskV conditions included arthritisRxRisk N stomach ulcers RxRiskN RxRiskV N strokeRxRiskV N heart attack RxRiskVN and other heart trouble RxRiskVN There was very good agreement between the selfreported TILDA measure of diabetes and the RxRiskand RxRiskV measures κ There was also good 0cMannion BMC Geriatrics Page of Table Characteristics of study participants by age years and ‰¥ years years N “Age‰¥ years N “GenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimarynoneSecondaryThirdHigher LevelPoor delayed recall YesDepression YesPolypharmacy Yes Data presented as N or mean CI unless otherwise statedagreement between selfreported measures of osteoporosis κ and glaucoma κ and the RxRiskV measure of these morbidities in the older cohort Despite the high prevalence of high cholesterolin both measures of morbidity there was only moderate agreement κ RxRisk κ RxRiskVbetween the two measures There was moderateagreement also for asthma κ RxRisk Parkinson™s κ RxRiskV and angina κ RxRisk V Agreement was fair for selfreported highblood pressure or hypertension RxRisk and RxRiskV heart attack RxRisk stroke RxRisk abnormalheart rhythm RxRiskV cancer RxRisk depression RxRisk and RxRiskV and pain RxRiskVand RxRisk measures of these conditions κ “ All other conditions had poor agreement κ “ including arthritis RxRisk chronic lungdisease and incontinence RxRiskV and emotionalnervous psychiatric problems anxiety and stomach ulcers RxRisk and RxRiskV Tables Patientlevel characteristics associated with discordancebetween the two measures of morbidityAge gender marital status education poor delayedrecall depression and polypharmacy were all associated with discordance between the two measures ofmorbidity Table Females were five times morelikely to have discordance in reporting osteoporosisOR Confidence Intervals CI P Females were also more likely to have discordance in reporting anxiety OR CI emotional problems OR CI and depression OR CI as well as use of pain medication OR CI and incontinence OR CI They were less likely to have discordance in reporting stroke and high cholesterol TablePatients who were never married were less likely tohave discordance in reporting a heart attack OR CI and stroke OR CI Patients with third level educationwere lesslikely to have discordance in reportinghypertension OR CI comparedto those with primary level education Table Patients with poor delayed recall and depression weremore likely to have discordance in reporting anxietyand depression In general discordance was higher inpatients with polypharmacy Table found thatagreement between patientDiscussionWithin a population based study of ageing in Irelandweselfreported morbidity and medicationbased measures ofmorbidity RxRisk and RxRiskV was generally notgood with most conditions achieving only moderateor fair agreement There was ˜very good™ agreementκ between selfreported diabetes and pharmacy dispensing records across both age cohortsThis was the only morbidity common to both age cohorts for which the level of agreement was found tobe ˜very good™ Many research studies confirm this 0cGlaucomaHigh CholesterolAsthmaHigh blood pressure orHypertensionCancer or a malignant tumourDepressionStroke cerebral vascular diseaseParkinsonHeart attack including myocardialinfarction or coronary thrombosisManic depressionEmotional nervous or psychiatricproblem such as depression oranxietyCirrhosis or serious liver damageStomach ulcersArthritis including osteoarthritis orrheumatismN Diabetes A10AB01A10BG03 A10BH A10BX Glaucoma S01EA01S01EB03 S01EC03S01EX Hyperlipidaemia C10AA01C10BX17 Asthma R03AAR03AL R03BAR03BX R03CAR03CC R03DAR03DX Hypertension C03AA01C03BA11 C03DA01C03EA01 C09BA02C09BA09 C09DA01C09DA07 C02AB01C02AC05 C02DB02C02KX01 Malignancies L01AA01L01XX31 Depression N06AA01N06AG02 N06AXAntiplatelet therapy B01AC04B01AC30Parkinson™s disease N04AA01N04BX02Antiplatelet therapy B01AC04B01AC30Bipolar disorder N05AN01 Anxiety N05BA01N05BA12Anxiety N05BA01N05BA12Liver disease A05AA01A05BA08 J05AF05 J05AF07 J05AF11 GORD Peptic ulcer A02B A02BB A02BC Rheumatoid Arthritis M01AAM01CX M02AAM02AX L01BA01L04AB01L04AB05 L04AD01 L04AX03Ischaemic heart diseasehypertension C07AA01C07FB07C08CA01C08DB01Anxiety Mannion BMC Geriatrics Page of Table Agreement kappa statistic and standard error between selfreported morbidity in TILDA and RxRisk algorithm yearsTILDAStandardErrorSelfreported morbidityDiabetes or high blood sugarRxRisk Pharmacy ClaimsMedicationbased Morbidity ATCKappaκNAny other heart troubleRheumatoid arthritis only Rheumatoid Arthritis M01AAM01CX M02AAM02AX L01BA01Ministroke or TIAL04AB01L04AB05 L04AD01 L04AX03Antiplatelet Anticoagulation therapya B01AC04B01AC30B01AA03B01AB06ATC Anatomical Therapeutic ChemicalGORD GastroOesophageal Reflux DiseaseaAnticoagulant counted if patient coprescribed antiarrhythmic for Atrial Fibrillation ie if patient not in sinus rhythm []same level of agreement for diabetes [ ] Thiswas expected given that previous research has demonstrated the reliability of reporting to be better inmorbidities for which there are clear diagnostic criteria eg diabetes [] Furthermore with many educational resources promoting selfmanagement of thiscondition patients with diabetes are more likely toplay an active role in managing their condition egregular selfmonitoring of blood glucose levels dietarymanagement recognising and dealing with symptomssuch as hypo and hyperglycaemia andor medication taking and are therefore more likely to selfreport accurately []There was ˜good™ agreement between both measures ofmorbidity for osteoporosis and for glaucoma in the olderage group A MultiCare cohort study of primary carepatients in Germany found only moderate agreement between patientreported and GPreported osteoporosis[] A retrospective cohort study of older patients in asecondarycare setting in Canada also found moderateagreement for glaucoma between physician and patientreports [] Similar to diabetes patients are required toplay an active role in the management of osteoporosiswhile glaucoma is very often a comorbidity of diabetes[]There was ˜moderate™ agreement between the measures of morbidity for asthma in the younger age cohort years Similar results have been reported for agreement between selfreported asthma and medical recorddata in older hospitalised patients [] There was also˜moderate™ agreement for high cholesterol in both agecohorts and for angina and Parkinson™s disease in the 0cMannion BMC Geriatrics Page of Table Agreement kappa statistic and standard error between selfreported morbidity in TILDA and RxRiskV algorithm ‰¥ yearsTILDASelfreported morbidityDiabetes or high blood sugarRxRiskV Pharmacy claimsMedicationbased Morbidity ATC KappaκNStandardErrorN Diabetes A10AB01A10BG03 A10BH A10BX Glaucoma S01EA01S01EB03 S01EC03S01EX OsteoporosisPaget™s disease M05BA01M05BB09 M05BX03Pain taking pain medication Pain Opioids N02AA01N02AX02 GlaucomaOsteoporosisParkinsonAnginaHigh CholesterolManic depressionHigh blood pressure orHypertensionG03XC01 A12AX92Parkinson™s disease N04AA01N04BX02 Angina C01DA02C01DA14 C01DX16 C01EB17C01EB18 Hyperlipidaemia C10AA01C10BX17 Hypertension C03AA01C03BA11 C03DA01C03EA01 C09BA02Bipolar disorder N05AN01C09BA09 C09DA01C09DA09 C02AB01C02AC05 C02DB02C02KX01PainAbnormal Heart RhythmDepressionDementiaChronic lung disease such aschronic bronchitis or emphysemaCancer or a malignant tumourEmotional nervous or psychiatricproblem such as depression oranxietyPain Inflammation M01AB01 M01AH06 Pain Opioids N02AA01N02AX02Pain Inflammation M01AB01 M01AH06 Arrhythmia C01AA05 C01BA01C01BD01 C01BD07 Depression N06AA01N06AG02 N06AX Dementia N06DA02 N06DA01Chronic airways disease R03AC02R03DC03 Malignancies L01AA01L01XX31 Anxiety N05BA01 N05BA12Congestive heart failureCirrhosis or serious liver damageHeart attack including myocardialinfarction or coronary thrombosis Chronic heart failure C03CA01C03CC01 C09AA01C09AA10C09CA01 C09CA03 C09CA06C09CA07Liver disease A05AA01A05BA08 J05AF05 J05AF07 J05AF11Antiplatelet therapy B01AC04B01AC30AnxietyStomach ulcersAlcohol or substance abuseAnxiety N05BA01N05BA12 GORD Peptic ulcer A02BA A02BCAny other heart trouble Stroke cerebral vascular diseaseMinistroke or TIA Alcohol dependence N07BB01 N07BB04Ischaemic heart diseasehypertension C07AA01C07FB07C08CA01C08DB01Antiplatelet therapy B01AC04B01AC30Antiplatelet Anticoagulation therapya B01AC04B01AC30B01AA03B01AB06 B01AB10Incontinence Neurogenic Bladder Urinary Incontinence V07ANPsychotic illness N05AA01 N05AX17PsychosisATC Anatomical Therapeutic ChemicalGORD GastroOesophageal Reflux DiseaseaAnticoagulant counted if patient prescribed antiarrhythmic for Atrial Fibrillation ie if patient not in sinus rhythm [] 0cMannion BMC Geriatrics Page of Table Odds ratios with confidence intervals for patientlevel characteristics associated with discordance between themeasures of morbidity selfreport and RxRisk and RxRiskVAge yearsGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecall YesDepression YesPolypharmacyYesAgeGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecallDepression YesPolypharmacyHypertension HeartAttack “ “StrokeTIAHigh Cholesterol “ “ “HeartTrouble “Cancer “EmotionalProblems “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Depressiononly “ “ “ “ “ “Stomachulcers “ “ “ “ “ “Asthma “ “ “ “ “ “Arthritisgeneral “ “ “ “ “ “RheumatoidArthritis only “ “ “ “ “ “ “ “ “Angina “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Congestive HeartFailure “Abnormal HeartRhythm “ “ “ “ ““ “ “ “ “ “ “ “ “ “ “ “ “ “ “ ““““ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ ““ “ “ “““““ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Anxiety “ “ “ “ “ “ “ “ “ “LungDisease “ “ “ “ “ “ “ “ “ “ 0cMannion BMC Geriatrics Page of Table Odds ratios with confidence intervals for patientlevel characteristics associated with discordance between themeasures of morbidity selfreport and RxRisk and RxRiskV ContinuedHypertension HeartAttackOsteoporosis “Psychosisonly “StrokeTIAHigh CholesterolHeartTroubleCancerEmotionalProblemsAnxietyIncontinence PainPain meds “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “AgeGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecallDepression YesPolypharmacyExcluded diabetes Parkinson™s disease manic depression cirrhosis glaucoma alcohol or substance abuse and dementia as number of patients misreporting wassmall N p older age cohort Other studies have reported loweragreement for high cholesterol and higher agreement forangina and Parkinson™s diseases [ ] Discordancehere may be explained by patients managing their cholesterol using nonpharmacological means eg lifestylemodifications[]Interestingly the prevalence of selfreported angina inTILDA was higher than the prevalence reported by RxRiskV This may reflect poor patient adherence if prescribed medications were not dispensedincluding cardioprotective dietThere was only ˜fair™ agreement between both measures of morbidity for hypertension despite hypertensionbeing the most prevalentselfreported morbidityacross both age cohorts Higher agreement betweenselfreported antihypertensive drug use and pharmacyrecords has been reported in a populationbasedstudy and a cohort study of older people in theNetherlands [ ] The discordance observed hereis likely attributable to the omission of a major group[]increasingantihypertensivesofcalciumchannelblockersCCBs in the current version of the RxRisk and RxRiskV algorithms [ ] This is significant giventhat CCBs are recommended as firstline therapy inpatients aged years [] Equally since hypertension is considered to be a condition without symptomsthis may influence patient adherence toantihypertensive medications and their proclivity tofill a prescription for these medications There wasalso ˜fair™ agreement for pain in the older age groupwith agreementsomewhat when selfreported pain specified ˜taking pain medication™ Theprevalence of selfreported pain was higher than themedicationbased RxRiskV prevalenceand thismay be due to patients managing their pain throughnonpharmacological or lifestyle interventions such asphysiotherapy and cognitive behavioural therapy []In both age cohorts there was œpoor to fair agreement between selfreporting of emotional problems 0cMannion BMC Geriatrics Page of poorfoundagreementeg depression anxiety and medicationbased measures These findings are consistent with previous research whichbetweenphysician diagnosis and patient selfreports of anxiety and depression [] This low level of agreementmay be due to a potential stigmatisation bias as only of patients regularly dispensed antidepressants selfreported as having depression in theolder age cohort [ ] Equallyit may be thatcertain antidepressants eg amitriptyline are beingused for other indications such as neuropathic pain[ ] There was also ˜poor™ agreement in bothage cohorts for stomach ulcers and for incontinenceand chronic airways disease COPD in the older cohort Like depression poor agreement here may bedue to gastrointestinal medications being used by patients for other indications such as preventative orsymptomatic reasons [] The poor agreementforchronic airways disease may reflect the nonspecificquestion used in TILDA to measure this selfreportedmorbidity as there is evidence in the literature thatquestionnaire design is an important determinant ofpatient recall In a US study the prevalence of selfreported COPD was found to increase when more explicit questions were asked about emphysema chronicbronchitis and COPD in combination [] The pooragreement between the two measures for incontinenceis most likely reflective of the current version of theRxRiskV which compares selfreported urinary incontinence with dispensed ˜diapers and pads supplies™ []agepoordelayedincreasingA number of factors were associated with discordance between the two measures of morbidity particularlyrecalldepression and polypharmacy A study determiningthe agreement between selfreported and diagnosisbased multimorbidity in older community dwellingwomen reported similar findings where agreementwas found to decrease with decreasing cognition andeducation increasing age and fo

Thyroid_Cancer

"Age is associated with the prognosis of glioma patients but there is no uniform standard of agegroup classification to evaluate the prognosis of glioma patients In this study we aimed to establish an age groupclassification for risk stratification in glioma patientsMethods patients diagnosed with gliomas at Nanfang Hospital between and were enrolled TheWHO grade of glioma was used as a dependent variable to evaluate the effect of age on risk stratification Theevaluation model was established by logistic regression and the Akaike information criterion AIC value of themodel was used to determine the optimal cutoff points for ageclassification The differences in gender WHOgrade pathological subtype tumor cell differentiation tumor size tumor location and molecular markers betweendifferent age groups were analyzed The molecular markers included GFAP EMA MGMT P53 NeuN Oligo2 EGFRVEGF IDH1 Ki67 PR CD3 H3K27M TS and 1p19q statusResults The proportion of men with glioma was higher than that of women with glioma vs Analysisof age showed that appropriate classifications of age group were “ years old pediatric group “ years oldyouth group “ years old middleaged group and ‰¥ years old elderly groupThe proportions ofglioblastoma and large tumor size “ cm increased with age p p respectively Analysis of thepathological molecular markers across the four age groups showed that the proportion of patients with larger than area of Ki67 expression or positive PR expression increased with age p p respectivelyConclusions Appropriate classifications of the age group for risk stratification are “ years old pediatric group“ years old young group “ years old middle age group and ‰¥ years old elderly group This agegroup classification is effective in evaluating the risk of glioblastoma in glioma patientsKeywords Glioma Age group classification Risk stratification Personalized treatment Correspondence hgl1020163com Zhiying Lin and Runwei Yang contributed equally to this work1Department of Neurosurgery Nanfang Hospital Southern MedicalUniversity No Guangzhou Avenue North Guangzhou Guangdong China2The Laboratory for Precision Neurosurgery Nanfang Hospital SouthernMedical University Guangzhou Guangdong 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 0cLin BMC Neurology Page of BackgroundOver the past years the incidence of primary malignant brain tumors has increased at an annual rate of “ with an especially higher rate in the elderly population [] Glioma accounts for approximately of allcentral nervous system CNS tumors and of malignant primary brain tumors [] According to the World Health anization WHO classification of tumors ofthe CNS gliomas were classified into fourgrades WHO grade I to IV based on histologic criteria[] WHO grades I and II gliomas are recognized as lowgrade gliomas LGG and grades III and IV are considered highgrade gliomas HGG [] In particular glioblastoma GBM WHO grade IV is the most commonmalignant tumor of the CNS accounting for ofprimary malignant the CNS tumors and of all gliomas [] The median survival of GBM patients is approximately months even after receiving multimodaltherapies that include maximal surgical resection withthe preservation of neurological functions followed byadjuvant radiotherapy and chemotherapy []Gliomas can occur at any age with various incidencesat different ages as reported in populationbased studies[ ] LGG is the most common brain tumor in children while HGG is the most frequent brain tumor inadults [] Tumors in the supratentorial areas of thebrain cerebral hemispheres and midline structuresabove the tentorium were most frequent in adults whilesubtentorial brainstem and cerebellum tumors weremore common in young children than in adolescentsand adults [] Besides increasing studies have assessedage a prognostic factor There are differences in prognosis among patients of different ages even with the samediagnosis A singlecenter review of patients withintracranial anaplastic oligodendroglioma showed thatthe median survival time of patients younger than years old was significantly longer than that of patientsolder than years old [] Other studies have shownthat age was an important prognostic factor in additionto KPS score surgical scope and histology [ ]Therefore for patients diagnosed with glioma by imaging examination and auxiliary examination it is necessary to consider the age of the patients to performpersonalized treatment for better outcomesHowever there is no uniform age criterion for groupingglioma patients for personalized treatment [] Some glioma patient cohorts were divided into different age groupsaccording to fixed age intervals [] some were dividedinto two groups based on a certain age point [] andothers were divided based on the overall survival OS ofthe patients [] Different criteria for age grouping haveled to inconsistent s regarding the prognosticvalue of age Some studies showed that age was not aprognostic factor in patients with glioma [ ] whileanother populationbased glioblastoma study with five agegroups years “ years “ years “ yearsand years showed that the OS of young patients years was significantly longer than that for elderly patients years median months vs months p [] Agerelated studies involving a large numberof glioma patients have yielded some relevant results [] but the age grouping criteria for these studies are influenced by several clinical factors such as the tendencyof clinical researchers Therefore there is an urgent needto establish a more appropriate age group classificationcriterion for better management of glioma patientsFor this purpose we conducted a retrospective studycollecting clinical data from patients with histologically proven gliomas in Nanfang Hospital between and Based on this cohort we established a methodof age group classification according to WHO grade forrisk stratification in glioma patients and investigated thecharacteristics of different age groups in terms of genderWHO grade pathological subtype tumor cell differentiation tumor size tumor location and pathological molecular markersMethodsData collectionA total of patients diagnosed with gliomas bypathological examination after surgery from to in Nanfang Hospital were enrolled in this studyThe clinical data for age gender pathological diagnosisaccording to the WHO Central nervous systemtumor Classification anatomic location of gliomatumor size and pathological molecular markers werecollected Supplementary Table S1The terminology of the anatomic location of gliomaused in this study was based on the Central BrainTumor Registry of the United States CBTRUS Brainand other Central Nervous System Tumor Site Groupings We recognize that with the WHO classification of central nervous system tumors many of thehistological diagnostic criteria have undergone majorchanges and steps have been taken to align their histological grouping scheme with the WHO standardsThe pathological diagnosis included histological classification WHO grade and molecular expression Thepathological molecular markers included GFAP EMAMGMT wtP53 NeuN Oligo2 EGFR VEGF IDH1 Ki and ATRXfluorescence in situhybridization FISH detection of 1p19q was also included All pathological information was collected fromthe hospital medical records systemIn additionCalculation of age group cutoff pointsDummy variables were established by age groups of 1Iyears old and I82 years old I any age between and 0cLin BMC Neurology Page of The established dummy variables were consideredas independent variables and a logistic regression modelwas established according to whether the patients werehighgrade glioma or WHO IV grade glioma whichwere set as dependent variables The AIC was calculatedto determine the best cutoff point for age among allmodels The model with the lowest AIC value wasregarded as the best model The results showed that thediagnostic age classification criterion was “ years oldand ‰¥ years old The probability of highgrade gliomaor WHO IV grade glioma in the age group ‰¥ years oldwas greater than that in the age group “ years old vs vs respectively Supplementary Table S2tolerance and treatmentOwing to the differences in the epidemiology betweenadults and pediatric glioma patients the differences insurgicalregimens betweenmiddleaged and elderly patients and the various prognoses of patients of different groups even with the samediagnosis only two age groups for the classification ofglioma patients were not sufficient in clinical practiceTherefore these two groups were subdivided into fourgroups First dummy variables were created by agegroups of 0I years old and I47 years old I any age between and The established dummy variables wereconsidered as independent variables and a logistic regression model was set up according to whether gliomapatients were high grade glioma or WHO IV grade glioma The AIC value for each model was calculated Themodel with the smallest AIC value was regarded as thebest model According to whether the patient sufferedfrom WHO IV glioma the diagnostic age classificationcriteria were “ years old pediatric group and “years old young group According to whether the patient was suffered from highgrade glioma the diagnosticage classification criteria were “ years old pediatricgroup and “ years old young group The evidencesuggeststhe difference between the biologicalspectrum of the disease may be reflected in the diagnostic age with the majority of the pediatric group belonging to the category described by Paugh et al[]Although some of the molecular abnormalities encountered in HGG in children are reminiscent of secondaryglioblastomas these tumors rarely originate from existing LGGs [] Finally years old was chosen as theage for distinguishing the pediatric group from the adultgroupthatSecond dummy variables as independent variableswere established by age groups of 48I years old and ‰¥ Iyears old I any age between and The cutoff ofthe model with the minimum AIC value was calculatedby the same method described above The resultingdiagnostic age classification criterion was “ yearsold middleaged group and ‰¥ years old elderlygroup The probability of highgrade glioma or WHOIV grade glioma in the age group ‰¥ years was greaterthan that of the age group “ years oldStatistical analysisThe SPSS statistical software package version IBMCorp was used for all analyses The statistical significance level was set as p Note that reported percentages may not add up to due to roundingCategorical variables are shown as numbers and percentages while continuous variables are shown as the meanand standard deviation SD Pearson™s chisquare testwas performed to compare the categorical dataResultsAnalysis of demographic and clinical characteristicsThe study population comprised male patients and female patients The ratio of malesto females was The age range was to years oldand the mean age was years old SD years oldThere were patients were classified as WHOgrade I patients were classified as WHOgrade II patients were classified as WHOgrade III and patients were classified asWHO grade IV Supplementary Figure S2According to the WHO classification of tumorsof the CNS the glioma patients diagnosed andtreated at Nanfang Hospital were subdivided into histologically distinct types of primary glioma Astrocytomas accounted for approximately n of allgliomas The average diameter of glioma was cmSD cm Gliomas mostly occurred in the frontallobe n and temporal lobe n GBM represented the majority of gliomas n The distribution of tumor sites showed that cases occurred in the brain casesoccurred in the spinal cord and cauda equina and cases involved the spinal cord cauda equina andbrain Detailed information for this cohort of glioma patients is recorded in Supplementary Table S1The median age at diagnosis for all primary glioma tumors was years old As shown by the cumulativecurves of the proportion of gliomas across four WHOgrades gliomas of higher grades tended to be diagnosedat older ages Fig 1a p The average age at diagnosis of WHO grade IV glioma was while WHOgrade I gliomas were diagnosed at years with anage gap of more than years Fig 1b The average agesat diagnosis of WHO grade II and III were and years respectively Fig 1b In addition we compared the average age at diagnosis of various pathological subtypes of glioma We found that anaplasticastrocytoma WHO grade III was diagnosed at an olderage than that ofindividuals diagnosed with diffuse 0cLin BMC Neurology Page of Fig Cumulative age distribution and T test of the average age at diagnosis of different types of glioma a Cumulative age distribution of WHOIIV grade glioma the mean age of glioma patients increases with the WHO grade WHO I years WHO II years WHO III years andWHO IV years respectively b The diagnosed age boxplot figure of WHO IIV grade glioma c Cumulative age distribution of anaplasticastrocytoma and diffuse astrocytoma there is likely for an earlier manifestation in diffuse astrocytoma d The average age at diagnosis ofanaplastic astrocytoma and diffuse astrocytoma e Cumulative age distribution of Oligodendroglioma and anaplastic oligodendroglioma most ofoligodendroglioma and anaplastic oligodendroglioma arise in adults with peak incidence in patients aged “ years f The diagnosed ageboxplot figure of oligodendroglioma and anaplastic oligodendroglioma g Cumulative age distribution of Oligoastrocytoma and anaplasticoligoastrocytoma the median ages of patients with oligoastrocytoma are years The median age of patients with anaplastic oligoastrocytomais years h The diagnosed age boxplot figure of oligoastrocytoma and anaplastic oligoastrocytomaastrocytoma WHO grade II Fig 1c and d vs years respectively p With a similar trendanaplastic oligodendroglioma WHO grade III was diagnosed at a median age of years and oligodendroglioma WHO grade II was diagnosed at a median age of years Fig 1e and f p Besides oligoastrocytoma WHO grade II and anaplastic oligoastrocytomaWHO grade III were diagnosed at average ages of and years respectively Fig 1g and h p Isocitrate dehydrogenase IDH1 is a vital marker for themolecular classification of glioma In this cohort whenanalyzing the average age at diagnosis of different IDH1phenotypes by using the whole cohort no significant differences were observed Supplementary Figure S1B andD however IDHwt GBM was diagnosed at an olderage than that of individuals diagnosed with IDHmutGBM Supplementary Figure S1A and C vs respectively p These results indicated that theage at diagnosis was closely correlated with the WHOgrade and pathological subtypes of gliomaEstablishment of age group classification cutoffAge and positive area of Ki67 and wtP53 showed greatvalue for the diagnosis of WHO grade IV glioma andhighgrade glioma Fig 2a and b The status of Ki67and P53 could be assessed only after surgery of biopsywhile the information of age could be obtained beforesurgery Therefore age could be an earlier factor for theevaluation of patients in clinical practice We thensought to establish an age group classification for bettermanagement of patients according to the AIC methodmentioned in the section of œmethod Glioma patientswere divided into four age groups “ years oldpediatric group “ years old youth group “years old middleaged group and ‰¥ years old elderlygroup of patients were “ years old pediatricgroup were “ years old middleaged group were “ years old youth group and were ‰¥ years old elderly group The proportion ofprimary WHO grade IV gliomas and larger tumor sizeslarger than cm increased with age Fig 2c and ghowever the proportions of glioma of astrocyte differentiation only include WHO grade IIII and ependymalcells differentiation decreased with age Fig 2d and fMost of the gliomas of oligodendrocyte differentiationwere found in “ age group Fig 2eTo examine the value of this age group classificationin risk stratification of GBM we collected data from patients in the Chinese Glioma Genome Atlas CGGAdatabase and calculate the proportion of different gliomagrade in four age groups respectively The sensitivity ofpredicting WHO grade IV was the specificity was 0cLin BMC Neurology Page of Fig ROC curve of the sensitivity and specificity for diagnosing WHO IV glioma a and high grade glioma b Age ki67 and positive area ofwtp53 have great value for the diagnosis of WHO grade IV glioma and highgrade glioma The proportion of WHO grade IV glioma c astrocytedifferentiation d oligodendrocyte differentiation e ependymal cells differentiation f and cm of tumor size g in four age groupsAccording to the discriminant classification of whether the pathological diagnosis of the patients was WHO grade IV or not the predictionprobability was taken as the discriminant dividing point and the total judgment rate was h and the total judgment rate was p Fig 2hAnalysis of the pathological subtypes of glioma acrossfour age groupsIn the pediatric group the proportion of pilocytic astrocytoma was while GBM accounted for the largestproportion in the youth group middleage group andelderly group and respectively Fig and Supplementary Figure S3 Pilocytic astrocytomapleomorphic xanthoastrocytoma ependymoma anaplastic ependymoma choroid plexus papilloma atypicalchoroid plexus papilloma and ganglioglioma are predisposed to patients in pediatric group Diffuse astrocytoma diffuse midline glioma H3K27Mmutant gliomaoligodendroglioma oligoastrocytoma and myxopapillaryependymoma commonly occurred in youth group Anaplastic astrocytoma anaplastic oligodendroglioma andanaplastic oligoastrocytoma were more likely to occur inmiddleage group GBM and anaplastic gangliogliomawere more likely to occur in elderly group p0001The proportions of anaplastic oligodendroglioma andanaplastic ganglioglioma increased with age Ependymoma gradually decreased in the younger age groupsFig Analysis of glioma cell differentiation size and anatomiclocation across four age groupsPatients aged ‰¥ years old were predisposed to gliomasof astrocyte differentiation Patients aged “ yearsold were predisposed to gliomas of oligodendrocyte andhybrid cell differentiation Patients aged “ years oldwere predisposed to gliomas of ependymal cell and othercells differentiation Supplementary Table S2 p The proportion of tumors with sizes of “ cm decreased with age however the proportion of tumorswith sizes ranging from to cm was larger in oldergroups Supplementary Table S2 p In the pediatric group the common locations of gliomas were the cerebellum and ventricle accountingfor and respectively Supplementary Tablein the youth and middleage groupsS3 Howeverlobe accounted for the largest proportionthe frontalSupplementary Table S3 p In the elderlygroup the proportion of tumors in the frontallobeand temporal lobe was higher than that in the otherlocations Supplementary Table S3 and respectivelyAnalysis of molecular marker expression in four agegroupsThe proportion of positive expression of glial fibrillaryacidic protein GFAP was more than in all agegroups Detailed information is recorded in Supplementary Table S2 The proportion of positive expression ofIDH1wt Ki67 and Oligodendrocyte transcription factor Oligo2 increased with age The proportion ofpositive expression of epithelial membrane antigenEMA vascular endothelial growth factor VEGF andMGMTO6methylguanineDNA methyltransferasewere maximalthein the pediatric group while 0cLin BMC Neurology Page of Fig Histological distribution by Age groups a Histological distribution by “ years old group b Histological distribution by “ years oldgroup c Histological distribution by “ years old group and d Histological distribution by ‰¥ years old group In the “ age group Theproportion of pilocytic astrocytoma in the histological distribution was however glioblastoma accounted for the largest proportion of theage group “ years old “ years old and ‰¥ years old with and respectivelyFig Composition changes of pathological subtypes across four age groups 0cLin BMC Neurology Page of proportion of positive expression of neuronal nucleiNeuN and epidermal growth factor receptor EGFRwere highest in the middleage group Fig Besideswe analyzed the expression of gliomaassociated genesin homogeneous groups including subgroups of different cell origins and different molecular subtypes suchas EGFRpositive and EGFRnegative gliomas The results revealed great heterogeneity across the four agegroups Supplementary Figure S4 S5 S6 S7 S8 S9S10 S11 Supplementary Table S4S5DiscussionClinical and biological data clearly indicate thatthecharacteristics and outcomes of malignant gliomas differsignificantly between adults and children [] A numberof studies have showed that the tumorprone locationshistopathology prognosis and some molecular markersare different in glioma patients of different ages [ ]Growing research has shown that the molecular characteristics of GBM in elderly patients are more aggressivethan those in young patients [] Childhood GBMFig The glioma heatmap of 10gene signatures by gene expression subtype Representative genes are shown for each subtype a Heatmap ofpediatric group b Heatmap of youth group c Heatmap of middleage group d Heatmap of elderly group 0cLin BMC Neurology Page of displayed on average considerably fewer DNA copynumber changes than histologically similar adult tumor[“] In addition the prognosis of glioma is particularly severe in older adults [ ] The clinical practicepatterns show that with increasing age the applicationof surgical resection radiotherapy and chemotherapy decreases [“] Nevertheless some elderly patients withglioblastoma can benefitfrom these therapies []These elderly patients will receive aggressive treatmentwith radiation or chemotherapy When consideringtreatment options for children with gliomas neurosurgeons will try to avoid the deleterious effects of radiotherapy on the developing brains of children Minimaldysfunction resulting from glioma and treatment shouldbe achieved as much as possible with the expectation ofchildren living to adulthood [] Moreover age isregarded as an important factor related to the prognosisof glioma patients Thereforefor patients diagnosedwith glioma age should be taken into consideration toperform personalized treatment for a better outcomeHowever the criterion for appropriately dividing agegroups of glioma patients remains an unresolved clinicalproblemA large number of studies used different age groupings and these studies led us to differential sabout the prognosis value of age in glioma patients [ ] These contradictory s could be partlyexplained by the difference in age classification criteriabetween different studies In one study a multivariateCox regression model with different cutoff points wasused to analyze the effect of age on OS but only threeage groups were compared and univariate analysis wasperformed using prognostic factors as a classification criterion [] OS is a good indicator for evaluating patientoutcomes but confounding factors such as tumor sizetumor location surgical resection extent and patientcompliance might impair the accuracy of the relationship between age and OSTo avoid the disturbance of confounding factors asmuch as possible our study used the WHO grade of glioma as a dependent variable to assess the prognosis ofglioma patients The classification criteria for glioma patients based on age were “ years old pediatric groupand “ years old youth group “ years oldmiddleaged group and ‰¥ years old elderly groupThis age group classification can be used for preliminaryevaluation of newlydiagnosed glioma patients and helpsto perform precise management in clinical practice according to age group Besides we found that EGFRpositive expression was more common in the middleage group and the EGFR expression in IDH1mut gliomas was more apparent Therefore patients withIDH1mut glioma aged “ years old might benefitfrom EGFR inhibitor therapy Based on this age groupclassification we further analyzed the characteristics ofWHO grade tumor size tumor histology and anatomical location among the four age groups We found thatthe proportion of WHO grade IV gliomas and positiveexpression of Ki67 Oligo2 and IDHwt increased significantly in elderly age groups In addition in the olderage group more patients suffered from a heavy tumorburden tumor size cm Regarding the histology ofglioma pilocytic astrocytoma is the most common inchildren while glioblastoma accounts for the largest proportion of adult groups Many studies have demonstrated that patients with a higher grade of glioma havea worse outcome [] Moreover a larger tumor burdenmight cause a higher risk of functional deficits includingmotor dysfunction impaired communication ability ordecline in neurocognitive function [] Therefore theprognosis of patients with gliomas can initially be evaluated according to age On the other hand patientsgrouping according to age has been widely used in clinical studies but there is no uniform standard of agegroup classification for patients with glioma The agegroup established on the basis of objective pathologicaldiagnosis in this study will be helpful for clinical trialsdesign in the futureGlioma especially glioblastoma is a highly heterogeneous malignancy In addition to the marked heterogeneity of tumor size and histopathology the heterogeneityof the molecular characteristics of tumors is becomingincreasingly important and is reported in several studiesAccording to the WHO classification glioma isfirst classified according to histologicalfeatures andthen more subtypes are classified according to molecularcharacteristics There are a variety of indicators that arewidely used in clinical practice such as GFAP EMAMGMT P53 NeuN Oligo2 EGFR VEGF IDH1 Ki671p19q and these indicators are highly correlated withthe prognosis of the patients [“] Agedependentoccurrence and the effects of different biological markershave been reported in malignancies [] For examplethe association between age and tumor grade Ki67markers apoptosis index EGFR expression and erbB2expression has been reported in breast cancer [] Astudy indicated that the prognostic effects of P53 1pand CDKN2Ap16 alterations are dependent on patientage [] Increasing translational studies have significantly advanced the understanding of glioma pathogenesis and have identified several prognostic factorsHigher tumor grade older age [] and increased expression of molecular biomarkers such as P53 []MGMT [] PR [] IDH1wildtype [] H3K27Mmutation of pediatric HGG [ ] and Ki67 []were related to poorer prognoses Analysis of the pathological molecular markers acrossfour age groupsshowed that the proportion of patients with larger than 0cLin BMC Neurology Page of area of Ki67 positive expression or PR positive expression increased with age Other molecular markersGFAP EMA NeuN EGFR IDH1 CD3 and H3K27Mshowed great heterogeneity among the four age groupsGender age anatomic location of the tumor size oftumor and molecular markers are simple and objectiveparameters that can be collected easily in clinical practice or clinical studies on patients with glioma Our research can provide clinicians with a simple method toevaluate the prognosis of glioma patients and help topromote the personalized management of glioma patients In addition for some clinical trials that need todivide participants of glioma into different groups thisage group classification based on WHO grade will bemore objective However this study was limited by thesample size and these data were retrospective Hospitalbased retrospective studies may lead to certain selectionbiases Another limitation of this study was that we didnot include patients with postoperative recurrence Further validation of our results will require multicenterprospective studies with larger sample sizesConclusionOur research indicated that the classification criteriabased on the age for glioma patients were “ years oldpediatric group “ years old youth group “years old middleaged group and ‰¥ years old elderlygroup Our cohort indicates that pilocytic astrocytomaaccounts for the largest proportion in the “ year agegroup while GBM accounts for the largest proportion inthe other three age groups Besides the proportion oftumors of “ cm in size or with Ki67 increaseswith WHO grade This age group classification will helpto improve the diagnosis personalized treatment andclinical trial design involved patients with gliomaSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1288302001888wAdditional file Figure S1 Cumulative age distribution and T test ofthe average age at diagnosis of glioma A Cumulative age distribution ofIDH1wt glioma and IDH1mut glioma B Cumulative age distribution ofIDH1wt glioma and IDH1mut glioma C The diagnosed age boxplot figure of IDH1wt GBM and IDH1mut GBM D The diagnosed age boxplotfigure of IDH1wt GBM and IDH1mut GBMAdditional file Figure S2 Constituent ratios of four age groups Theproportion of patients in the four age groups “ “ “ and ‰¥ years oldAdditional file Figure S3 Distribution by Age groups of otherhistology A “ years old B “ years old C “ years old D‰¥ years old subependymal giant cell astrocytoma subependymomaangiocentric glioma chordoid glioma of the third ventricle anaplasticganglioglioma desmoplastic infantile astrocytoma and gangliogliomawere not analyzed because the total number of patients was no morethan threeAdditional file Figure S4 Heatmap of 10gene signatures by geneexpression subtype Representative genes are shown for each subtype AHeatmap of all glioma B Heatmap of all GBM C Heatmap of pediatricgroup D Heatmap of youth old group E Heatmap of middleage groupF Heatmap of elderly groupAdditional file Figure S5 The heatmap of glioma derived fromastrocyte differentiation only including WHO grade I III A Heatmap ofpediatric group B Heatmap of youth group C Heatmap of middleagegroup D Heatmap of elderly groupAdditional file Figure S6 The heatmap of glioma derived fromoligodendrocyte differentiation A Heatmap of pediatric group BHeatmap of youth group C Heatmap of middleage group D Heatmapof elderly groupAdditional file Figure S7 The heatmap of glioma derived fromependymal cells differentiation A Heatmap of pediatric group BHeatmap of youth group C Heatmap of middleage group D Heatmapof elderly groupAdditional file Figure S8 The heatmap of glioma with EGFRpositive expression A Heatmap of pediatric group B Heatmap of youthgroup C Heatmap of middleage group D Heatmap of elderly groupAdditional file Figure S9 The heatmap of glioma with EGFRnegative expression A Heatmap of pediatric group B Heatmap ofyouth group C Heatmap of middleage group D Heatmap of elderlygroupAdditional file Figure S10 The heatmap of IDH1mut glioma AHeatmap of pediatric group B Heatmap of youth group C Heatmap ofmiddleage groupAdditional file Figure S11 The heatmap of IDH1wt glioma AHeatmap of pediatric group B Heatmap of youth group C Heatmap ofmiddleage groupAdditional file Table S1 Clinical and molecular characteristics ofpatients with gliomas n Additional

Thyroid_Cancer

"attention has been paid to whether snoring frequency is associated with body composition inmenopausal women particularly in China This study objected to investigate the association between selfreportedsnoring and body composition in peripost menopausal Chinese women as well as metabolic indicatorsMethods This crosssectional study enrolled participants aged “ years from the Menopause Clinic in theShanghai Sixth People™s Hospital Participants were categorized into four subgroups stratified by selfreportedsnoring frequency never rarely night per week occasionally “ nights per week regularly ‰¥ nights perweek while body composition was measured using bioelectrical impedance analysis BIA Besides blood samplewere collected to test the glycolipid indicatorsResults In our sample of investigation regular snoring ‰¥ nights per week was found to be an independent riskfactor for higher fat mass total upper limbs trunk with the highest risk of times for fat mass of trunk afteradjusting for metabolic confoundersp Meanwhile regular snoring was independently associated withhigher fat mass total and each segment only in menopausal transition p Conclusions We suggested that selfreported regular snoring may be taken as a simple alternative to predict higherfat mass ‰¥ kg upper quartile in menopausal women Similarly body composition should be attached to thegreat importance to those who in menopausal transition in order to help to prevent obstructive sleep apnea OSAKeywords Body composition Snoring Menopausal transitionBackgroundSnoring the manifestation of increased upper airway resistanceis commonly regarded as a reliable proxymarker of obstructive sleep apnea OSA [ ] Moreover regular snoring has been suggested to be correlatedwith obesity [] hypertension [] and diabetes mellitus[] OSA is supposed to be more prevalent in men than Correspondence yctengsjtueducn taomfsjtueducn Yang Zhou and Fei Liu contributed equally to this work1Department of Gynecology and Obsterics Shanghai Jiao Tong UniversityAffiliated Sixth People™s Hospital Yishan Road Shanghai ChinaFull list of author information is available at the end of the women however the gap was narrowed when womenapproach menopause [ ] Women in menopause transition are more likely to report perspective poor sleepsnoring [] which largely affected quality life of menopausal women In addition previous studies have reported that menopause was an important risk factor forsnoring mainly due to the declining ovarian hormones[ ] Thus it is important to combat snoring in peripost menopausal womenMeanwhile menopause is a vital window for variationsin the body composition and rising in the body weightcaused by hormonal alterations [] However body 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 0cZhou BMC Women's Health Page of in menopausal women [] Changesmass index BMI is not a valid measure of true obesitystatusinmenopauserelated body composition may be coveredand underestimated by stable BMI since the counteractive effect of loss of lean mass and gain of fat masswhen aging Therefore body composition by bioelectrical impedance analysis BIA may be a more representative and precise instrument rather than BMI amongmenopausal Chinese women []So far current studies on the association of snoringand obesity have focused primarily on men and children[ ] while underrepresented women In addition anyassociation between snoring and body composition inmenopausal women has received little attention Sinceit™s possible that glycolipid metabolism may confoundthe association and whether snoring is associated withbody composition in menopausal women independentlyof glycolipid metabolism confounders remains unknownGiven the evidence of the cross interplay among snoringobesity and menopause we aim to explore the association with snoring and body composition in menopausalwomenMethodsStudy participantsThis crosssectional study enrolled participants who visited the Menopause Clinic in the Shanghai Sixth People™s Hospital HanChinese woman aged “ yearspassing through the menopause were recruited Exclusion criteria were with rhinitis having severe internalsuch as myocardialinfarction stroke and cancer current smoking atleast once per week for the previous months excessive alcohol drinking at least one pack per month forthe previous months suffering from thyroid disease having tubercle and cachexy missing dataUltimately715 participants were recruited in this studyillnesses andor diseasesGeneral questionnaireBaseline sociodemographic information was collectedfrom a questionnaire through facetoface interviewwhich has been previously employed [] seen in supplementary file Variables included age marital statusemployment status education level income per monthmenopausal age menopausal status history of chronicdisease ie hypertension diabetes mellitus rhinitisother diseases besideslifestyle ie smoke alcoholconsumption were recorded Guiding by the Stages ofReproductive Aging Workshop STRAW []participants were divided into three different menopausalsubgroups namely menopausal transition group consecutive irregularities for over days of menstrual cycleearly postmenopausal group absence of menstrual periods for months ˆ’ years and late postmenopausalfor ‰¥ yearsgroup absence of menstrual periodsHypertension was defined by any prior diagnosis fromthe questionnaire or by the criteria recommended by theseventh report of the Joint National Committee on Prevention Detection Evaluation and Treatment of HighBlood Pressure JNC7 [] While diabetes mellitus wasidentified by FPG ‰¥ mmolL or received any treatmentfor diabetes according to the WHO criteria []Snoring frequency assessmentParticipants were asked by the question to assess thesleep snoring frequency which was applied previously[ ] œOver the past weeks did you snore And ifdid how many times per week and the options for responses were œnever œrarely œoccasionally and œregularly corresponding to œnever œ night per weekœ“ nights per week and œ‰¥ nights per week respectively seen in supplementary file Anthropometric and lab testsWe measured and recorded participants™ weight heightBody mass index BMI was computed by dividingweight in kilograms by the square of their height in meters We took the blood pressure for all participants onthe right arm three consecutive times after 5min sittingsystolic blood pressure SBP diastolic blood pressureDBP Blood samples were collected for the detectionof serum concentration of triglyceride TG cholesterolTC highdensity lipoprotein HDL lowdensity lipoprotein LDL and fasting blood glucose FBG after anovernight fastBody compositionWe measured the body composition by BIA TBF418Banalyzer TANITA of lean mass LM fat mass FMand fatfree mass FFM and each segment includedupper lower limbs and trunk We also recorded basalmetabolic rate BMR concurrently [] The welltrained staff guided the participants to take off heavyclothes socks and shoes and hold the hand electrodesstanding barefoot in contact with footpad electrodes[] Fat mass total and each segment and lean masstotal and each segment were stated in the dichotomized form with a cutoff of the highest quartile as thehigher one comparing the highest to the lower two tertiles We defined ‰¥ kg ‰¥ kg and ‰¥ kg ashigher total fat mass higher fat mass of upper limbs andhigher fat mass of trunk respectivelyStatistical analysesAll statistical analyses were taken by SPSS IBMCorporation Armonk NY USA Data were tested fornormal distribution bythe Kruskal WallisHtestLevene™s test of homogeneity of variance was also 0cZhou BMC Women's Health Page of performed Variables were presented as mean ± standarddeviation SD when they showed normal distributionswhereas medians inter quartile range or values Oneway ANOVA normal distributions the KruskalWallis Htest skewed continuous variables and χ2 testcategorical variables were carried out to compare thedifferences among the four groups Snoring was analyzedas a categorical variable with never as the referencegroup Relationship between body composition andsnoring frequency was computed by multiple logistic regression analysis Covariates included TG TC HDLLDL FBG SBP DBP age marital status employmentstatus education level income per month menopausalage menopausal status hypertension diabetes mellitusTwosided p was considered significantResultsCharacteristics of the study participants based on snoringfrequencyA total of participants were finally entered into thestudy The basic characteristics among the four groupsdivided by the snoring frequency never rarely occasionally regularly were presented in Table Participants were on average ± years of age with amean weight of ± kg and the average BMIwas ± kgm2 The mean lean mass fat massand fat free mass were ± kg ± kgand ± kg respectively Compared with nonsnorers rare and occasional snorers regular snorerstended to be older showed higher triglyceridelowerHDLC and had less income p Moreover therewas an ascending trend in the incidence of hypertensionin different snoring frequency subgroupswith innonsnorers increasing to in regular snorersp However we did not observe the differenceamong three menopausal status respect to the snoringfrequencyBody composition of the study participants distributed bysnoring frequencyAs presented in Table compared with nonsnorersrare and occasional snorers regular snorers had higherfat mass upper limbs trunk lower limbs In additionwe found that there was an increasing trend in the fatmass of upper limbs trunk and lower limbs and also inlean mass of upper limbs with the increase of the sleepsnoring frequency p Odds ratio of snoring frequency for body composition bymultiple logistic regression analysisWe next investigated the odds ratio of snoring frequencyin predicting for body composition p in univariate analysis after adjusting for potential confounders Asdepicted in Fig compared with nonrare andoccasional snoring regular snoring was the risk predictor for higher total fat mass ‰¥ kg OR 95CI P higher fat mass of upperlimbs ‰¥ kg OR 95CI P higher fat mass of trunk ‰¥ kg OR 95CI P while other segmentsshowed no significance after adjustments In additionregular snoring increased the highest odds ratio OR of for fat mass of trunk among the other statisticallysignificant body compositionsIndependent determinants for regular snoring stratifiedby menopausal statusWe also investigated the independent roles of body composition for predicting regular snoring in multivariate logistic regression analysis in Table however we did notobserve any significance of body composition in predicting for regular snoring after adjusting confoundersInterestingly when the participants were stratified bydifferent menopausaltransitionearly postmenopause late postmenopause we observedthat fat mass segments were independently associatedwith regular snoring in menopausal transition but notpostmenopause TotalOR 95CI P fat mass of upper limbs OR 95CI P fat mass of trunkOR CI P fat mass oflower limbs OR 95CI P were independent indicators for regular snoring afteradjusting for confounders in menopausal transitionmenopausalstatusfat massDiscussionTo our knowledge this is the first study to document associations of snoring and body composition as well asmetabolic indicators in women with regard to menopausal status The main finding was that regular snoring‰¥ nights per week was an independent risk factor forhigher fat mass total trunk upper limbs in menopausalwomen after adjusting for wellestablished metabolicvariables Of special concern was that regular snoringhad a times significantly higher odds of higher fatmass of trunk which was the highest among other significant body composition This finding was in concordant with the previous study that OSA was more inclinedto a centralobesity phenotype than a wholeobesity pattern []Several mechanisms can interpretthis associationUpper airway resistance and collapsibility caused byregular snoring could result in intermittent hypoxia andsympathetic activation thus leading to the aggravationof obesity especially for abdominal fat [] In additionprotective role of progesterone and estrogen in respiratory control vanished after menopause which was associated with continuum from increased airway resistance 0cZhou BMC Women's Health Page of Table Body composition and characteristics of the women distributed by snoring frequencyVariablesRarelyn ± ± ± ± Occasionallyn ± ± ± ± Regularlyn ± ± ± ± Totaln ± ± ± ± ± “ ± ± “ ± ± “ ± ± “ ± ± ± ± ± ± ± “ ± ± “ ± ± “ ± ± “ ± ± “ ± ± ± ± ± ± Snoring FrequencyNevern ± ± ± ± ± “ ± Age yearsWeight KgHeight cmBMI Kgm2BMRTG mmollTC mmollHDLC mmollLDLC mmollFPG mmolLSBP mmHgDBP mmHgChronic disease n HypertensionDiabetesMarital status n MarriedSingleSeparatedDivorcedWidowedMenopausal status n Perimenopause Early postmenopause Late postmenopause Employment n Education n Junior or belowSenior highCollege or aboveIncome RMBmonth n “““ Lean mass kgUpperTrunkLowerFat mass kgUpperTrunkLower ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Fatfree kg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± P value 0cZhou BMC Women's Health Page of Table Body composition and characteristics of the women distributed by snoring frequency ContinuedVariablesRarelyn ± ± ± Occasionallyn ± ± ± Regularlyn ± ± ± Totaln ± ± ± P valueSnoring FrequencyNevern ± ± ± Upper limbsTrunkLower limbsHigher fat massHigher fat mass of upper limbsHigher fat mass of trunkHigher fat mass of lower limbsHigher lean massHigher lean mass of upper limbsOR OR OR OR OR OR Higher fat massHigher fat mass of upper limbsHigher fat mass of trunkHigher fat mass of lower limbsHigher lean massHigher lean mass of upper limbsRare snoringOR OR OR OR OR OR Occasional snoringHigher fat massHigher fat mass of upper limbsHigher fat mass of trunkHigher fat mass of lower limbsHigher lean massHigher lean mass of upper limbsOR OR OR OR OR OR Fig Odds ratios 95CI of snoring frequency for body composition in women analyzed by multivariate logistic regression Covariates age BMITG TC HDL LDL FBG SBP DBP hypertension diabetes mellitus menopause income education employment status means p0005Regular snoring 0cZhou BMC Women's Health Page of Table Odds ratio of body composition for regular snoring stratified by menopausal status by logistic regressionFat mass kgRegular snoringPerimenopause n Odds ratio CI Pvalue Fat mass of upper limbs kg Fat mass of trunk kg Fat mass of lower limbs kg manifested as snoring [“] Taken together menopause make women lose protective effects against snoring and further augmentsnoreobesity associationespecially snorecentralobesity associationInterests in obesity and OSA as regards to œwhich isthe chicken or the egg has existed since the dawn ofhistory [ ] Thus to identify the mutual effect ofsnoring and obesity we also assessed the role of bodycomposition in predicting the snoring We found thatfat mass was an independent risk factor for regular snoring only in menopausal transition not postmenopause ina multivariable model Taken together we suggestedthat the rise in obesity may serve as a key contributor tothe burgeoning prevalence of snoring in women whilemenopausal transition not postmenopause period maymark this relationshipThe reason can be explained by the fact that menopausal transition is more concerned with fluctuation ofsex hormone than postmenopause which predisposed tomodulate sleep regulation and breathing thus leading tothe snoringBesides other independent factors for regular snoringsuch as higher TG lower LDL were compatible with onestudy [] Although selfreported snoring was closelyrelated with hypertension and diabetes Unexpectedlywe did not find that hypertension was related with regular snoring after multiple adjustments These divergentfindings may be attributed to difference in sample sizeethnicity culture and the definition of hypertension anddiabetes etc Another possible explanation is that manyprevious studies did not consider menopause status in toaccount which may aggravate the snoreobesity association thus overshadow the snorehypertensiondiabetesassociationHowever our study should be interpreted in light ofthe following limitations First one limitation of thepresent study is that the crosssectional design does notpermit conclusion of causality further prospective studies are needed to verify the association between snoringfrequency and body composition Second selfreportedsnoring frequency but not polysomnography the goldstandard for diagnosing OSA which could bring aboutthe statistical error However precious study has suggestthat selfreport is a reliable measure []Early postmenopause n Odds ratio CI Pvalue Late postmenopause n Odds ratio CI Pvalue ConclusionsRegular snoring ‰¥ times per week may be an independent strong predictor for fat mass of trunk in menopausal women while fat mass in turn serves as a strongpredictor for regular snoring only in menopausal transition Taken together early detection and interventionsof participant showing regular snoring and higher fatmass in menopause could have important preventiveimplicationsSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12905020010252Additional file AbbreviationsBIA Bioelectrical impedance analysis OSA Obstructive sleep apneaTG Triglyceride TC Cholesterol HDL Highdensity lipoprotein LDL Lowdensity lipoprotein FBG Fasting blood glucose BMI Body mass indexFM Fat mass LM Lean mass FM Fat mass FFM Fatfree mass BMR Basalmetabolic rateAcknowledgementsThe authors would like to acknowledge all women who consented to takepart of this study We are also thankful for the support and cooperation fromstaff members of obstetrics and gynecology in Shanghai Jiao TongUniversity Affiliated Sixth People™s HospitalAuthors™ contributionsMT conceived the study and YT designed the study [YZ] drafted andcritically revised the manuscript FL designed the questionnaire and analyzedthe data CL [YZ] JH [YZ] LG and SJ administered the questionnairesurvey and managed the data All authors read and approved the finalmanuscriptFundingThis study was supported by grants from the Science and TechnologyCommission of Shanghai Municipality The role of thefunding body in the design of the study and collection analysis andinterpretation of data and in writing the manuscript should be declaredAvailability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateAll participants had provided verbal informed consent after full explanationbecause the study posed no threat to the health of patients This study wassubmitted to and approved by the ethics committees of Institutional ReviewBoard in Shanghai Sixth People™s Hospital affiliated for Shanghai JiaotongUniversity No2016R07 0cZhou BMC Women's Health Page of Consent for publicationNot applicableCompeting interestsThe authors declare that they have no conflict of interestAuthor details1Department of Gynecology and Obsterics Shanghai Jiao Tong UniversityAffiliated Sixth People™s Hospital Yishan Road Shanghai China2Reproductive medicine center Shanghai Jiao Tong University Affiliated SixthPeople™s Hospital Yishan Road Shanghai ChinaReceived July Accepted July ReferencesLee YH Kweon SS Choi JS A GenderSpecific Association betweenSelfReported Snoring and Hemoglobin A1c Levels in a General Populationwithout Type Diabetes Mellitus Yonsei Med J “ httpsdoi103349ymj20175861152 PubMed PMID PubMedCentral PMCID PMCPMC5653480Song J Wang C Ma A Selfreported snoring is associated with chronickidney disease independent of metabolic syndrome in middleaged and elderlyChinese J Diabetes Investig “ httpsdoi101111jdi12855PubMed PMID PubMed Central PMCID PMCPMC6319474 engBiggs SN Tamanyan K Walter LM Overweight and obesity add to behavioralproblems in children with sleepdisordered breathing Sleep Med “httpsdoi101016jsleep201709001 PubMed PMID Shivashankar R Kondal D Ali MK Associations of Sleep Duration andDisturbances With Hypertension in Metropolitan Cities of Delhi Chennaiand Karachi in South Asia CrossSectional Analysis of the CARRS StudySleep httpsdoi101093sleepzsx119 PubMed PMID PubMed Central PMCID PMCPMC5806550 eng Wu HB Wang H Hu RY The association between sleep durationsnoring and prevalent type diabetes mellitus with regard to gender andmenopausal status the CKB study in Zhejiang rural area China ActaDiabetol “ httpsdoi101007s0059201609181PubMed PMID Phillips BA Collop NA Drake C Sleep disorders and medicalconditions in women Proceedings of the Women Sleep WorkshopNational Sleep Foundation Washington DC March “ J WomensHealth Larchmt “ httpsdoi101089jwh20070561PubMed PMID engKapsimalis F Kryger M Sleep breathing disorders in the US femalepopulation J Womens Health Larchmt “ httpsdoi101089jwh20081054 PubMed PMID PubMed Central PMCIDPMCPMC2825714 engZhou Y Yang R Li C Sleep disorder an independent risk associatedwith arterial stiffness in menopause Sci Rep httpsdoi101038s41598017014897 PubMed PMID PubMed CentralPMCID PMCPMC5432489Young T Finn L Austin D Menopausal status and sleepdisorderedbreathing in the Wisconsin Sleep Cohort Study Am J Respir Crit Care Med“ httpsdoi101164rccm2002091055OC PubMedPMID engLi QY Huang SG Li M BMI is an independent risk factor for snoring inChinese women aged over years Sleep Breath “httpsdoi101007s1132500802360 PubMed PMID engStenholm S Harris TB Rantanen T Sarc ic obesity definitioncause and consequences Curr Opin Clin Nutr Metab Care “ httpsdoi101097MCO0b013e328312c37d PubMed PMID PubMed Central PMCID PMCPMC2633408 eng Banack HR WactawskiWende J Hovey KM Is BMI a valid measure ofobesity in postmenopausal women Menopause “ httpsdoi101097gme0000000000000989 PubMed PMID PubMedCentral PMCID PMCPMC5821529 engThomson R Brinkworth GD Buckley JD Good agreement betweenbioelectrical impedance and dualenergy Xray absorptiometry forestimating changes in body composition during weight loss in overweightyoung women Clin Nutr “ httpsdoi101016jclnu PubMed PMID eng Marin JM Carrizo SJ Vicente E Longterm cardiovascular outcomes inmen with obstructive sleep apnoeahypopnoea with or without treatmentwith continuous positive airway pressure an observational study LancetLondon England “ httpsdoi101016s0140 PubMed PMID eng Harlow SD Gass M Hall JE Executive summary of the Stages ofReproductive Aging Workshop addressing the unfinished agenda ofstaging reproductive aging Fertil Steril “ httpsdoi101016jfertnstert201201128 PubMed PMID PubMed CentralPMCID PMCPMC3340904 engLenfant C Chobanian AV Jones DW Seventh report of the JointNational Committee on the Prevention Detection Evaluation andTreatment of High Blood Pressure JNC resetting the hypertension sailsHypertension “ httpsdoi10116101hyp000007579033892ae PubMed PMID engLu B Yang Y Song X An evaluation of the International DiabetesFederation definition of metabolic syndrome in Chinese patients older than years and diagnosed with type diabetes mellitus Metabolism “ httpsdoi101016jmetabol200604003 PubMed PMID engEndeshaw Y Rice TB Schwartz AV Snoring daytime sleepiness andincident cardiovascular disease in the health aging and body compositionstudy Sleep “ httpsdoi105665sleep3140PubMed PMID PubMed Central PMCID PMCPMC3792392 eng Wang HB Yan WH Dou JT Association between Selfreported Snoringand Prediabetes among Adults Aged Years and Older without DiabetesChin Med J “ httpsdoi10410303666999202741PubMed PMID PubMed Central PMCID PMCPMC5381312 eng Zhou Y Zheng Y Li C Association of body composition with menopausalsymptoms in perimenopausal women Climacteric “ httpsdoi1010801369713720181428295 PubMed PMID engTanaka NI Hanawa S Murakami H Accuracy of segmental bioelectricalimpedance analysis for predicting body composition in pre andpostmenopausal women J Clin Densitometry “ httpsdoi101016jjocd201407002 PubMed PMID eng Gasa M LopezPadros C Monasterio C Anthropometrical phenotypesare important when explaining obstructive sleep apnea in female bariatriccohorts J Sleep Res 2019e12830 httpsdoi101111jsr12830 PubMedPMID eng Pillar G Shehadeh N Abdominal fat and sleep apnea the chicken or the eggDiabetes Care 200831Suppl 2S303“ httpsdoi102337dc08s272PubMed PMID PubMed Central PMCID PMCPMC2453667 eng Behan M Kinkead R Neuronal control of breathing sex and stresshormones Comprehensive Physiology “ httpsdoi101002cphyc100027 PubMed PMID eng Behan M Zabka AG Thomas CF Sex steroid hormones and the neuralcontrol of breathing Respir Physiol Neurobiol ““PubMed PMID eng Bixler EO Vgontzas AN Lin HM Prevalence of sleepdisordered breathing inwomen effects of gender Am J Respir Crit Care Med Pt “httpsdoi101164ajrccm16339911064 PubMed PMID eng Carneiro G Zanella MT Obesity metabolic and hormonal disordersassociated with obstructive sleep apnea and their impact on the risk ofcardiovascular events Metabolism “ httpsdoi101016jmetabol201803008 PubMed PMID eng Zhang N Chen Y Chen S SelfReported Snoring Is Associated withDyslipidemia High Total Cholesterol and High LowDensity LipoproteinCholesterol in Obesity A CrossSectional Study from a Rural Area of China Int JEnviron Rese Public Health httpsdoi103390ijerph14010086PubMed PMID PubMed Central PMCID PMCPMC5295337 engTelakivi T Partinen M Koskenvuo M Periodic breathing and hypoxia insnorers and controls validation of snoring history and association withblood pressure and obesity Acta neurologica Scandinavica “ PubMed PMID engPublisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

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

This study was performed to explore the effective management of bleeding associated with radiofrequency ablation RFA of benign thyroid nodulesMethods Thirtyfive patients with benign thyroid nodules who were treated with ultrasoundguided RFA from July to December at the Third Affiliated Hospital of Sun YatsenUniversity were retrospectively reviewed The technique efficacy bleeding and other complications were assessed during the followup periodResults The mean technique efficacy was 06 at month and 06 at months after the procedure One case of an intranodular haematoma and two cases of voicechange month were observed All patients recovered with corresponding treatmentConclusion Although the incidence of haemorrhage is low serious haematomas are lifethreatening Therefore having a comprehensive understanding of the potential complicationsan accurate clinical strategy and adequate technical skills may prevent or help to properly managethese complicationsKeywordsRadiofrequency ablation benign thyroid nodules haemorrhage management haematomaultrasoundDate received January accepted June 1Department of Medical Ultrasound The Third AffiliatedHospital of Sun Yatsen University Guangzhou China2General Surgery Department The Third AffiliatedHospital of Sun Yatsen University Guangzhou ChinaThese authors contributed equally to this workCorresponding authorsBo Liu General Surgery Department The Third AffiliatedHospital of Sun Yatsen University Tianhe RoadGuangzhou City Guangdong Province China Jie RenDepartment of Medical Ultrasound The Third AffiliatedHospital of Sun Yatsen University Tianhe RoadGuangzhou City Guangdong Province ChinaEmails renjieguangzhou126com liubojake126comCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cIntroductionAssociationfor AdultThyroid nodules are extremely commonand the associated morbidity rate rangesfrom to according to highresolution ultrasound US findings12Mostthyroid nodules are benign andrequire no intervention other than clinicalfollowup According to the AmericanThyroidManagementGuidelinesPatients withThyroid Nodulesand DifferentiatedThyroid Cancer thyroidstimulating hormone suppression therapy for benign thyroid nodules BTNs is not recommendedbecause the potential harm outweighs thebenefit3 Radioiodine therapy was historically an effective treatment for thyroid hotnodules and a possible alternative to surgery Howeverthis technique has beenproven to have uncertain efficacy andsome adverse effects such as hypothyroidrecurrence4“ Surgery may beism orconsideredgrowing BTNs withpressurerelated symptoms neck discomfort cosmetic concerns or decreased quality of life3 At present partialtotal thyroidsurgery is considered the gold standardtreatment Surgeryassociated withnumerous complications such as nerveinjury anaesthesiarelated problemslonghospital stays conspicuous scars haemorrhage and lesions ofthe parathyroidglands78 In addition hypothyroidism isinevitable after totalthyroidectomy andrequires lifelong hormone supplementationHenceincreasingly minimally invasivetherapeutic strategies are currently used totreat BTNs In most cases several thermalablation techniques such as laser ablationmicrowave ablation radiofrequency ablation RFA and highintensity focused UShave been shown to be effective in BTNsAmong these thermal ablation techniquesRFA is the most widely applied910forisRFA of thyroid diseases first reported in“ is considered efficacious and safeJournal of International Medical Researchfor treatment of BTNs1415 To date no lifethreatening complications related to RFAhave been reported Howeverseveralcases of haemorrhagerelated to fineneedle aspiration FNA or core needlebiopsy CNB have been reported16“Although a microinvasive procedure suchas FNA can result in massive uncontrolledbleeding resulting in upper airway respiratoryuncontrolledbleeding is a rare but lifethreatening complication of RFA Thus management ofbleeding associated with RFA of BTNs isof vitalimportance This study was performed to explore the effective managementof bleeding associated with RFA of BTNsobstructionsuchMaterials and methodsofThis study was approved by the EthicsCommitteethe Third AffiliatedHospital of Sun Yatsen University andwritten informed consent was obtainedfrom all patients prior to the performanceof USguided FNA or CNB and RFA Therequirement to obtain informed consent forpublication was waived because of the retrospective nature of the studyPatientsAll consecutive patients who underwentRFA of BTNs at our institution from July to December were analysed Thefollowing inclusion criteria were appliedconfirmation of benignancyBethesdaClass II by FNA cytology or CNB complaints of pressure symptoms compressivesymptoms neck discomfort orforeignbody sensation or cosmetic problems a2cm maximum diameter of the indexnodule anxiety about a malignancy unsuitability for surgery or unwillingness toundergo surgery and a normal serum thyrotropin concentration normal completeblood counts and normal blood coagulation test results The exclusion criteria 0cHu et alwere nodules showing malignant featuresie taller than wide spiculated marginmarked hypoechoic appearance or microcalcifications on US imaging19 abnormalthyroid function performance of othertreatments for the thyroid nodules within months before the procedure pregnancyand age of years For the presentstudy only patients with 15 months offollowup after the procedure were included Thirtyfive patients met the inclusioncriteriaPretreatment assessmentBefore the procedure conventional USfindings USguidedFNA findingscontrastenhanced US CEUS findingsand laboratory and clinical results wereevaluated Two radiologists TW and JR with and years of thyroid US experiencerespectively performed the USUSguided FNA and CEUS examinationsusing a Logiq E9 US device GE MedicalSystems Milwaukee WI USA equippedwithtransducerwith a MHzfrequency range “ MHz The USexamination included characterisation ofthe location shape size margins solidcystic proportions echogenicity calcification status and internal vascularity ofeach nodulefrequency ofan ML615centrelinearLaboratory tests included the levels ofthyroidstimulating hormone free triiodothyronine free thyroxin and thyrotropina complete blood cell count and a coagulation test prothrombin time and activatedpartial thromboplastin time The nodulevolume was calculated using the followingvolume¼ length 02 width 02equationdepth 02 In addition all patientsunderwent vocal cord function assessmentsby an experienced laryngologist before theablation procedure Atenrolment allpatients were asked to rate their pressuresymptoms on a 10cm visual analoguescale grade “ cm and the cosmeticgrading score was assessed by the physicianas described in the consensus statement20Procedures and equipmentpreventsignificantAll RFA procedures were performed by oneradiologist JR with years of experienceperforming RFA in an outpatient clinic Weused an RF generator VIVA RF SystemVR STARmed Gyeonggisi South Korea andan internally cooled 18gauge 70mmlength or 10mm activetip electrodeStar RF ElectrodeVR STARmed Localanaesthesia with lidocaine was appliedto the puncture site The hydrodissectiontechnique was used under US guidance glucose and norepinephrine weremixed and injected into the surroundingthyroid capsule which provided a safe distance between the needle tip and adjacentcritical structures During the procedurewe paid special attention to the preservation of surrounding important structurestocomplicationsTherefore two essential techniques werethe transisthmic approach andappliedtechnique2122 Ablationthe movingshotwas suspended when the index nodule wascovered by hyperechoic zones The technique efficacy TE was then evaluated byCEUS at to minutes after RFA untilthedisappearedTechnicalthechange of an entire nodule to a noenhancement zone on realtime CEUSFor nodules with an enhancement zonean additional ablation was performed todestroy the nodule as much as possibleComplications were monitored immediatelyafter the procedure and during the followup period Major and minor complicationsand adverse effects were defined accordingto the criteria established by the Society ofInterventional Radiology2324success was defined ashyperechoiczones 0cJournal of International Medical ResearchFollowup evaluationatandperformedserum thyroidAny specific complaints or concerns wererecorded for month Postproceduralfollowup wasand months after treatment At each followup visit a US examination CEUS examinationhormonemeasurements were performed pressuresymptoms and the cosmetic grading scorewere evaluated and the volume ofthetreated nodule was calculated The TE wascalculated using the following equationTE¼ final nodule volumeinitial nodule volume 02 Statistical analysisstatistical analyses were performedAllusing SPSS software version IBMCorp Armonk NY USA Continuousvariables are expressed as mean 06 standarddeviation Quantitative data for volumeand TE were analysed using a pairedttest A P value of 14 was consideredstatistically significantResultsThe patients™ characteristics are summarised in Table Thirtyfive patients underwent RFA including male and femalepatients mean age years The meanlargest BTN dimension was 06 mmrange “ mm and the mean BTNvolume was 06 mL Twentytwototal complications minor and majorcomplications were observed among thetreated patients None of these complications was lifethreatening and all occurredwithout sequelaeNodule volumeAfter treatment the overall volume of thesignificantly decreased 06nodules 06 mL at mL at month and 06 mL at monthsbaselineTable Patients™ baseline characteristics n¼ Characteristics 06 06 06 06 06 06 06 06 Age at treatment yearsMalefemale ratioBody weight kgBody height cmBody mass index kgm2Symptom score “Cosmetic score “Cosmetic score of Cosmetic score of Cosmetic score of Preablation serum FT4 level pmolLPreablation serum TSH level mIULIndex nodule on ultrasoundRight sideLeft sideLargest dimension mm to to 15Data are presented as mean 06 standard deviation ornumber of patientsFT4 free thyroxin TSH thyroidstimulating hormoneP and the TE was 06 at month and 06 at months P Table Figure shows the shrinkage of the nodules at and months after the procedure comparedwith baseline no hypoechoic blood supplywas observed within the area ofthenodulesBleeding complicationsTwelve patients developed bleeding complicationsincluding a perithyroidal haematoma minor complication in patientsand an intranodular haematoma majorcomplication in patient as shown inTable The haematomas were detectedby US scans which revealed gradualenlargement of a hyperechoic mass in oraround the nodules Figure For thepatient with intranodular haemorrhage 0cHu et alTable Changes in volume before RFA and at each followup visitParameterInitial month laterLargest diameter mm 06 “ 06 “Volume mLTechnique efficacy ”Data are presented as mean 06 standard deviation range 06 “ 06 “ 06 “ months laterP value 06 “ 06 “ 06 “ Figure a c e Ultrasound examination and b d f contrastenhanced ultrasound examination of a39yearold woman treated with radiofrequency ablation a b Ultrasound and contrastenhanced ultrasound revealed a cysticsolid nodule before ablation c d One month after ablation ultrasound showed ahypoechoic nodule with a decreased volume d e Six months after ablation the volume of the nodule haddecreased further and no blood supply was observed within the area of the nodule 0cJournal of International Medical ResearchTable Complications and adverse effects in patients who underwent RFA of thyroid nodulesComplication or adverse effectAdverse effectsFeverPainDizzinessSensation of heatMinorPerithyroidal haematomaVomitingnauseaOedemaswellingVoice change for monthMajorVoice change for monthIntranodular haemorrhageData are presented as n the haematoma was controlled throughtimely use of the ablation needle to coagulate the injured blood vessel and by injecting lyophilising thrombin powder into thehaematoma Figure Most of the perithyroidalseriesrequired only observation with or withoutcompression and disappeared within to weeks after the procedure None of the patientssubscapularhaematomahaematomasdevelopedthisinaOther complications and adverse effectsThe adverse effects of RFA included fevern¼ pain n¼ dizziness n¼ and a sensation of heatn¼ Minor complications includedoedemaswelling n¼ and a voicechange for month n¼ vomitingnausean¼ DiscussionImageguided thermal ablation techniquessuch as laser ablation ethanol ablationmicrowave ablation highintensity focusedUS and particularly RFA have recentlybecome more widely used to treat thyroidthe creation ofnodules Briefly the basic mechanism ofRFA involvesthermaldamage by friction and heat conductionwhich is generated from an oscillatinghighfrequency alternating electric currentproduced by the RFA generator and thentransferred through the electrode tip Theenergy of RFA is powerful and accurate2526 RFA is considered an effectiveand safe treatment for control of BTNsIn most cases the incidence of haemorrhage and other complications is low20However haemorrhage is sometimes lifethreatening because serious haematomasmay compress the upper airways Manyreports have described active bleedingduring FNA of thyroid nodules and RFAof hepatocellular carcinomas14““ andsome reports have described fatalities14ThusimportantcomplicationhaemorrhageanisThree types of haemorrhage may occurperithyroidal subcapsular and intranodular121427“ The mechanism of haemorrhage is thoughtto be related to themechanical or thermal injuries induced bythe RFA electrode tip3031 Thyroid nodulesreportedly have abundant capsular vesselsthat are usually anastomosed with vesselspenetrating into the core32 These numerousvessels are abnormal thinwalled and susceptible to rupture Large thyroid nodulesare another cause of haemorrhage becausemultiple insertions are often required totreat such nodules In additionthepatient cannot coordinate with the physician during the RFA procedure the perithyroidal orintrathyroidal vessels mayeasily be damaged by movement of theneedle tip or production of heat energyifIt is important to manage bleeding associated with RFA of BTNs Based on ourexperience we suggest several steps to preventshouldobtain a thorough medical history of eachpatient before the procedure All risk factorsdrugssuch bleeding Physiciansincludingbleedingfor 0cHu et alFigure Ultrasound examination and contrastenhanced ultrasound examination of patients with intranodular haemorrhage and perithyroidal haemorrhage a Ultrasound and contrastenhanced ultrasoundrevealed a hyperechoic mass lesion in the nodule b Ultrasound and contrastenhanced ultrasound revealedperithyroidal haemorrhagedrugsnonsteroidalantiplateletantiinflammatory drugs and anticoagulantsand diseases affecting coagulation shouldbe recorded33 In addition the patient™scoagulation function should be thoroughlyevaluated All patients with clinical coagulation disorders should be excluded Evenwhen coagulation indices are normalinpatients with high risk factors for bleedingsuch as liver cirrhosis endstage renal disease anticoagulant use or hypertension34sufficient preoperative preparation shouldbe emphasised A patient with active bleedingthesein the presentstudy metconditions Although his coagulation indices were normal he had a subclinical coagulation disorder due to endstage liverdisease Fresh frozen plasma or human prothrombin complex should be used inpatients with liver cirrhosis and anticoagulants should be withdrawn in these patientswhich will help to improve coagulation function before the procedure If a possibility ofbleeding exists Reptilase haemocoagulaseatrox forinjection Pentapharm BaselSwitzerland can be used preoperativelyDuring the RFA procedure an effectiveclinical strategy and adequate technical 0cJournal of International Medical ResearchFigure Ultrasound examination and contrastenhanced ultrasound examination of patients with intranodular haemorrhage or perithyroidal haemorrhage during ablation a Ultrasound revealed a hyperechoicmass lesion in the nodule b Ultrasound showed an ablation needle inserted into the nodule to coagulatebleeding vessels c After lyophilising thrombin powder was injected into the hematoma ultrasound andcontrastenhanced ultrasound showed disappearance of the hyperechoic mass lesion and microbubbleextravasation d Ultrasound showed a hyperechoic mass lesion around the thyroid and contrastenhancedultrasound showed no microbubble extravasation around the thyroid e After lyophilising thrombinpowder was injected into the haematoma no microbubble extravasation was observedskills are both essential Patient cooperationis the first requirement When the needle tipis in the patient™s body any uncooperativemotion of the patient may lead to injury ofvessels or other structures Most patientscan endure the procedure under local anaesthesia however anxious patients mayrequire general anaesthesia to achieve cooperation If possible smallbore electrodesshould be chosen to decrease the risk ofbleeding35 It is necessary to cauterise thesupplying vasculature of nodules to avoidrecurrence and residue Howeverthepuncture route should be carefully designedto avoid pericapsular vessels and the electrode tip should be closely monitoredActive bleeding during needle puncture isvisible as a rapidly expanding hypoechoicor anechoic signal Locating the haemorrhagic focus is not difficult with CEUSguidance The bleeding pointcan beblocked by RF electrode tip insertion anddirect ablation When the bleeding is toorapid to control with the RF electrode tipby increasing the power drug injection is asuitable alternative Lyophilised thrombin 0cHu et alpowder can be dissolved in normal salineand then injected at the bleeding pointthrough a syringe with US guidance Onereport also described haemorrhage treatedby local injection of hypertonic saline andepinephrine solution in a patient with hepatocarcinoma36 Mildbleeding whichappears as a hypoechoic layer can mostlybe controlled using ice and compression ofthe neck for several minutes after the procedure30 All bleeding can be controlled byconservative methodsthus no surgicalintervention is needed Ecchymosis can befound after the procedure and usually disappears in approximately to weeksPostprocedure CEUS is indispensablefor all patients regardless of whether bleeding occurs CEUS is an objective evaluationtool for active bleeding37 Close clinicalobservation for hours postoperatively isrecommended in our department becausemost bleeding occurs during the firstlobectomy38Observation of the neck can help to detecta haematoma early and may aid in preventing serious adverse effectsthyroidhoursafterConclusionAcute thyroid bleeding is one possible complication of RFA although rare it is potentially lifethreatening Proper selection ofpatients and sufficient preparation areessential During the RFA procedureboth an effective clinical strategy and adequate technical skills are indispensable Thephysician should trace the electrode tipusing realtime US and sufficiently managebleeding Mild bleeding has limited morbidity and can be easily controlled by compression Active bleeding tends to be rarehowever it may be disastrous if the operator is unaware or careless Direct ablationwith the RF electrode tip and drug injectioninto the bleeding focus are effective modalities for active bleeding CEUS and closeobservation are also recommended afterthe procedureto detect abnormalitiesearly RFA is an effective and relativelysafe alternative for selected patients withBTNs if performed by skilled physiciansAuthor contributionsI Conception and design Jie Ren and Bo LiuII Administrative support Jie RenIIIstudy materials or patientsProvision ofKunpeng Hu and Yufan Lian IV Collectionand assembly of dataJinfen Wang andWenchao Li V Data analysis and interpretation Wenchao Li and Zhicheng YaoVIManuscript writing All authors VII Finalapproval of manuscript All authorsData availabilityData regarding the patients™ characteristics usedto support the funding are shown in Table Declaration of conflicting interestThe authors declare that there is no conflict ofinterestFundingthe NaturalThis work was supported by the NationalNatural Science Foundation of China CNNoScienceFoundation of Guangdong ProvinceNo2016A030313200 the Science and TechnologyProject of Guangzhou City No the Hengrui Foundation of Hepatobiliary andPancreaticNoCXPJJH1180000120183331NaturalScience Foundation of Guangdong ProvinceNothe FundamentalResearch Funds for the Central UniversitiesSun Yatsen University No 17ykpy67 andthe Clinical Research Project of Sun Yatsen University No 2017A030313580theCancerResearchORCID iDKunpeng Huorcid00000001 0cReferences Guth S Theune U Aberle J et al Very highprevalence of thyroid nodules detected byhigh frequency MHz ultrasound examination Eur J Clin Invest “ Tan GH and Gharib H Thyroid incidentalomas management approaches to nonpalpable nodules discovered incidentally onthyroid imaging Ann Intern Med “ Haugen BR Alexander EK Bible KC et al AmericanThyroid AssociationManagement Guidelines for Adult Patientswith Thyroid Nodules and DifferentiatedThyroid Cancer The American ThyroidAssociation Guidelines Task Force onThyroid Nodulesand DifferentiatedThyroid Cancer Thyroid “ Ceccarelli C Bencivelli W Vitti P et alOutcome ofradioiodine131 therapy inhyperfunctioning thyroid nodules a years™ retrospective study Clin EndocrinolOxf “ Reiners C and Schneider P Radioiodinetherapy of thyroid autonomy Eur J NuclMed Mol Imaging S471“S478 Nieuwlaat WA Hermus AR SivroPrndeljF et al Pretreatment with recombinanthuman TSH changes the regional distribution of radioiodine on thyroid scintigramsof nodular goiters J Clin Endocrinol Metab “ LinosDEconomopoulosKPKiriakopoulos A et al Scar perceptionsafter thyroid and parathyroid surgery comparison of minimaland conventionalapproaches Surgery “ Jeannon JP Orabi AA Bruch GA et allaryngeal nervethyroidectomy a systematicDiagnosis ofpalsy afterreview Int J Clin Pract “recurrent Lang B Woo YC and Chiu KW Identifyingpredictive factors for efficacy in high intensity focused ultrasound HIFU ablationof benign thyroid nodules “ a retrospectiveInt J Hyperthermia analysis“ Mauri G Pacella CM Papini E et alImageguided thyroid ablation proposalforterminology andstandardization ofJournal of International Medical Researchreporting“criteria Thyroid Sato M Tateishi R Yasunaga H et alMortality and hemorrhagic complicationsassociated with radiofrequency ablation fortreatment of hepatocellular carcinoma inendstagepatients on hemodialysissurveyrenalJ“Gastroenterol Hepatolnationwidediseasefora Krokidis M Spiliopoulos S Jarzabek Met al Percutaneous radiofrequency ablationof small renal tumours in patients with asingle functioning kidney longterm resultsEur Radiol “ Lim HK Lee Dupuy DE Monchik JM Decrea C et alRadiofrequency ablation of regional recurrence from welldifferentiated thyroid malignancy Surgery “JH Ha EJalRadiofrequency ablation of benign nonfunctioning thyroid nodules 4year followup results for patients Eur Radiol “et Braga M Cavalcanti TC Collaco LM et alEfficacy of ultrasoundguided fineneedleaspiration biopsy in the diagnosis of complex thyroid nodules J Clin EndocrinolMetab “ Kakiuchi Y Idota N Nakamura M et alA fatal case of cervical hemorrhage after fineneedle aspiration and core needle biopsy ofthe thyroid gland Am J Forensic Med Pathol “ Donatini G Masoni T Ricci V et al Acuterespiratory distress following fine needleaspiration of thyroid nodule case reportand review of the literature G Chir “ Roh JL Intrathyroid hemorrhage and acuteupper airway obstruction after fine needleaspirationthyroidglandLaryngoscope “theofofAssociation Gharib H Papini E Garber JR et alAmericanClinicalEndocrinologists American College ofEndocrinology and Associazione MediciEndocrinologi medical guidelines for clinicalpractice for the diagnosis and managementthyroid nodules“ update EndocrofPract “ 0cHu et al Na DG LeeetJHJung SLalRadiofrequency ablation of benign thyroidnodules and recurrent thyroid cancers consensusstatement and recommendationsKorean J Radiol “ Ha EJ Baek JH and Lee JH Movingshotversus fixed electrode techniques for radiofrequency ablation comparison in an exvivo bovine liver tissue model Korean JRadiol “ Jeong WK Baek JH Rhim H et alRadiofrequency ablation of benign thyroidnodules safety and imaging followup in“patients Eur Radiol Cardella JF Kundu S Miller DL et alSociety of Interventional Radiology clinicalpractice guidelines J Vasc Interv Radiol S189“S191 Sacks D McClenny TE Cardella JF et alSociety of Interventional Radiology clinicalpractice guidelines J Vasc Interv Radiol S199“S202 Goldberg SN Radiofrequency tumor ablation principles and techniques Eur JUltrasound “ Rhim H Goldberg SN Dodd GR et alEssential techniques for successful radiofrequency thermal ablation of malignanthepatic tumors Radiographics Spec No S17“S35 S36S39 Korkusuz Y Erbelding C Kohlhase Ket al Bipolar Radiofrequency Ablation ofBenign Symptomatic Thyroid Nodules initial Experience Rofo “ Garberoglio R Aliberti C Appetecchia Met al Radiofrequency ablation for thyroidnodules which indications The first Italianopinion statement J Ultrasound “ Baek JH LeeJH Sung JYet alComplications encountered in the treatmentof benign thyroid nodules with USguidedradiofrequencya multicenterstudy Radiology “ablation Chen MH Dai Y Yan KalIntraperitoneal hemorrhage duringandafter percutaneous radiofrequency ablationof hepatic tumors reasons and managementChin Med J Engl “et Rhim H Dodd GR Chintapalli KN et alRadiofrequency thermal ablation of abdominal tumors lessons learned from complications Radiographics “ Terry WI Radium emanations in exophthalmic goiter”blood vessels of adenomas ofthyroid J Am Med Assoc “ Hor T and Lahiri SW Bilateral thyroidhematomas after fineneedle aspiration causing acute airway obstruction Thyroid “ Minami Y Hayaishi S and Kudo MRadiofrequency ablation for hepatic malignancies is needle tract cauterization necessary for preventing iatrogenic bleeding DigDis “ Baek JH Kim YS Lee D et al Benign predominantly solid thyroid nodules prospective study of efficacy of sonographicallyguided radiofrequency ablation versus control condition AJR Am J Roentgenol “ Koda M Murawaki Y Hirooka Y et alComplications of radiofrequency ablationfor hepatocellular carcinoma in a multicenter study an analysis of treated nodules in patients Hepatol Res “ Wiggermann P Wohlgemuth WA Heibl Met al Dynamic evaluation and quantificationof microvascularization during degradablestarch microspheres transarterial chemoembolisation DSMTACE of HCC lesionsusingultrasoundCEUS a feasibility study Clin HemorheolMicrocirc “enhancedcontrast Rosenbaum MA Haridas M and McHenryCR Lifethreatening neck hematoma complicating thyroid and parathyroid surgeryAm J Surg “ 0c'

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

The Adler grade by Doppler ultrasound isassociated with clinical pathology of cervicalcancer Implication for clinical managementDehong Che1 Zhirong Yang2 Hong Wei3 Xuedong Wang4 Jiayin GaoID1 Department of Obstetrics and Gynecology The Second Affiliated Hospital of Harbin Medical UniversityHarbin China Department of Ultrasound Harbin Red Cross Center Hospital Harbin China Departmentof Ultrasound The Second Affiliated Hospital of Harbin Medical University Harbin China Department ofObstetrics and Gynecology Longnan Hospital Daqing General Hospital Daqing Chinaa1111111111a1111111111a1111111111a1111111111a1111111111 dsp082901163comAbstract ACCESSCitation Che D Yang Z Wei H Wang X Gao J The Adler grade by Doppler ultrasound isassociated with clinical pathology of cervicalcancer Implication for clinical management PLoSONE e0236725 101371journalpone0236725Editor Linus Chuang University of Vermont LarnerCollege of Medicine UNITED STATESReceived February Accepted July Published August Copyright Che This is an access distributed under the terms of the CreativeCommons Attribution License which permitsunrestricted use distribution and reproduction inany medium provided the original author andsource are creditedData Availability Statement All relevant data arewithin the paper and its Supporting InformationfilesFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existAbbreviations FIGO International Federation ofObstetrics and Gynecology TVCDFI transvaginalObjectiveTo analyze the relationship of Adler grade by transvaginal color Doppler flow imaging TVCDFI and the clinical pathological parameters of patients with cervical cancer and to identify the value of Adler grade in the diagnosis and treatment of cervical cancerMethodsPatients with cervical cancer diagnosed pathologically in our hospital from January to December were included All patients underwent TVCDFI examination and theimages were divided into to III grades according to the Adler grades and the correlationsbetween the Adler classification and clinical pathological parameters clinical stage masssize pathological type squamous cell carcinoma subtype CA125 CA199 were analyzedResultsA total of patients with cervical cancer were included With the increase of Adler severity the clinical stage of cervical cancer increased accordingly the cancer size differed significantly in patients with different Adler grade p There were significant differences inthe level of CA125 CA199 between the squamous cell carcinoma and adenocarcinoma allp005 the Adler grade was positively related with the clinical stage pathological type andsquamous cell carcinoma subtypes of cervical cancer all p005 no correlations werefound among the Adler grade and the cancer size CA125 CA199 all p005 The areaunder ROC curve of the cervical squamous cell carcinoma predicted by Adler grade basedon FIGO results and pathological results was 0811and respectively all p005ConclusionsAdler grades are closely associated with the clinical pathology of cervical cancer which maybe a convenient and effective approach for the assisting assessment of cervical cancerPLOS ONE 101371journalpone0236725 August PLOS ONE 0ccolor Doppler ultrasound WHO World HealthanizationUltrasound cervical cancerIntroductionCervical cancer is one of the most common malignant tumors in gynecology it has beenreported that the morbidity and mortality of cervical cancer ranks second among gynecological malignancies second only to breast cancer [ ] The number of new cases of cervical cancer has been increased every year around the world and the onset age of cervical cancer hasbecome much younger [] Cervical cancer is a malignant tumor which occurs at the junctionof squamous epithelial cells in the cervical vagina or the transitional zone and columnar epithelial cells in the endometrium of the cervical canal [] The etiology is not totally clear and itmay be related to sexual behavior frequency of deliveries and the infection of human papillomavirus [ ] The early detection and treatment of cervical cancer is very essential to theprognosis of patients with cervical cancerIn recent years with the popularization of many effective screening methods the overallmortality rate of cervical cancer patients has declined [] The clinical diagnosis mainlydepends on clinical manifestations and gynecological examinations which requires the combination of multiple auxiliary diagnostic methods including cervical cytology film cervicalmultipoint biopsy The International Federation of Obstetrics and Gynecology FIGO hasdeveloped a clinical staging standard for cervical cancer which referred to the colposcopypathological biopsy [] However when comparing the early diagnosed stage with the patient™spostoperative medical examination results the stage of cervical cancer has been much underestimated [“] More sophisticated radiological methods such as ultrasound computertomography and magnetic resonance imaging hasn™t been included in the stage of FIGO butthey have been the routine examination methods in clinical practice [] Many scholars [] have studied the characteristics of those examinations but so far there is no asto which is the best examination method for assessing cervical cancer staging It™s necessary toconduct more studies to identify the role of those tools for early diagnosis of cervical cancerPrevious studies [“] have reported that the transvaginal color Doppler ultrasoundTVCDFI can accurately reflect the size of the lesion the extent of infiltration and the bloodsupply and it is widely used in the detection of cancers in breast and thyroid but not in thecervical parts Besides it is well known that vascular patterns such as density of vessels is different for squamous and adenocarcinomas [] Therefore it™s necessary to show the difference to provide insights into clinical treatment Therefore in order to make clinicians moreclear about the blood supply of cervical cancer and facilitate communication between ultrasound doctors and clinicians we attempted to conduct a preliminary investigation on the correlation between the Adler grade and clinical pathology of cervical cancer to provide insightsinto the diagnosis and treatment of cervical cancer The study design was participantspatients with cervical cancer 2intervention TVCDFI detection and Adler grading 3comparison no applicable 4outcomes Adler secores and antigens CA125 and CA199MethodsEthical considerationsThe study was approved by the Medical Research Ethics Committee of our hospitalNo20180038 and written informed consents were obtained from all the patientsPatientsPatients with cervical cancer diagnosed pathologically in our hospital from January toDecember were selected as study objects in this present study The inclusion criteriawere as follows there were surgical pathology or biopsy detections verifying small cellPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerneuroendocrine tumors by our experienced clinicans and the FIGO cervical cancer stagingwere used for severity differentiation [] no history of other malignant tumors or historyof radiotherapy and chemotherapy complete clinical data no history of cervical surgerysuch as we excluded patients undergone microwave freezing laser electrocautery surgeryThe exclusion criteria were as follows patients with congenital malformations of theuterus patients with coagulopathy patients with severe heart liver and kidney dysfunction patients with inability to cooperate with research or couldn™t followupTVCDFI detectionThe color doppler ultrasound system Philips E80 equipped with ultrasonographic probeDC58D were selected for ultrasound detection Before the detection all patients were askedto empty the rectum and bladder When performing the doppler ultrasound detection thepatients took the lithotomy positions Firstly we performed a transvaginal ultrasound examination of the uterus and bilateral accessory areas to determine the location number size shapeboundary echo of the cervical area and its relationship with surrounding tissues Secondly wechose the color Doppler blood flow imaging mode to observe the blood flow inside and aroundthe cervical area related parameters such the blood flow cancer size have been calculated andcollected The staging and TVCDFI done in the same setting and in different datesAdler grade classificationAdler grade classifications were conducted based on the detected blood flow [ ] We classified the blood flow signal of the lesion into four levels Adler refers to that no obvious bloodflow signal Adler I refers to or small blood vessels with a diameter of mm are detectedAdler II refers to that or small blood vessels are detected Adler III refers to that more than blood vessels or the blood vessels are intertwined into a network are detected The Adlergrade classifications were made by one radiologist and one gynecologist coherently and ifthere were any disparity further discussions were conducted for consensusThe detection of carbohydrate antigens CA125 and CA199Before any drug treatment ml venous blood without anticoagulation in the early morningwere collected from patients the blood specimens were left at room temperature for 15minthen centrifuged it and separated the serum then we stored it at environment with ˚C for further examination The levels of serum CA125 and CA199 were detected by professional stallwith electrochemical luminescence method The electrochemical luminometer and detectionkit used in the test were produced by Roche and the operation process was strictly performedaccording to the instructionsStatistical analysisSPSS statistical software were used for data analysis The measurement data for normal distribution was expressed as mean± standard deviation and the measurement data for skeweddistribution was expressed as median P25 P75 The tumor size CA125 CA199 were compared using the KruskalWallis H rank test of multiple sample comparisons of which Wilcoxon rank test was further used for pairwise comparisons And t tests were conducted in thecancer size between pathological types or squamous cell carcinoma subtypes Spearman correlation analysis was conducted for correlation analysis And receiver operating characteristicROC curves were drawn for evaluating the diagnosis value of Adler grade for cervical cancer[] p005 was considered as being statistically different in this present studyPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerResultsThe ultrasound characteristics of cervical cancerAs Fig presented the ultrasound characteristics differed hugely among the ultrasoundimages with different Alder grades For Alder the cervical morphology was normal andno obvious blood flow signal were found in the images of color Doppler For Alder I thecervical morphology was slightly thickened and more enhanced intracervical echo couldbe collected one or two small spotlike blood flow signals could be detected For Alder IIuneven or thickened cervical echo distribution could be detected and strip blood flow signals at two to four locations could be seen For Alder III parauterine and extrauterineinvasion and blood metastasis could be found and reticular blood flow signals could bedetectedThe distribution of Adler classification and clinical stage of cervical cancerWe identified potential candidates firstly and patients were excluded As Table presented a total of patients were included for data analysis With the increase of Adler severity classification the clinical stage of cervical cancer increased accordinglyFig The Doppler ultrasound images on cervical cancer with different Adler grade a Adler b Adler I c Adler II d Adler III101371journalpone0236725g001PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerTable The distribution of Adler classification and clinical stage of cervical cancerAdler gradeFIGO gradeGrade IGrade IIGrade IIIIVAdler Adler IAdler IIAdler IIIIn total101371journalpone0236725t001In totalThe comparison on the cancer size CA125 CA199 with Adler grade andpathological parametersAs Table showed the cancer size differed significantly among the different Adler gradesp but there were no significantly differences in the level of CA125 CA199 amongthe different Adler grades all p005 There were significant differences in the level ofCA125 CA199 between the squamous cell carcinoma and adenocarcinoma all p005 nosignificant difference was found in the cancer size between the squamous cell carcinoma andadenocarcinoma p No significant differences were found in the cancer size CA125and CA199 between the keratinized and nokeratinized squamous cell carcinoma all p005Furthermore as Table showed the Adler grade was positively related with the FIGO stagingpathological type and squamous cell carcinoma subtypes of cervical cancer all p005 nocorrelations were found among the Adler grade and the cancer size CA125 CA199 allp005The diagnosis value of Adler grade for cervical cancerAs Fig showed based on FIGO results the area under the ROC curve of the cervical squamous cell carcinoma predicted by Adler grade was p005 and its sensitivityTable Relationship analysis on the cancer size CA125 CA199 with Adler grade and pathological parametersItemsAdler gradeAdler Adler IAdler IIAdler IIItzpPathological typeSquamous cell carcinomaAdenocarcinomatzpsquamous cell carcinoma subtypesKeratinizedNonkeratinizedtzpCasesCancer sizemmCA125[MP25 P75 UmL]CA199[MP25 P75 UmL]±±± ± ± ± ± 101371journalpone0236725t002PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerTable Correlation analysis between Adler grade and clinicopathological parametersClinicopathological parametersFIGO gradeCancer sizeCA125CA199Pathological typesquamous cell carcinoma subtypes101371journalpone0236725t003rpspecificity positive predictive value negative predictive value and Youden index were respectively And based on pathological results the area underthe ROC curve of the cervical squamous cell carcinoma subtype predicted by Adler grade was p005 and its sensitivity specificity positive predictive value negative predictivevalue and Youden index were respectively Thoseresults indicated that Adler grade could provide valuable reference for the diagnosis of cervicalcancerDiscussionsTVCDFI has the advantages of being noninvasive cheap with convenient operation [ ]It can not only clearly display the structure of each layer of the uterus accurately locate andqualitatively diagnose the lesion but also can observe the blood flow of detected area [ ]Dynamic ultrasound is currently one of the most widely used and mature imaging methods inthe diagnosis and treatment of obstetrics and gynecology diseases [] In this present studythe changes of cervical morphology and echo in Adler grade to I were small and Dopplerblood flow signals were less displayed but the blood flow signals in Adler grade II and IIIincreased significantly and the flakelike low echoes were seen inside With the increase ofAdler classification cervical masses continue to increase and color Doppler blood flow signalsare also displayed The results of this present study have indicated that Adler grade is expectedto be useful to reflect the richness of blood flow in cervical cancer which can provide important insights into the diagnosis and treatment of cervical cancerFig The ROC curve on the Adler grade for cervical cancer101371journalpone0236725g002PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerThe occurrence proliferation invasion and metastasis of malignant tumors may largelydepend on tumor neovascularization [ ] Cervical cancer has characteristics of rapid proliferation and active cell division and growth which is closely related to the proliferation oftumor vessels in it [] On color Doppler ultrasound images there are often abundant different types of blood flow signals in tumor tissues which correspond to its rich vascular networkand are related to the special structure and blood flow characteristics of tumor blood vessels[ ] The Alder grades are mainly concentrated on the blood flow signals which to someextent can reflect the proliferation of vessels and the characteristics of microvessel densityaround the cervical cancer Previous studies [“] have reported that the blood signals maypredict the ability of tumor invasion and metastasis to a certain extent which is consistentwith the results of our studySquamous carcinoma is more common in the pathological and histological types of cervicalcancer [] We have found that there was correlation between Adler grade and the histopathological type The World Health anization WHO classifies cervical squamous cell carcinoma into two subtypes keratinized and nonkeratinized according to the presence ofkeratinized morphological features in the tissue [] Currently the studies on the relationshipof keratinized cervical squamous cell carcinoma and patient prognosis has been controversialIt™s been reported [“] that compared with nonkeratinized cervical squamous cell carcinoma the survival rate of keratinized cervical squamous cell carcinoma is significantlyreduced and it is not sensitive to radiotherapy and the prognosis is very poor for untreatedpatients or advanced patients Therefore differentiate the various subtypes of squamous cellcarcinoma of cervix is very helpful in clinical managementIn this study Spearman rank correlation analysis showed that Adler grade was positivelycorrelated with squamous cell carcinoma subtypes Therefore this technique is expected to bea reliable indicator for predicting the prognosis of patients with cervical cancerWith the continuous progress of cancer marker research the role of cancer related markerin the diagnosis and treatment of cancers has become increasingly important [ ] Severalstudies have shown that cervical cancer is also closely related to the marker CA125 and CA199It has been reported [“] that the carbohydrate antigens CA125 and CA199 are expressedat high levels in a variety of malignancies In this study the levels of CA125 and CA199 inpatients with adenocarcinoma were significantly higher than those in patients with squamouscell carcinoma The results were consistent with the findings of previous studies [ ] Eventhrough the Spearman correlation analysis shows that there is no correlation between Adlergrade and CA125 and CA199 it may be explained that the samples in this present study is notlarge enough to powerfully detect the difference future studies with larger population samplesare warranted to identify the role of CA125 and CA199 in cervical cancerSeveral limitations must be concerned in this present study Firstly the sample size of thisstudy is not large enough and the clinical staging is relatively rough biases may be existed inthis present study Secondly we did not use the cervical cancer specific markers such as squamous cell carcinoma tumor marker for reference it should be further elucidated in the futurestudies Thirdly it must bring to our attentions that the subjective factors in the Adler classification different operators and different parameter settings may bias the test results Howeverwe have performed related trainings on the TVCDFI before this study to avoid unnecessaryerrors in the process of ultrasound detection and analysisIn the results of this study have favored the value of Adler grade in the diagnosisand treatment of cervical cancer With the continuous development of ultrasound technologyand popularization of clinical ultrasound applications Adler grade should be promoted in theapplication of color Doppler ultrasound for the diagnosis and treatment of cervical cancerPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerSupporting informationS1 ChecklistDOCXS2 ChecklistDOCXAuthor ContributionsConceptualization Dehong Che Zhirong Yang Jiayin GaoData curation Dehong Che Xuedong Wang Jiayin GaoFormal analysis Dehong Che Zhirong Yang Hong Wei Jiayin GaoInvestigation Dehong Che Zhirong Yang Hong Wei Xuedong Wang Jiayin GaoMethodology Xuedong WangResources Xuedong Wang Jiayin GaoSoftware Dehong CheValidation Dehong Che Jiayin GaoWriting “ original draft Dehong CheReferences Shrestha AD Neupane D Vedsted P Kallestrup P Cervical Cancer Prevalence Incidence and Mortality in Low and Middle Income Countries A Systematic Review Asian Pac J Cancer Prev “ Epub 1022034APJCP2018192319 PMID PubMed Central PMCID PMC5980914 Kalliala I Athanasiou A Veroniki AA Salanti G Efthimiou O Raftis N Incidence and mortalityfrom cervical cancer and other malignancies after treatment of cervical intraepithelial neoplasia a systematic review and metaanalysis of the literature Ann Oncol “ Epub 101016jannonc201911004 PMID Murfin J Irvine F MeechanRogers R Swift A Education income and occupation and their influenceon the uptake of cervical cancer prevention strategies A systematic review J Clin Nurs ““ Epub 101111jocn15094 PMID Coutlee F Viscidi RP SaintAntoine P Kessous A Yolken RH The polymerase chain reaction a newtool for the understanding and diagnosis of HIV1 infection at the molecular level Mol Cell Probes “ Epub 101016089085089190046m PMID Loopik DL IntHout J Ebisch RMF Melchers WJG Massuger L Siebers AG The risk of cervicalcancer after cervical intraepithelial neoplasia grade A populationbased cohort study with women Gynecol Oncol Epub 101016jygyno202001023 PMID Wang R Pan W Jin L Huang W Li Y Wu D Human papillomavirus vaccine against cervical cancer Opportunity and challenge Cancer Lett “ Epub 101016jcanlet201911039 PMID Jansen EEL Zielonke N Gini A Anttila A Segnan N Voko Z Effect of anised cervical cancerscreening on cervical cancer mortality in Europe a systematic review Eur J Cancer Epub 101016jejca201912013 PMID Wright JD Matsuo K Huang Y Tergas AI Hou JY KhouryCollado F Prognostic Performance ofthe International Federation of Gynecology and Obstetrics Cervical Cancer Staging GuidelinesObstet Gynecol “ Epub 101097AOG PMID Xiao C Zhou J Yang C Li J Pan Y Dai Y The Comparison of Vascular Characteristics of ThyroidMicrocarcinoma By Conventional Colour Doppler Ultrasonography and Superb Microvascular ImagingChinese Journal of Ultrasound in Medicine “PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerInfante F Espada Vaquero M Bignardi T Lu C Testa AC Fauchon D Prediction of Tubal EctopicPregnancy Using Offline Analysis of 3Dimensional Transvaginal Ultrasonographic Data Sets An Interobserver and Diagnostic Accuracy Study J Ultrasound Med “ Epub 101002jum14489 PMID Haldorsen IS Lura N Blaakaer J Fischerova D Werner HMJ What Is the Role of Imaging at PrimaryDiagnostic WorkUp in Uterine Cervical Cancer Curr Oncol Rep Epub 101007s1191201908240 PMID PubMed Central PMCID PMC6663927 Ngan HYS Seckl MJ Berkowitz RS Xiang Y Golfier F Sekharan PK Update on the diagnosisand management of gestational trophoblastic disease Int J Gynaecol Obstet Suppl “ Epub 101002ijgo12615 PMID PatelLippmann K Robbins JB Barroilhet L Anderson B Sadowski EA Boyum J MR Imaging of Cervical Cancer Magn Reson Imaging Clin N Am “ Epub 101016jmric201703007 PMID Cibula D Potter R Planchamp F AvallLundqvist E Fischerova D Haie Meder C The EuropeanSociety of Gynaecological OncologyEuropean Society for Radiotherapy and OncologyEuropean Society of Pathology guidelines for the management of patients with cervical cancer Radiother Oncol “ Epub 101016jradonc201803003 PMID Wang P Sun W Wang L Gao J Zhang J He P Correlations of p53 expression with transvaginal colorDoppler ultrasound findings of cervical cancer after radiotherapy J BUON “ Epub PMID Wang HR Lin Y Zhang XY Ma XT Transvaginal color doppler sonography combined with colposcopyfor diagnosis of early stage cervical cancer and precancerous lesions J Biol Regul Homeost Agents “ Epub PMID Palsdottir K Fischerova D Franchi D Testa A Di Legge A Epstein E Preoperative prediction of lymphnode metastasis and deep stromal invasion in women with invasive cervical cancer prospective multicenter study using 2D and 3D ultrasound Ultrasound Obstet Gynecol “ Epub 101002uog14643 PMID Wang S Yang W Fu JJ Sun Y Zhang H Bai J Microflow imaging of contrastenhanced ultrasound for evaluation of neovascularization in peripheral lung cancer Medicine Baltimore 32e4361 Epub 101097MD0000000000004361 PMID PubMed Central PMCID PMC4985302 Bhatla N Berek JS Cuello Fredes M Denny LA Grenman S Karunaratne K Revised FIGO staging for carcinoma of the cervix uteri Int J Gynaecol Obstet “ Epub 101002ijgo12749 PMID Adler DD Carson PL Rubin JM QuinnReid D Doppler ultrasound color flow imaging in the study ofbreast cancer preliminary findings Ultrasound Med Biol “ Epub 101016030156299090020d PMID Yang WT Tse GM Lam PK Metreweli C Chang J Correlation between color power Doppler sonographic measurement of breast tumor vasculature and immunohistochemical analysis of microvesseldensity for the quantitation of angiogenesis J Ultrasound Med “ Epub 107863jum200221111227 PMID Hickey GL Dunning J Seifert B Sodeck G Carr MJ Burger HU Statistical and data reportingguidelines for the European Journal of CardioThoracic Surgery and the Interactive CardioVascular andThoracic Surgery Eur J Cardiothorac Surg “ Epub 101093ejctsezv168 PMID Jurado M Galvan R MartinezMonge R Mazaira J Alcazar JL Neoangiogenesis in early cervical cancer correlation between color Doppler findings and risk factors A prospective observational studyWorld J Surg Oncol Epub 101186147778196126 PMID PubMed Central PMCID PMC2611993 Exacoustos C Zupi E Piccione E Ultrasound Imaging for Ovarian and Deep Infiltrating EndometriosisSemin Reprod Med “ Epub 101055s00361597127PMID Arya S Kupesic Plavsic S Preimplantation 3D ultrasound current uses and challenges J Perinat Med “ Epub 101515jpm20160361 PMID Romosan G Valentin L The sensitivity and specificity of transvaginal ultrasound with regard to acutepelvic inflammatory disease a review of the literature Arch Gynecol Obstet “Epub 101007s0040401330916 PMID PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerTariq M Zhang J Liang G Ding L He Q Yang B Macrophage Polarization AntiCancer Strategies toTarget TumorAssociated Macrophage in Breast Cancer J Cell Biochem “Epub 101002jcb25895 PMID Huang Z Zhao B Qin Z Li Y Wang T Zhou W Novel dual inhibitors targeting CDK4 andVEGFR2 synergistically suppressed cancer progression and angiogenesis Eur J Med Chem Epub 101016jejmech201907044 PMID Cohen PA Jhingran A Oaknin A Denny L Cervical cancer Lancet “ Epub 101016S014067361832470X PMID Ebina Y Mikami M Nagase S Tabata T Kaneuchi M Tashiro H Japan Society of GynecologicOncology guidelines for the treatment of uterine cervical cancer Int J Clin Oncol “ Epub 101007s101470181351y PMID The L Cervical cancer unequal progress Lancet Epub 101016S0140673619300030 PMID Ma G Zhang J Jiang H Zhang N Zhu Y Deng Y Microvessel density as a prognostic factor inesophageal squamous cell cancer patients A metaanalysis Medicine Baltimore e7600 Epub 101097MD0000000000007600 PMID PubMedCentral PMCID PMC5521944 Ruscito I Cacsire CastilloTong D Vergote I Ignat I Stanske M Vanderstichele A Characterisation of tumour microvessel density during progression of highgrade serous ovarian cancer clinicopathological impact an OCTIPS Consortium study Br J Cancer “ Epub 101038s414160180157z PMID PubMed Central PMCID PMC6070919 Hu X Liu H Ye M Zhu X Prognostic value of microvessel density in cervical cancer Cancer Cell Int Epub 101186s1293501806473 PMID PubMed Central PMCID PMC6169003 Yang Y He L Liu Y Xia S Fang A Xie Y Promising Nanocarriers for PEDF Gene TargetingDelivery to Cervical Cancer Cells Mediated by the Overexpressing FRalpha Sci Rep Epub 101038srep32427 PMID PubMed Central PMCIDPMC5006243Lekskul N Charakorn C Lertkhachonsuk AA Rattanasiri S Israngura Na Ayudhya N The Level ofSquamous Cell Carcinoma Antigen and Lymph Node Metastasis in Locally Advanced Cervical CancerAsian Pac J Cancer Prev “ Epub 107314apjcp PMID Xie X Song K Cui B Jiang J Yang X Kong B A comparison of the prognosis between adenocarcinoma and squamous cell carcinoma in stage IBIIA cervical cancer Int J Clin Oncol “ Epub 101007s1014701712258 PMID Grjibovski AM Dubovichenko D Saduakassova S Zhatkanbayeva G Omarova G Shalgumbayeva G Incidence mortality and determinants of survival from cervical cancer in Northwest Russia a registrybased cohort study Int Health “ Epub 101093inthealthihx068 PMID Charakorn C Thadanipon K Chaijindaratana S Rattanasiri S Numthavaj P Thakkinstian A The association between serum squamous cell carcinoma antigen and recurrence and survival of patients withcervical squamous cell carcinoma A systematic review and metaanalysis Gynecol Oncol “ Epub 101016jygyno201803056 PMID Kori M Yalcin Arga K Potential biomarkers and therapeutic targets in cervical cancer Insights from themetaanalysis of transcriptomics data within network biomedicine perspective PLoS One e0200717 Epub 101371journalpone0200717 PMID PubMedCentral PMCID PMC6051662 Wang W Xu X Tian B Wang Y Du L Sun T The diagnostic value of serum tumor markers CEACA199 CA125 CA153 and TPS in metastatic breast cancer Clin Chim Acta “ Epub 101016jcca201704023 PMID Laengsri V Kerdpin U Plabplueng C Treeratanapiboon L Nuchnoi P Cervical Cancer Markers Epigenetics and microRNAs Lab Med “ Epub 101093labmedlmx080 PMID Zhou X Wang H Wang X Preoperative CA125 and fibrinogen in patients with endometrial cancer arisk model for predicting lymphovascular space invasion J Gynecol Oncol 282e11 Epub 103802jgo201728e11 PMID PubMed Central PMCIDPMC5323282 Yu J Zheng Q Ding X Zheng B Chen X Chen B Systematic reanalysis strategy of serum indices identifies alkaline phosphatase as a potential predictive factor for cervical cancer Oncol Lett PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancer“ Epub 103892ol201910527 PMID PubMedCentral PMCID PMC6676666Fujii S Konishi I Nanbu Y Nonogaki H Kobayashi F Sagawa N Analysis of the levels of CA125carcinoembryonic antigen and CA199 in the cervical mucus for a detection of cervical adenocarcinoma Cancer “ Epub 1010021097014219880801623541aidcncr282062031730co24 PMID 24555900co2“ Mitsuhashi A Matsui H Usui H Nagai Y Tate S Unno Y Serum YKL40 as a marker for cervicaladenocarcinoma Ann Oncol “ Epub 101093annoncmdn552 PMID PLOS ONE 101371journalpone0236725 August PLOS ONE 0c'

Thyroid_Cancer

"Lactation has a negative effect on female sexual function Hormonal changes during lactation causechanges which might lead to dyspareunia lack of libido and anasmia There are various pharmacological andnonpharmacological approaches to treat sexual dysfunction While pharmacological treatment has multipleunwanted side effects nonpharmacological therapies such as complementary medicine are a potential saferalternative The aim of this study is to evaluate the effect of ear acupressure on sexual function of lactating womenMethodsdesign This is a randomized clinical trial with a parallel sham control group In this study lactatingwomen between months and year after childbirth were referred to health care centers in Qazvin City andwould be invited to participate Participants will be divided into intervention n and control n groupsusing simple block randomization Both intervention and sham control groups will be visited over sessionswithin a 4day interval At each visit the adhesives containing Vaccaria seed will be adhered for the interventiongroup while nonlatexbased adhesives with no Vaccaria seeds will be placed on the same ear acupoints for thesham control group Selected ear acupoints include genitalia two ear points pelvic point master shoulder andposterior pituitary gland The women will be asked to hold the seeds on their ears for days and press each earpoint three times a day for s After days they will be asked to remove the seeds from their ears and rest for day Sexual function as primary outcome in both groups will be assessed using the Female Sexual Function Indexbefore and immediately after and months after the intervention Also Sexual Quality of Life as secondaryoutcome will be assessed using Sexual Quality of LifeFemale SQOLF before and months after intervention Datawill be analyzed using repeated measure ANOVA at the significant level of Discussion This study is expected to support the impact of ear channel ear acupressure on sexual function inlactating womenTrial registration Iranian Clinical Trial Registration Center IRCT20190626044028N1 Registered on August Keywords Ear acupressure Sexual function Lactation Correspondence nbahramiqumsacir2Social Determinants of Health Research Center Research Institute forPrevention of NonCommunicable Diseases Qazvin University of MedicalSciences Shahid Bahonar Blvd Qazvin 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 0cBarghamadi Trials Page of BackgroundThe postchildbearing period is a crucial stage in thewoman™s life that involves physical hormonal mentalsocial and cultural changes [] Fatigue insomnia somestressors such as child care and changes in the bodyimage along with hormonal changes can reduce interestin sexual life and decrease the number of sexual intercourses during this period [] Endocrine function of lactation has a negative effect on sexual function becauseprolactin decreases sexual hormones such as androgenand estrogen This mechanism reduces sexual functionbecause of vaginal dryness vaginal epithelium atrophyand dyspareunia [] About two thirds of women duringlactating period experience at least one sexual problemsuch as decreased libido lack of sexual pleasure dyspareunia and vaginal dryness which usually are resolvedwithin year after childbirth [ ] The prevalence ofsexual dysfunction increases from to in the prepartum period [] to to in the postpartum period[] The results of various Iranian studies confirmed thehigh prevalence to of sexual dysfunctionduring lactation [ ] Therefore sexual dysfunction is acommon problem during lactation that needs moreattention from health care providersIn the postchildbearing period most women try to return to their sexual life within to weeks after childbirth Howeverit may take up to year to resumesexual relationships with the same quality of beforepregnancy [] Psychotherapy and pharmacotherapy aretwo main methods for treating female sexual dysfunctionFSD [] However the US Food and Drug Administration FDA does not recommend any foods or medicines for the treatment of FSD [] Therefore nonpharmacological therapies such as complementary medicine are a potentially safer alternative to pharmacologicaltherapy Acupressure is one of the noninvasive complementary methods oftraditional Chinese medicineTCM based on the principles of acupuncture []Acupuncture stimulates medical points and meridiansthat are distributed throughout the body to regulatephysiologic reactions [] Acupressurelike acupuncture modulates a person™s vital energy by stimulatingacupoints and meridians that are distributed throughoutthe body []The external ear is one of the several somatotopicmicrosystems that can be used in acupressure Ear acupressure also known as auriculotherapy or auricularacupressure [ ] was first presented in ancient Chinese medicine to years BC but Dr Paul Nogierwas the first Western physician who put forward auriculotherapy in a scientific way [ ] Accordingto this theory each part of the body is associated with aspecific part of the ear that reflects the physiological orpathological state of the body [] In this method theouter surface of the ear auricle can be stimulated to reduce pathological conditions in other parts of the body[] Ear stimulation can be performed in various wayssuch as manual finger pressure electrical stimulation lasers differenttypes of needles magnetic seeds andseeds to strengthen neural connections [] The WorldHealth anization WHO has recognized ear acupuncture as a promising therapeutic approach becauseof its efficacy in managing health disorders [] Ear acupuncture has been recognized as a form of acupuncturethat can affect all body ans [] Ear acupressure isan easy noninvasive and safe therapy [ ] It is easyto use for mothers who need to take care of their childin the postchildbearing period []There are several studies supporting the effects of earacupressure on insomnia [ ] menstrual cramps anddysmenorrhea [ ] premenstrual pain []chemotherapyinduced nausea and vomiting [] smoking cessation [] obesity [] and shortness of breath[] However the effect of ear acupressure on sexualfunction has not been studiedResearch aimThis study is designed to investigate the effect of earacupressure on the sexual function of lactating womenMethodsDesign and settingThis is a randomized clinical trial with a parallel shamcontrolled group The study is designed in accordancewith the CONSORT standards Lactating women referred to health care centers in Qazvin City Iran will beinvited to participate Figure shows the CONSORTflow diagram The current protocol is anized basedon the SPIRITTCM extension []ParticipantsIn this study breastfeeding women between monthsand year after childbirth will be referred to health carecenters in Qazvin City Iran and they will be invited toparticipate in the study Inclusion criteria will be willingness to participate in the study being literate beingprimiparous breastfeeding fullterm singleton deliverylesionfree auricle no ulcer and pain in the ear and ability to present for all intervention sessions Exclusion criteria will be being away from their spouse for more than month having complications during pregnancy orhaving postpartum depression in the postchildbearingperiod These criteria were diagnosed using the Edinburgh Postpartum Depression Scale substance abuse inthe individual or the spouse and history of illnessesaffecting sexual function in a woman or her spouse egpremature ejaculation cardiovascular mental health disorders thyroid diseases any form of cancer or injuries 0cBarghamadi Trials Page of Fig The CONSORT flow diagramof the genital area The use of drugs affecting sexualfunction such as psychotropic cardiovascular neurological and hormonal drugs will also be excluded Afterthe initial screening the Demographic and ObstetricQuestionnaire and Female Sexual Function Index FSFIwill be completed by the participants before anyinterventionRole and qualification of practitionersThe research team consists of one specialist in auriculotherapy TO two specialists in reproductive health ZAand NB and one midwifery postgraduate student Theprotocol of intervention was designed based on the literature review and expert opinion of TO who is an expert inauriculotherapy and is a journal editor for several international journals Other team members have been qualified in this technique before designing present researchParticipant screening and care providing will be done bySB and ZA and NB will monitor her during the first sessions to ensure the fidelity of providing interventionSample size calculationSample size is estimated according to the study ofBokaie with the mean and standard deviation of 0cBarghamadi Trials Page of total sexual function score has been reported as ± and ± in the intervention telephonecounseling group and control groups respectively []type error α Therefore considering the first confidence the second type error β power and error d and the possibility of loss tofollowup individuals for each group are requiredSo the total sample size will be peopleInterventionEar acupressure groupThe researcher will clean the auricle using alcoholand then place Vaccaria seeds using nonlatexbased adhesives on ear acupoints for genitalia pelvis shoulderand the posterior pituitary gland Figure illustrates theintended ear acupoints Intervention is designed for sessions within a 4day interval During these sessionsthe seeds are placed on the designated ear acupressurepoints The women will be instructed to keep the seedson their ears for days during which each point shouldbe compressed three times a day for s The compression should be performed with moderate stimulationthrough pressing steadily and slightly tighter until feelinga slight tingling and discomfort After days they willbe asked to remove the seeds from their ears and restfor a day [] The women will be reminded daily bysending the text and will be reminded by phone for thenext interventional session This procedure will be performed for ten sessions for each participant It should benoted that if a participant has problems during the useof the method for any reason displacement of seedsdiscomfort etc she can remove the previous seed andask the researcher to reattach new seedsSham control groupThe control group will have sessions that will be thesame as the intervention group except that no Vaccariaseeds will be placed on the ear acupointsMeasuresIn this studycollectionfour measures will be used for dataDemographic and Obstetric QuestionnaireThe demographic section includes questions such asage education level occupation place of residence economic status age at marriage and length of marriageThe obstetric section includes questions regarding thenumber of pregnancies type of contraceptive methoddelivery date type of delivery perinealinstrumental delivery history of painful intercourse in prenatalperiod infant gender birth weight first lactation number of intercourse per month before pregnancy onset offirst intercourse after delivery and average number ofintercourses per week during lactation Validity of thisquestionnaire will be confirmed by some of the facultymembers ofthe midwifery department of QazvinUniversity of Medical Sciences IraninjuryFig The selected ear acupoints 0cBarghamadi Trials Page of Female Sexual Function Index FSFIIt has been designed by Rosen to evaluate sexualfunction in women over the past weeks [] It consistsof items subdivided into six subcategories of sex desire items arousal items lubrication items asm items satisfaction items and pain itemsThese subcategories have response ranges from or to with higher scores indicating better sexual performance [] The questionnaire has been used in manystudies abroad and has shown to have a high degree ofinternal consistency reliability and validity [] Thefindings of Fakhri and Mohammadi™s study showed thatthe Farsi version of FSFI FSFIIV is a reliable and validinstrument with appropriate psychometric properties toevaluate women™s sexual function [ ] Reliability ofthe scale has been calculated through stability analysisor calculation of the internal consistency coefficient TheCronbach™s alpha coefficient was and was higher forall domains and for the whole scale [] In addition reliability was confirmed by the testretest method and thecorrelation coefficient was reported high indicating thatFSFIIV is reusable within a 4week interval [ “]The maximum score for each domain is and for thewhole scale is Also any score less than will beused to indicate sexual dysfunction []The Edinburgh Postpartum Depression Rating ScaleThis 10item scale has been designed to detect depression from weeks after delivery The Edinburgh Scalescores vary between and with cutoff point andabove to detect postpartum depression [] Psychometric evaluation of the Farsi version of this scale is carriedout in [] The Cronbach™s alpha for the Edinburgh Scale has been reported as Validity of the Edinburgh Beck Scale has been reported as Thefindings of the study confirmed the high validity of theFarsi version of the Edinburgh Scale for the diagnosis ofpostpartum depression []The Sexual Quality of LifeFemale SQOLFIt is a short tool that specifically assesses the relationshipbetween sexual function and women™s quality of life Ithas been developed by Symonds [] This 18itemquestionnaire focuses on sexual selfesteem emotionalaffairs and relationships Each item is responded with aLikert scale of strongly agree score to strongly disagree score Higher scores indicate better quality ofsexual life for women [] Validity and reliability of theFarsi version ofthis scale have been confirmed byMaasoumi []Primary outcomePrimary outcome of this study is to investigate changesof sexual function using the FSFISecondary outcomeThe secondary endpoint is investigating the quality ofwomen™s sexuallife which will be assessed using theSexual Quality of Life Questionnaire Any harm or sideeffects will be also reportedStudy procedureAfter obtaining the necessary permissions from the Ethics Committee of Qazvin University of Medical Sciencesregistration at the Iranian Clinical Trial RegistrationCenter IRCT and obtaining the permission from Qazvin Nursing and Midwifery School the researcher willattend for recruitment in Qazvin Health Centers Thelactating women of each center will be invited for participation The process and purpose of the research willbe explained to those who meet the inclusion criteriaAlso they will be ensured that their information will bekept confidential and that the questionnaires will be anonymous and coded in accordance with the researchethics They will enter into the study after signing thewritten consent form The researcher will ensure thatthey can leave the study at any time After enrolling eligible individuals a simple block randomization will beused Participants will be randomly assigned to the intervention and control groupsThe questionnaires will be filled out by the participants before the intervention Then the interventionacupressure will be performed as mentioned aboveEvaluation of sexual function in both the interventionand control groups will be performed using the FSFIquestionnaire immediately and months after theintervention Figure provides the timeline of recruitment allocation and assessmentStatistical analysisData will be analyzed using the chisquare test Fisherexact test and independent t test via the SPSS v24 software The KolmogorovSmirnov and Shapiro tests willalso be used to check for the normal distribution of sexual function scores Descriptive statistics number meanand standard deviation will be used to describe thedemographic characteristics of the participants The repeated measure ANOVA will be used to compare themean sexual function of women before and after theintervention The significance level of all tests is set asp Methods to protect against biasRandomization and allocationSampling will be made using a twostep samplingmethod In the first step Qazvin City will be consideredas five geographical districts Next two health centerswill be randomly selected in each of these five districtsof Qazvin City Secondly women referred to selected 0cBarghamadi Trials Page of STUDY PERIODEnrolment AllocationPostallocationCloseoutTIMEPOINT1t1t2t3t4t5t6t7t8t9t10t11t12t13tXENROLLMENTEligibility screenInformed consentRandomized by independent personAllocationINTERVENTIONSGroup A Auricular acupressureGroup B ControlASSESSMENTSFSFISQOLXXXXXXX X XXFig Schedule of enrolment interventions and assessmentshealth centers will be assessed for eligibility and in caseof willingness to participate in the study will be recruited They will be randomly assigned to two groupsas follows One letter is assigned to each group A intervention group B control group and all possible conditions for the 4block will be written and numbered asfollows 1AABB 2ABAB 3BBAA 4BABA 5ABBA6BAAB In a simple block randomization method using thetable of random numbers numbers of blocks will be selected until the specified sample size is achieved Therandom allocation sequence is specified For example ifthe numbers given are and respectively theallocation sequence will be as follows AABB ABABABAB BBAA As a result each participant will have aunique code n Allocation concealmentFor this purpose after preparing the allocation sequencethe sequence is annotated on paper and placed inopaque sealed envelopes which will be numbered consecutively The questionnaires will be coded in the samemanner A questionnaire with the same code will becompleted by the person who receives code intervention The assignment sequence and its concealment willbe performed by someone outside the research teamBlindingParticipants outcome assessor and statistical analyzerwill be blind to which group a participant was placedThe individual entering the data from the participantquestionnaires on demographic information postpartumdepression female sexual functioning and female qualityof life will be blind to which participant was assigned tothe auricular acupressure seed group or the sham control groupTreatment fidelityOne of the researchers SB is responsible to performintervention She has participated specific course on auriculotherapy and is supervised by acupuncturist MHAabout how to perform the intervention Approximately of intervention sessions will be run under supervision of the acupuncturist to ensure that all acupoints arechosen correctlyData managementOne of the researchers SB will be responsible for collecting data at each study stage and will assign anotherindividual for the task of data entry into the SPSS software This process will be performed under supervisionof NB and ZA 0cBarghamadi Trials Page of Ethical and safety issuesThe research protocol will be reviewed and approved bythe Ethics and Human Research Committee of QazvinUniversity of Medical Sciences decree code IRQUMSREC1398056 It is registered on the Iranian ClinicalTrial Registration Center underofIRCT20190626044028N1 In addition the following ethical considerations will be considered throughout this research obtaining written informed consent the volunteernature of participation in the study ability to withdrawfrom the study at any time and confidentiality of information even during the publication of findings All ethical issues related to clinical trials will be considered Informedconsent during recruitment phase will be obtained by SBdecreecodeDiscussionTo the best of our knowledge this study is the first randomized clinical trial investigating the effect of ear acupressure on the sexual function of lactating women Thestrength of this study is the randomized design with parallel treatment procedures and a large sample size butdue to the importance of the treatment practitionerknowing whether they are placing an adhesive patchwith a Vaccaria seed or without one a true double blindstudy is not possible Acupressure has no known side effects is noninvasive and is easy to use [ ] Therefore the therapist and clients can develop a sense ofclosenesstrust and confidence [] Several studieshave shown promising results about the effect of earacupressure on insomnia [ ] menstrual cramps anddysmenorrhea [ ] premenstrual pain []chemotherapyinduced nausea and vomiting [] smoking cessation [] obesity [] and shortness of breath[] In the study of Oakley acupuncture wasaffective in premenopausal women with sexual function[] In line with previous literature this study wouldprovide insights about the impact of ear acupressure onsexual function in lactating womenAbbreviationsCONSORT Consolidated Standards of Reporting Trials SQOLF Sexual Qualityof LifeFemaleAcknowledgementsNot any in this stageTrial statusThe recruitment has not yet begun and necessary permissions are acquiredthe estimated date of recruitment is November The expected timefor completing the recruitment is March Protocol version registered on August Authors™ contributionsAll authors SB ZA NB and TO contributed to the design of the study SBNB and ZA drafted the preliminary manuscript TO revised the manuscriptand prepared the final version of the manuscript All authors revised themanuscript agreed to be fully accountable for ensuring the integrity andaccuracy of the study and read and approved the final version of themanuscript to be published All the authors met the criteria for authorshipand listed as coauthors on the title pageFundingThe research deputy of Qazvin University of Medical Sciences will providefinancial support Study funders have no role in the study design thecollection management analysis and interpretation of data the writing ofthe report and the decision to submit the report for publicationAvailability of data and materialsAnalyzed data and materials will be deidentified and publishedEthics approval and consent to participateResearch protocol is approved by the Ethics and Human ResearchCommittee of Qazvin University of Medical Sciences decree codeIRQUMSREC1398056 Permissions from responsible authority will beobtained before recruitment Informed consent will be acquired beforeparticipationConsent for publicationNot applicableCompeting interestsNone to declareAuthor details1Student Research Committee Qazvin University of Medical Sciences QazvinIran 2Social Determinants of Health Research Center Research Institute forPrevention of NonCommunicable Diseases Qazvin University of MedicalSciences Shahid Bahonar Blvd Qazvin Iran 3Emperor™s College of TraditionalOriental Medicine Santa Monica CA USAReceived November Accepted August ReferencesAbdelhakm EM Said AR Elsayed DMS Effect of PLISSIT model sexualcounseling program on sexual quality of life for postpartum women Am JNurs Sci Avery MD Duckett L Frantzich CR The experience of sexuality duringbreastfeeding among primiparous women J Midwifery Womens Health“Acele EÖ Karaçam Z Sexual problems in women during the firstpostpartum year and related conditions J Clin Nurs ““ Mohammed YF Hassan HM AlDinary AM Rashed NS Sexual function afterchild birth according to the mode of delivery AAMJ “Si H Kumamoto Y Sato Y Masumori N Horita H Kato R Prevalenceof female sexual dysfunction symptoms and its relationship to quality of lifea Japanese female cohort study Urology “ Williams A HerronMarx S Carolyn H The prevalence of enduring postnatalperineal morbidity and its relationship to perineal trauma Midwifery “Ahmad Shirvani M Bagheri NM Sexual dysfunction and related factorsamong breast feeding women Iran J Obstet Gynecol Infertility “Tork Zahrani S Banaei M Ozgoli G Azad M Investigation of the postpartumfemale sexual dysfunction in breastfeeding women referring to healthcarecenters of Bandar Abbas Iran J Obstet Gynecol Infertility “Yörük F Karaçam Z The effectiveness of the PLISSIT model in solvingpostpartum sexual problems experienced by women Athens J Health “ Davis SR Van Der Mooren M van Lunsen RH Lopes P Ribot J Rees M et alEfficacy and safety of a testosterone patch for the treatment of hypoactivesexual desire disorder in surgically menopausal women a randomizedplacebocontrolled trial Menopause “ Belkin ZR Krapf JM Goldstein AT Drugs in early clinical development forthe treatment of female sexual dysfunction Expert Opin Investig Drugs“ Ozgoli G Mobarakabadi SS Heshmat R Majd HA Sheikhan Z Effect of LI4and BL32 acupressure on labor pain and delivery outcome in the first stage 0cBarghamadi Trials Page of of labor in primiparous women a randomized controlled trial ComplementTher Med “ Oleson T Auriculotherapy manual Chinese and Western systems of ear Mohammadi KH Heydari M Faghihzadeh S The Female Sexual FunctionIndex FSFI validation of the Iranian version Health Monit J Iran Inst HealthSci Res Burri A Cherkas L Spector T Replication of psychometric properties of theFSFI and validation of a modified version FSFILL assessing lifelong sexualfunction in an unselected sample of females J Sex Med “Sidi H Abdullah N Puteh SEW Midin M The female sexual function indexFSFI validation of the Malay version J Sex Med “Sun X Li C Jin L Fan Y Wang D Development and validation of Chineseversion of female sexual function index in a Chinese population”a pilotstudy J Sex Med “Ter Kuile MM Brauer M Laan E The female sexual function index FSFI andthe female sexual distress scale FSDS psychometric properties within aDutch population J Sex Marital Ther “ Cox JL Holden JM Sagovsky R Detection of postnatal depressiondevelopment of the 10item Edinburgh Postnatal Depression Scale Br JPsychiatry “ Ahmadi kani Golzar A GoliZadeh Z Validation of Edinburgh PostpartumDepression Scale EPDS for screening postpartum depression in Iran J NursEduc “Symonds T Boolell M Quirk F Development of a questionnaire on sexualquality of life in women J Sex Marital Ther “ Maasoumi R Lamyian M Montazeri A Azin SA AguilarVafaie ME HajizadehE The sexual quality of lifefemale SQOLF questionnaire translation andpsychometric properties of the Iranian version Reprod Health Oakley SH WaltherLiu J Crisp C Pauls R Acupuncture in premenopausalwomen with hypoactive sexual desire disorder a prospective cohort pilotstudy Sexual medicine 201643e176“e81Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsacupuncture Yeh ML Chang YC Huang YY Lee TY A randomized controlled trial ofauricular acupressure in heart rate variability and quality of life forhypertension Complement Ther Med “Tan JY Molassiotis A Wang T Suen LK Current evidence on auriculartherapy for chemotherapyinduced nausea and vomiting in cancer patientsa systematic review of randomized controlled trials Evid BasedComplement Alternat Med Yeh CH Chien LC Chiang YC Lin SW Huang CK Ren D Reduction innausea and vomiting in children undergoing cancer chemotherapy byeither appropriate or sham auricular acupuncture points with standard careJ Altern Complement Med “ Abbate S Chinese auricular acupuncture Florida Routledge Yeh TL Chen HH Pai TP Liu SJ Wu SL Sun FJ The effect ofauricular acupoint stimulation in overweight and obese adults a systematicreview and metaanalysis of randomized controlled trials Evid BasedComplement Alternat Med Yeh CH Chiang YC Hoffman SL Liang Z Klem ML Tam WW Efficacyof auricular therapy for pain management a systematic review and metaanalysis Evid Based Complement Alternat Med Kim JY Ryu HS Nam SH Park KS Effects of auricular acupressure therapy onnocturia and insomnia in the elderly Korean J Rehabil Nurs “ Wang YJ Hsu CC Yeh ML Lin JG Auricular acupressure to improvemenstrual pain and menstrual distress and heart rate variability for primarydysmenorrhea in youth with stress Evid Based Complement Alternat MedShergis JL Ni X Jackson ML Zhang AL Guo X Li Y A systematicreview of acupuncture for sleep quality in people with insomniaComplement Ther Med “ Dantas KKdL Auriculoterapia chinesa com o uso de sementes de colza nadismenorreia primaria relato de caso Universidade Federal do Rio Grandedo Norte Portman DJ Bachmann GA Simon JA Group OS Ospemifene a novelselective estrogen receptor modulator for treating dyspareunia associatedwith postmenopausal vulvar and vaginal atrophy Menopause “ Oleson T Flocco W Randomized controlled study of premenstrualsymptoms treated with ear hand and foot reflexology Obstet Gynecol“Kwon SJ Park JS Effects of auricular acupressure on chemotherapyinducednausea vomiting and serum serotonin level Korean J Adult Nurs “ Di YM May BH Zhang AL Zhou IW Worsnop C Xue CC A metaanalysis ofearacupuncture earacupressure and auriculotherapy for cigarette smokingcessation Drug Alcohol Depend “ Huang ETY Di PhD YM Acupuncture therapies for chronic obstructivepulmonary disease a systematic review of randomized controlled trialsAltern Ther Health Med Dai L Cheng CW Tian R Zhong LL Li YP Lyu AP Standard ProtocolItems for Clinical Trials with Traditional Chinese Medicine recommendations explanation and elaboration SPIRITTCM extension Chin J Integr Med “ Bokaie M Hajimaghsoudi S Dehghani A Hosseini F The effect of sexualhealth counselling on the sexual function and satisfaction of breastfeedingwomen in the form of group consultation and telephone consultancyJ Adv Pharm Educ Res 20199S2191“ Rosen CB Heiman J Leiblum S Meston C Shabsigh R Ferguson DD'Agostino R The Female Sexual Function Index FSFI a multidimensionalselfreport instrument for the assessment of female sexual function J SexMarital Ther “Fakhri A Pakpour AH Burri A Morshedi H Zeidi IM The Female SexualFunction Index translation and validation of an Iranian version J Sex Med“ Wiegel M Meston C Rosen R The female sexual function index FSFI crossvalidation and development of clinical cutoff scores J Sex Marital Ther“ 0c"

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